α-Crystallin B prevents apoptosis after H2O2 exposure in mouse neonatal cardiomyocytes
demonstrated that overexpression of cryAB has potent cardioprotective properties in cultured rat cardiomyocytes (CMs) (28) or in hearts of transgenic mice (32), protecting them from ischemia-reperfusion (I/R) damage. Additionally, using in vitro models, cryAB has been shown to prevent apoptosis induced by various insults, including H 2O 2 treatment (25).In contrast, silencing of cryAB increases sensitivity to I/R injury and leads to increased cell death in mouse hearts (29). Recently, our group (5) has shown that cryAB is protective against apoptosis in a mouse model of calcineurin hypertrophy, which presents with elevated levels of endoplasmic reticulum (ER) stress (5).The protective mechanism of cryAB in response to stress has not been fully characterized,but a previous study (3) has indicated that cryAB binds myofilament proteins, thereby preserving contractile protein integrity and myocardial function. CryAB also translocates to the mitochondria (27) and is phosphorylated on Ser 59 (18) in response to ischemia and other stresses (17), which is required for limiting myocardial cell apoptosis (28), although the precise interactions mediating this protection have not been elucidated.Apoptosis in cells occurs by two pathways: the intrinsic pathway involving mitochondria or the extrinsic pathway downstream of death receptors. The mitochondrial and death receptor pathways activate distinct apical caspases (caspase 9 or caspase 8, respectively) that activate the downstream executioner caspase 3 (43). Ischemia or other stresses that mimic ischemia activate the intrinsic apoptotic pathway, leading to the opening of the mitochondrial permeability transition pore (MPTP) and further downstream apoptotic events, culminating in cellular demise (11). Global ischemia as well as reperfusion have been associated with significant increases in ROS, including myocardial H 2O 2 content (37), which plays a significant role in oxidative stress injury (39). H 2O 2 also leads to apoptosis in CMs, by activating the intrinsic pathway of apoptosis (41), and thus makes H 2O 2 a very good in vitro model of I/R injury.
The same stress stimuli that trigger apoptosis, such as oxidative stress (25, 41), induce the
synthesis of diverse HSPs, which confer a protective effect against a wide range of cellular
stresses. Recent evidence indicates that many HSPs are anti-apoptotic by inhibiting one or
more components in the apoptotic cascade (2, 8). In this regard, cryAB has been shown to
directly interact with precursors of caspase 3 to suppress its activation in an immortalized
rabbit lens epithelial cell line (25) and in breast carcinoma cells (20). In human lens
epithelial cells, cryAB has also been shown to interact with the proapoptotic proteins Bax
and Bcl-X S , preventing their translocation from the cytosol to the mitochondria, thus leading
to decreased apoptosis (26). The antiapoptotic mechanisms of cryAB in CMs, however, have
not been fully characterized. Thus, the purpose of this study was to elucidate the
mechanisms by which cryAB prevents apoptosis in CMs with a specific focus on the
mitochondrial pathway in response to H 2O 2-induced oxidative stress, an in vitro model that
mimics I/R (37).
MATERIALS AND METHODS
Neonatal CM isolation and culture
Neonatal mice were euthanized using isoflurane, in accordance with procedures approved by
our institutional Animal Care and Ethics Committee. Hearts were harvested and placed in
ice-cold Hanks’ solution [containing 136 mM NaCl, 4.2 mM KCl, 5.6 mM dextrose, 0.44
mM KH 2PO 4, 0.34 mM NaH 2PO 4, 4.2 mM NaHCO 3, 5 mM HEPES (pH 7.4), and 100 U/
ml penicillin-streptomycin (Invitrogen)]. Atria were removed and discarded, and ventricles
were cut into small pieces (2– 4 pieces/heart) and washed several times with Hanks’
solution. After washes, tissues were incubated in fresh Hanks’ solution with 1 mg/ml
collagenase type II (Worthington Biochemicals) and subjected to gentle rocking for 2 h at
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room temperature. The suspended cells were pelleted by centrifugation at 800 g for 5 min.Fibroblasts were removed through preplating in 10% FBS-containing media for 1 h.Lentivector production and transduction of neonatal CMs Lentivector compatible short hairpin (sh)RNA clones targeting mouse cryAB were obtained from Open-Biosystems (Thermo Scientific). The scrambled shRNA construct used as a negative control was a kind gift from Stephane Angers (University of Toronto, Toronto, ON,Canada). Lentivector production and transduction of neonatal CMs were performed as previously described (5). Plasmids were isolated using Qiagen Maxi preparations according to the manufacturer’s instructions. The packaging plasmid (pCMV-R8.74psPAX2, 2.5 μg),envelope plasmid (VSV-G/pMD2.G, 0.3 μg), and target construct plasmid (pLKO.1, 2.7 μg)expressing either the shRNA or scrambled (Scram) shRNA (as a negative control) were simultaneously transduced into human embryonic kidney (HEK)-293T cells using FuGene (Roche) diluted in Optimem (Invitrogen). Neonatal CMs were incubated with supernatant from transduced HEK-293T cells for 21 h, after which the medium was replaced daily.Transduced CMs were selected by an incubation with 2 μg/ml puromycin for 48 h to remove all nontransduced cells to ensure a homogenous population of transduced cells.Subcellular fractionation and sucrose gradient separation Adult mice were euthanized by CO 2 asphyxiation. Hearts were harvested, and ventricular tissue was isolated. The tissue was rinsed with ice-cold PBS to remove any remaining blood and placed in ice-cold lysis buffer [containing 250 mM sucrose, 50 mM Tris·HCl (pH 7.4), 5mM MgCl 2, 1 mM DTT, and 1 mM PMSF]. The tissue was dounce homogenized, and differential centrifugation was carried out to isolate nuclear, cytosolic, microsomal, and mitochondrial fractions, as previously described (9). The homogenized sample was also used for sucrose gradient separation, as previously described (34).
CMs from neonatal mice were cultured as described above. Cells were maintained in culture
for 5 days, and on the sixth day they were either maintained in culture or stressed with H 2O 2(ranging in concentration from 0 to 200 μM) for 24 h. CMs were rinsed with PBS and
collected in lysis buffer as described above. CMs in lysis buffer were dounce homogenized,
and differential centrifugation was carried out to isolate cytosolic and organellar (including
the mitochondria) fractions, as previously described (9).
Sample preparation for transmission electron microscopy
CM fixation was performed at the Department of Pathology and Laboratory Medicine of
Mount Sinai Hospital (Toronto, ON, Canada). Briefly, neonatal CMs transduced with either
Scram shRNA or cryAB-targeting shRNA [cryAB knockdown (KD)] for transmission
electron microscopy (TEM) were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate
buffer, rinsed in buffer, postfixed in 1% osmium tetroxide in buffer, dehydrated in a graded
ethanol series followed by propylene oxide, and embedded in Quetol-Spurr resin. Sections
(100 nm thick) were cut on an RMC MT6000 ultramicrotome, stained with uranyl acetate
and lead citrate, and viewed in an FEI Tecnai 20 TEM.
Immunogold labeling
Immunogold labeling of cryAB was performed at the Department of Pathology and
Laboratory Medicine of Mount Sinai Hospital. Whole hearts from adult mice were fixed in
4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium cacodylate buffer, rinsed in
buffer, dehydrated in a graded ethanol series with progressive lowering of temperature, and
embedded in LR white resin. Sections (100 nm thick) were cut on an RMC MT6000 ultra-
microtome, labeled with anti-cryAB and anti-phosphorylated cryAB (PcryAB) antibodies
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followed by 10-nm gold-conjugated secondary antibodies, stained with uranyl acetate and lead citrate, and viewed in an FEI Tecnai 20 TEM. Negative controls consisted of gold-conjugated secondary antibody alone.Viability assays Neonatal CMs were subjected to 60 μM H 2O 2 for 24 h. Viability assays were carried out using a cell counting kit (CCK-8, Dodinjo) according to the manufacturer’s instructions.Early apoptosis was determined based on the dissipation of mitochondrial membrane potential, as measured by JC-1 fluorescent dye (Abcam), a mitochondrial dye, which was added at a concentration of 2.0 μM (10) according to the manufacturer’s instructions.Apoptosis was measured by detecting caspase 3 activity using an assay (R&D Systems) as per the manufacturer’s instructions and by labeling of TUNEL-positive nuclei (PRP-Histology Laboratory, University Health Network, Toronto, ON, Canada). The caspase 3inhibitor Z-D(OMe)-E(OMe)-V-D(OMe)fluoromethyl ketone (Z-DEVD-FMK; R&D Systems) was used at a concentration of 100 μM (7) according to the manufacturer’s instructions.ROS detection and inhibition The presence of ROS in cultured wild-type (WT) and KD neonatal CMs was detected using CellROX deep red reagent (Invitrogen), a fluorogenic probe, according to the manufacturer’s instructions. Briefly, CMs were seeded on 96-well plates and transduced with Scram shRNA or shRNA for cryAB and maintained in culture or stressed with 60 μM H 2O 2. The dye was then added at a concentration of 5 μM, and fluorescence was measured with a Perkin-Elmer plate reader. The ROS scavengers tiron (41) and sodium pyruvate (42)(Sigma) were used at concentrations of 1.0 mM.Coimmunoprecipitation
Immunoprecipitations were carried out using protein A/G-agarose beads (Thermo
Scientific). Briefly, heart tissue homogenates were obtained from WT control hearts or
hearts exposed to 100 μM H 2O 2 for 1 h (41). The tissue was collected in lysis buffer, as
described above. The lysate was cleared by centrifugation for 15 min at 8,000 g at 4°C. To
allow antibody-protein complex formation, the cleared lysate was incubated at 4°C under
continuous rotation with antibody in binding buffer [140 mM NaCl and 14 mM KCl (pH
7.4)] and 0.1% Triton X-100 with 0.01% BSA for 2 h. Protein A/G-agarose beads were
blocked in 0.1% BSA in binding buffer for 2 h. The beads were pelleted, added to the
protein sample, and rotated overnight at 4°C. Samples were washed three times and eluted in
0.1 M glycine (pH 2.4).
Immunoblot analysis
Total cellular protein was harvested from control cardiac ventricular homogenates from WT
animals or from cardiac ventricular homogenates stressed with 100 μM H 2O 2 for 1 h and
was subjected to standard immunoblot analysis. Protein concentrations were determined by
Bradford assay, and equal protein levels were loaded. The following antibodies were used to
target specific proteins: rabbit polyclonal to cryAB (Stressgen, 1:1,000), Ser 59 PcryAB
(Stressgen, 1:1,000), rabbit polyclonal to caspase 3 (Abcam, 1:1,000), rabbit polyclonal to
caspase 12 (Abcam, 1:1,000), mouse monoclonal to caspase 9 (Cell Signalling
Technologies, 1:1,000), rabbit polyclonal to cytochrome c (Cell Signalling Technologies,
1:1,000), goat polyclonal to voltage-dependent anion channel (VDAC; Santa Cruz
Biotechnology, 1:100), mouse monoclonal to translocase of outer mitochondrial membranes
20 kDa (TOM 20; Santa Cruz Biotechnology, 1:1,000), and mouse monoclonal to cryAB
(Santa Cruz Biotechnology, 1:100).
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Immunofluorescence Adult CMs were isolated as previously described (33) and fixed in ice-cold 90% methanol.Staining was performed as previously described (34). Briefly, cells were dissociated in modified Hanks’ solution (Hanks’ solution containing 10 mM taurine, 0.1 mM EGTA, 10mM 2,3-butanedione monoxime, 1 mg/ml BSA, and 1 mg/ml collagenase) with a magnetic stir bar added to the tube and rotated very gently at 37°C for 5 min to dissociate cells. Once dissociated, cells were fixed in 90% ice-cold methanol. For immuno-fluorescence,nonspecific interactions were first suppressed with 5% horse serum in permeabilization buffer [0.2% Tween 20 and 0.5% Triton X-100 in PBS (pH 7.0)] for 30 min, and samples were then incubated with primary antibodies in permeabilization buffer overnight at 4°C.Samples were then washed three times with PBS and incubated with either Alexa fluor 488or Alexa fluor 633. Two-dimensional images were collected using a Leica DM IRBE inverted microscope equipped with a Leica TCS SP laser scanning confocal system. Spectra for Alexa fluor 488 were collected by excitation at 488 nm and emission between 490 and 510 nm; images for Alexa fluor 633 were collected by excitation at 633 nm and emission between 640 and 670 nm. Three-dimensional (3-D) images were collected using a Quorum Angstrom Grid Axiovert 200M inverted structured illumination microscope system. For 3-D region of interest analyses, sequential sections of stained cells were acquired for 3-D image reconstruction and colocalization measurements. A 3-D volume was constructed from sequential z -sections of cells using Imaris software (version 7.4.2, Bitplane, Zurich,Switzerland). Colocalization statistics were calculated using Imaris software.Statistical analysis Statistical differences were determined by ANOVA and an unpaired Student’s t -test. Post hoc Tukey tests were performed when ANOVA was significant. Two-way ANOVA was used to determine interactions between independent variables, cryAB KD and viability, or
cryAB KD and apoptosis. Results were considered significant at P < 0.05.
RESULTS
Expression pattern of cryAB in mouse cardiac muscle
CryAB protein is diffusely present in the cytosol under normal physiological conditions in
CMs (23). To determine the endogenous distribution of cryAB protein among different
cellular compartments, subcellular fractionation of adult mouse hearts was performed by
differential centrifugation and sucrose gradient separation. Differential centrifugation was
used to generate nuclear, cell organellar (including the mitochondria), microsomal, and
cytosolic fractions. The positive fraction markers used were GAPDH for the cytosol, histone
H3 for the nucleus, Na +-K +-ATPase for the microsomes, and VDAC for the organellar/
mitochondrial fraction. As shown in Fig. 1A , fractions were relatively pure, as GAPDH was
exclusively found in the cytosolic fraction, VDAC was found restricted to the mitochondrial
fraction, histone H3 was preferentially found in the nuclear fraction, and Na +-K +-ATPase
was predominately found in the microsomal fraction with some expression in the organellar/
mitochondrial fraction. Immunoblot analysis for the expression of cryAB in each subcellular
fractions showed the highest levels (83% of total levels) seen in the cytosol, in agreement
with the literature (23). The remaining levels were 4% of total levels in the microsomal
fraction, 12% of total levels in the nuclear fraction, and negligible levels (<1%) in the
nuclear fraction (Fig. 1A ). PcryAB, on the other hand, was predominately found in the
mitochondrial fraction (75% of total levels) and at lower levels in the cytosol (25% of total
levels). The cytosolic localization of cryAB was further confirmed by sucrose gradient
separation of whole heart lysates. As shown in Fig. 1B , cryAB was restricted to fractions 4 –
6, with some expression in fraction 7. These data are consistent with GAPDH, a known
cytosolic protein, which was also detected in fractions 4 – 6. VDAC, a mitochondrial
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marker, was mainly isolated with fractions 2– 4 and showed some expression in fractions 5and 6. PcryAB was mainly isolated in fractions 2 and 3, consistent with VDAC, and to a lower extent in fraction 4, consistent with GAPDH, suggesting a mainly mitochondrial localization of PcryAB. Na +-K +-ATPase, a plasma membrane protein normally found within the microsome biochemical fraction (22), was isolated within the light fractions (with some expression in fractions 8 –12). Finally, electron microscopy images showed the distribution of cryAB in the cytosol and PcryAB predominantly in the mitochondria in adult mouse cardiac muscle. The representative images shown in Fig. 1C demonstrate that ~75% of gold-labeled cryAB (111 of 149 particles in 10 separate images) were found in the cytosol, 11%in the mitochondria, and 14% associated with the sarcomeres. When similar experiments were performed using gold-labeled PcryAB antibody, 75% of the gold particles were found in the mitochondria, whereas 10% were found in the cytosol and 15% were in the sarcomeres (73 total particles were observed in 10 separate images). Negative controls were labeled with gold-conjugated secondary antibody alone.CryAB silencing induces loss of viability in CMs To determine whether cryAB expression in mouse CMs affects cell viability, we silenced cryAB using lentivector-mediated transduction of shRNA targeting cryAB expression.Figure 2A shows the reduced levels of cryAB expression after the transduction of cryAB-specific shRNA lentivirus relative to a Scram shRNA (control vector). The transduction resulted in reduced cryAB expression to 28 ± 2% in KD CMs compared with Scram shRNA CMs (P < 0.05; Fig. 2, A and B ).Since cryAB has antiapoptotic properties (25, 28, 32) and a previous study (25) has shown that it prevents apoptosis induced by oxidative insult, neonatal CMs transduced with either cryAB-targeting lentivectors or Scram shRNA control lentivectors were analyzed after the administration of 60, 100, or 200 μM H 2O 2 for 24 h (41) to explore the role of cryAB on the
viability of CMs exposed to H 2O 2. CryAB KD neonatal CMs showed a significant reduction
in viability compared with neonatal CMs transduced with the Scram shRNA control
construct at all H 2O 2 concentrations used (Fig. 2C ). As expected, in Scram shRNA CMs,
there was a progressive decrease in viability with increasing H 2O 2 concentrations relative to
reference levels at baseline. In the absence of H 2O 2, viability in cryAB KD CMs was 50 ±
6% compared with levels seen in Scram shRNA CMs (P < 0.05). Decreases in viability were
observed in KD CMs with increasing H 2O 2 concentrations, and these were significantly
more pronounced at all H 2O 2 concentrations tested. Altogether, cryAB KD appears to
promote cell death at baseline and after exposure to H 2O 2, and exposure to H 2O 2 seems to
cause reductions in viability in Scram shRNA and cryAB KD CMs, which are more
pronounced in KD CMs. Since cryAB KD alone induced significant cell death and treatment
with 60 μM H 2O 2 led to significant reductions in viability in both Scram shRNA and cryAB
KD CMs, subsequent experiments were performed using 60 μM H 2O 2 to exacerbate the cell
death initially observed at baseline in KD CMs.
To confirm the activation of apoptosis at baseline and after exposure to H 2O 2, we measured
caspase 3 activity in control and cryAB KD CMs under resting conditions and after
exposure to H 2O 2 and in the absence or presence of caspase 3 inhibitors (Fig. 2D ). Caspase
3 activity levels in Scram shRNA CMs under baseline conditions were set as a reference
level of 1 arbitrary unit (AU). In cryAB KD CMs, caspase 3 activity levels were
significantly higher than in Scram shRNA control CMs (130 ± 2%) at rest (P < 0.05). The
addition of caspase 3 inhibitors had negligible effects on caspase 3 activity levels in Scram
shRNA CMs under basal conditions, bringing the levels down to 90 ± 2% compared with
baseline conditions. In KD CMs, however, caspase 3 activity levels were significantly
lowered to 70 ± 2% of their baseline levels after the addition of caspase 3 inhibitors (P <
0.05). As expected, after exposure to H 2O 2, caspase 3 activity levels increased to 240 ± 4%
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in control CMs (P < 0.05), whereas in KD CMs, they increased approximately fivefold with the addition of H 2O 2 (460 ± 5% relative to Scram shRNA CM baseline levels). The addition of H 2O 2 together with caspase 3 inhibitors resulted in caspase 3 activity levels close to those observed at basal conditions (110 ± 3%) in Scram shRNA CMs and KD CMs (120 ± 3% of reference levels). We confirmed the levels of apoptosis in Scram shRNA CMs and cryAB KD CMs at baseline and after exposure to 60 μM H 2O 2 by TUNEL staining. Analysis of TUNEL-positive nuclei (Fig. 2E ) showed a significantly higher percentage of TUNEL-positive nuclei in cryAB KD CMs compared with Scram shRNA CMs at baseline (30 ± 3%vs. 9 ± 2%, P < 0.05) and after exposure to H 2O 2 (67 ± 6% vs. 30 ± 5%, P < 0.05). These results suggest that KD CMs underwent apoptosis by activation of caspase 3 more readily compared with Scram shRNA CMs at baseline, an effect that was exacerbated after exposure to H 2O 2, suggestive of a protective role for cryAB against apoptosis.To determine whether cryAB KD and exposure to H 2O 2 are interacting variables affecting CM viability, we performed two-way ANOVA and found that, although cryAB KD had significant effects on lowering viability (P < 0.05) and exposure to H 2O 2 also significantly lowered viability at all concentrations tested in Scram shRNA and cryAB KD CMs (P <0.05), the effects of cryAB KD and H 2O 2 exposure combined on CM viability seemed to be additive. In other words, the interaction of cryAB KD and H 2O 2 exposure only approached statistical significance (P < 0.1). However, as shown in Fig. 2, A and B , cryAB silencing was not 100% efficient, which could account for the higher than expected viability that was observed in cryAB KD CMs in the presence of extrinsic oxidative stress. We performed two-way ANOVA to determine whether cryAB KD and exposure to H 2O 2 were interacting variables affecting CM apoptosis. CryAB KD and H 2O 2 were interacting variables (P <0.05) that had synergistic effects on caspase 3 activity levels as well as on TUNEL staining (P < 0.05). These results suggest that although cryAB KD does not seem to have a significant effect on CM viability after H 2O 2, it does, however, have a significant effect on
CM apoptosis, as measured by caspase 3 activity levels and TUNEL staining.
Upregulation and translocation of cryAB to the mitochondria under stress conditions
Expression levels of cryAB have been shown to increase in response to H 2O 2 exposure (35).
To determine whether similar upregulation occurs in our CM model, CMs isolated from
adult mice were treated with 100 μM H 2O 2, based on the literature (41), and cryAB levels
were determined. Compared with untreated conditions, after exposure to H 2O 2, total cryAB
levels were significantly increased (2.1-fold over control levels, P < 0.05). PcryAB levels
were also significantly increased under H 2O 2 treatment, with a 1.8-fold increase (P < 0.05)
in protein levels (Fig. 3, A and B ).
Under conditions of I/R, cryAB translocates to the mitochondria and contractile units (19).
After I/R, phosphorylation of cryAB on the Ser 59 residue enhances protection against
apoptosis, and it has been suggested that cryAB and PcryAB may have different protective
effects in CMs (19). To determine the distribution of cryAB and PcryAB in H 2O 2-induced
oxidative stress, subcellular fractionation of cultured control neonatal mouse CMs and CMs
exposed to 60 μM H 2O 2 for 24 h was performed. Figure 3, C and D , shows higher levels of
cryAB expression in the cytosol and lower levels in the mitochondria. Under normal culture
conditions, cryAB levels observed in the cytosol were 4.8-fold higher than the levels
observed in the mitochondria. In contrast, upon exposure to 60 μM H 2O 2, cryAB
translocated to the mitochondria, resulting in a 2.1-fold increase in cryAB levels associated
with the mitochondria compared with control conditions (P < 0.05). PcryAB, on the other
hand, was found at very low levels in the cytosol compared with the levels observed in
mitochondria. Expression ratio levels of PcryAB were calculated to be 1:5 cytosolic to
mitochondrial. Under stress conditions, PcryAB was almost exclusively associated with the
mitochondria, with only < 5 ± 0.2% of total PcryAB detected in the cytosol (P < 0.05; Fig.
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3, C and D ). GAPDH and VDAC were used as fraction markers for the cytosol and
mitochondria, respectively. Under control conditions, GAPDH and VDAC were exclusively
found in the cytosolic and mitochondrial fraction, respectively. Taken together, these
findings suggest that part of the protective mechanism of cryAB after exposure to oxidative
stress may involve its translocation to the mitochondria.
CryAB silencing induces mitochondrial dysfunction
Since CM viability was significantly decreased under control conditions in cryAB KD CMs,it was of interest to determine the cause for the increased cell death in the absence of a stressor. The association of cryAB and PcryAB with the mitochondria after exposure to oxidative stress suggested potential mitochondrial involvement in cryAB KD-induced apoptosis. Furthermore, previously, cryAB R120G transgenic mice were demonstrated to exhibit desmin-related cardiomyopathy where mitochondrial respiration was compromised,leading to alterations in the permeability transition pore, compromised inner membrane potential, and elevated levels of apoptosis (24). To test whether reduced levels of cryAB might affect the mitochondria by the induction of oxidative stress, the levels of ROS in cryAB KD and Scram shRNA CMs were determined at baseline, after exposure to 60 μM H 2O 2, and in the presence of ROS scavengers (Fig. 4A ). At baseline, ROS levels in cryAB KD CMs were 1.9-fold higher than levels observed in Scram shRNA CMs (P < 0.05). With the addition of ROS scavengers, ROS levels were decreased in Scram shRNA CMs to 75 ±2% of the levels observed in Scram shRNA cells without ROS inhibition. In cryAB KD CMs treated with ROS scavengers, ROS levels were decreased to near control levels of 110± 3% relative to baseline levels observed in Scram shRNA CMs (significantly different compared with untreated cryAB KD CMs, P < 0.05). These results suggest that some oxidative stress was associated with the culture process but that there was higher production of ROS by the mitochondria in cryAB KD CMs with or without scavengers. After the
addition of H 2O 2, there was a 2.4-fold increase in ROS levels in Scram shRNA CMs (P <
0.05) and much greater 5.8-fold increase in cryAB KD CMs (P < 0.05) relative to reference
levels in Scram shRNA CMs. In the presence of H 2O 2 and ROS scavengers, cells with
Scram shRNA showed a 54 ± 7%, decrease in ROS levels compared with H 2O 2 treatment
alone, whereas in KD CMs, ROS levels were decreased to 55 ± 4% compared with levels
observed with H 2O 2 treatment alone but still remained elevated compared with Scram
shRNA CMs (P < 0.05; Fig. 4A ). These results suggest that the oxidative stress observed at
baseline in Scram shRNA and KD CMs is exacerbated by the addition of exogenous H 2O 2.
To determine the effect of reduced expression of cryAB and ROS production on
mitochondrial function, we assessed the dissipation of mitochondrial membrane potential, an
indicator of early apoptosis, using the mitochondrial dye JC-1. Increased dissipation of the
mitochondrial membrane potential in cryAB KD CMs was observed compared with Scramb
shRNA/control CMs at baseline. This effect was illustrated by the significant loss of JC-1
red fluorescence in cryAB KD CMs to 63 ± 8% of the levels that were observed in Scram
shRNA/control CMs at baseline (P < 0.05). In the presence of ROS scavengers under control
conditions, JC-1 fluorescence was increased by 12 ± 7% in control CMs relative to initial
levels at rest (P < 0.05) and by 2 ± 0.5% in KD CMs (P < 0.05) relative to their initial levels
at rest. These results suggest that both control and KD CMs were exposed to oxidative stress
under cell culturing conditions but that dissipation of membrane potential only occurred in
KD CMs in the absence or presence of ROS scavengers. The increased ROS observed in KD
CMs may occur alongside mitochondrial structure alterations that cannot be salvaged by the
removal of ROS alone, pointing to an involvement of cryAB at the mitochondrial level.
After exposure to 60 μM H 2O 2, JC-1 fluorescence decreased to 32 ± 2% in Scram shRNA
CMs (P < 0.05) and to only 9 ± 1% (P < 0.05) in cryAB KD CMs (Fig. 4B ). In the presence
of H 2O 2 together with ROS scavengers, JC-1 fluorescence was 63 ± 3% of its original value
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in control CMs. In KD CMs, JC-1 fluorescence was 26 ± 3% of its original value (P < 0.05).Together, these results suggest that the increased ROS levels in cryAB KD CMs contributed to the loss of mitochondrial membrane potential, which cannot be overcome with free radical scavengers (Fig. 4B ).Since we observed mitochondrial dysfunction in cryAB KD CMs, we investigated whether mitochondrial ultrastructure was disturbed by cryAB silencing. Electron microscopy images showed that in CMs transduced with Scram shRNA, the mitochondria were intact, with dense, well-ordered cristae (Fig. 5, A and B ). However, in cryAB KD CMs, approximately half of the observed mitochondria exhibited altered morphology and loss of density and organization of cristae, suggestive of mitochondrial swelling and rupture of the inner membrane (Fig. 5, C and D ). These results are consistent with our earlier results of mitochondrial dysfunction and increased ROS production (Fig. 4, A and B ).Identification of cryAB interactions with proteins involved in apoptosis after exposure to H 2O 2Our results suggest an involvement of cryAB in preventing ROS-induced apoptosis at the level of the mitochondria. Furthermore, in response to oxidative stress, cryAB and PcryAB have been observed to associate with the mitochondria (19); thus, an interaction with mitochondrial proteins (such as VDAC and TOM 20) may stabilize the mitochondria and,ultimately, prevent mitochondria-induced apoptosis. We hypothesized that cryAB also potentially regulates the apoptotic cascade downstream of the mitochondria by interacting with cytochrome c , caspase 9, caspase 3, and caspase 12. To mimic and exacerbate the oxidative stress observed after cryAB KD, we exposed adult WT CMs to 100 μM H 2O 2(40). Colocalization of cryAB with these proteins was shown by qualitative fluorescence images of adult CMs under control conditions and after exposure to 100 μM H 2O 2 imaged
for cryAB, in the green channel at 488 nm, and TOM 20, VDAC, cytochrome c , or caspase
3, imaged in the red channel at 633 nm (Fig. 6, A and B ). The right images in Fig. 6, A and
B , show the overlap of cryAB with TOM 20, VDAC, cytochrome c , and caspase 3,
suggestive of a potential interaction of cryAB with these proteins. Colocalization statistics
and Pearson and Manders’ coefficients (with M 1 being indicative of the fraction of cryAB
green staining overlap with red staining of TOM 20, VDAC, cytochrome c or caspase 3 and
M 2 being indicative of the fraction of TOM 20, VDAC, cytochrome c , or caspase 3 red
staining overlap with cryAB green staining) were determined for the two-dimensional
images using ImageJ JACoP (Table 1). This analysis indicated a significant degree of
colocalization between cryAB and TOM 20, between cryAB andVDAC, between cryAB
and cytochrome c , and between cryAB and caspase 3. Colocalization was significantly
increased after treatment with 100 μM H 2O 2 for cryAB and TOM 20, cryAB and VDAC,
cryAB and cytochrome c , and cryAB and caspase 3. In addition, we performed a
comprehensive 3-D analysis of ~100-μm-thick z -stacks to assess colocalization of cryAB
with TOM 20, VDAC, cytochrome c , and caspase 3 under control conditions (Fig. 6C ) and
after 100 μM H 2O 2 (Fig. 6D ). This analysis indicated a significant degree of colocalization
between cryAB and TOM 20, cryAB and VDAC, cryAB and cytochrome c , and cryAB and
caspase 3. The extent of colocalization was enhanced after treatment with 100 μM H 2O 2 for
all proteins (Fig. 6D ).
After exposure to 100 μM H 2O 2, immunoblot analysis of cellular lysates showed significant
increases in the levels of cryAB (82.7 ± 2.9 AU in H 2O 2 vs. 24.2 ± 7.7 AU in controls, P <
0.05) and PcryAB (17.9 ± 1.1 AU in H 2O 2 vs. 5.1 ± 1.5 AU in controls, P < 0.05; Fig. 7A ).
Levels of cleaved caspase 3 (9.7 ± 0.8 AU in H 2O 2 vs. 2.6 ± 0.3 AU in controls, P < 0.05)
and cleaved caspase 12 (56.4 ± 4.3 AU in H 2O 2 vs. 13.3 ± 0.9 AU in controls, P < 0.05)
showed significantly increased expression levels. However, expression levels of TOM 20
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(39.7 ± 6.7 AU in H 2O 2 vs. 36.9 ± 5.0 AU in controls) and VDAC (36.2 ± 0.5 AU in H 2O 2vs. 35.0 ± 4.6 AU in controls) were not significantly different from controls.Next, we immunoprecipitated cryAB and PcryAB under control conditions from adult mouse cardiac tissue. CryAB and PcryAB both formed complexes with TOM 20, VDAC,un-cleaved and cleaved caspase 3, caspase 9, and caspase 12 (Fig. 7B ). We performed similar experiments using cardiac tissue exposed to 100 μM H 2O 2 and observed that cryAB and PcryAB appeared to precipitate greater amounts of TOM 20, VDAC, uncleaved and cleaved caspase 3, and caspase 12 in the eluate from CMs exposed to 100 μM H 2O 2compared with control conditions (results not shown), suggesting a potential increase in the level of interaction after exposure to oxidative stress.We made numerous attempts to determine interactions of either cryAB or PcryAB with Bcl-X S and Bax (26) but were unable to detect signals under these conditions. Neither cryAB nor PcryAB were found to precipitate with cytochrome c , suggesting that their colocalization (Fig. 6) was not indicative of an interaction between these proteins.Since cryAB and PcryAB appeared to precipitate caspase 12 under control conditions (Fig.7B ), we hypothesized that this potential interaction led to decreased caspase 12 activation.Cleaved caspase 12 levels (30) were assessed in control CMs and KD CMs in the absence or presence of H 2O 2. Immunoblot analysis and quantification showed a significant sixfold increase in levels of cleaved caspase 12 in cryAB KD CMs compared with Scram shRNA control CMs at baseline (P < 0.05; Fig. 7, C and D ). After H 2O 2, cleaved caspase 12 showed a 2.1-fold increase in Scram shRNA CMs and an ~10-fold increase in cryAB KD CMs,suggestive of an exacerbation of caspase 12 activation in cryAB KD CMs after exposure to H 2O 2.DISCUSSION
This study provides evidence of a cytosolic to mitochondrial translocation of cryAB and
PcryAB in adult mouse CMs in response to H 2O 2-induced oxidative stress. Upregulation of
total cryAB levels after H 2O 2 exposure and the significant reduction in viability in mouse
neonatal cryAB KD CMs suggest that cryAB is protective against apoptosis. Immuno-
precipitation assays indicated that cryAB intervenes at multiple points in the intrinsic
apoptotic cascade by interacting with TOM 20, VDAC, caspase 3, and caspase 12 [activated
by ER stress (30)] and that these interactions may be part of the protective mechanism of
cryAB in CMs.
One of the conclusions of this study is that the protective mechanism of cryAB in ROS-
induced cell death may involve its stabilization of the mitochondria. Our results extend
earlier findings regarding cryAB localization (28). PcryAB was associated with the
mitochondria even under control conditions (Fig. 1), and this association increased
significantly under conditions of oxidative stress (Fig. 3). It is possible that through this
association, PcryAB binds and stabilizes or modulates the activity of MPTP proteins,
preventing pore opening and ensuing apoptosis. Furthermore, our results indicate that the
interaction of cryAB or PcryAB with VDAC may be important for the protective
mechanism, as it was significantly increased under stress conditions (Fig. 7 and results not
shown). VDAC is a protein located in the outer mitochondrial membrane and mediates the
transport of anions, cations, ATP, Ca 2+, and many metabolites between the mitochondria
and the cytosol (36). VDAC has also been recognized as having a key role in mitochondria-
mediated apoptosis by interacting with both anti- and proapoptotic proteins to regulate
cytochrome c release and thus mitochondria-mediated apoptosis (36). By interacting with
VDAC, cryAB and/or PcryAB may stabilize the mitochondrial membrane, thus blocking the
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first step in the mitochondrial pathway of apoptosis. In agreement with this, a previous study (1) has shown that VDAC directly interacts with Bcl-x L in HEK-293 cells and that this interaction results in decreased channel conductivity and decreased staurosporine-induced apoptosis. The interaction of cryAB and PcryAB with TOM 20 can also act as a stabilizing force. TOM 20 is a member of the outer mitochondrial membrane protein translocase involved in transporting preproteins into the mitochondria (13). A previous study (6) has shown that TOM 20 is prone to unfolding in response to I/R in the myocardium. By maintaining the structure of TOM 20, HSPs (including Hsp70) have been suggested to contribute to protection of the myocardium in ischemic preconditioning (6). Furthermore,the unfolding of proteins when transported through TOM 20 may act as a stimulus to attract cryAB and PcryAB to the mitochondria. We have shown by electron microscopy that PcryAB was present in the mitochondria; thus, an interaction with proteins found in the outer and inner mitochondrial membrane would be feasible. Furthermore, a previous study (8) has shown that cytosolic HSPs can localize to the interior of the mitochondria, for instance, Hsp25 binds to and protects mitochondria cytochrome complex I from oxidative stress in PC12 cells. Therefore, the interaction of cryAB with proteins in the inner mitochondrial membrane may be a central mechanism by which cryAB prevents apoptosis.Interestingly, cryAB has been shown to interact with the proapoptotic proteins Bax and Bcl-X S , preventing their translocation from the cytosol into mitochondria, thus maintaining mitochondrial integrity and leading to decreased apoptosis (26).A second conclusion that can be drawn from the present study is that while cryAB KD does not seem to have a significant effect on CM viability after H 2O 2, it does, however, interact with oxidative stress-mediated apoptosis. This effect can be explained by the fact that viability measures are not sensitive enough to detect interactions between cryAB KD and H 2O 2 exposure, as they take into account various types of cell death (autophagy, necrosis,and apoptosis), not apoptosis alone. Our results suggest that cryAB is protective against
mitochondrial oxidative stress-induced apoptosis in CMs at baseline and after extrinsically
exacerbated oxidative stress. Much like other HSPs that confer a protective effect against a
wide range of cellular stresses, cryAB is induced by cellular stresses (9), such as oxidative
stress, and is involved in protection against such stress. We found that at baseline, cryAB
KD CMs were already subject to oxidative stress by increased ROS production, which
decreased their viability. Previously, it has been shown that in heat shock factor 1 knockout
mice, which have reduced expression of Hsp70, Hsp25, and cryAB, there is increased
mitochondrial oxidative damage (44), and, here, we show that silencing of cryAB alone
induced increased oxidative damage at the mitochondrial level as well as loss of
mitochondrial membrane potential. The mitochondria are a major source of ROS and
oxidative stress, and they trigger ROS-induced activation of the intrinsic apoptotic pathway
by releasing cytochrome c (12). Hence, KD of cryAB could contribute to increased
mitochondria-induced apoptosis by ROS-induced ROS production (45). Increased ROS
production as a result of alterations to the mitochondrial membrane potential and electron
transport chain would trigger a feedforward, subsequent increased level of ROS production,
leading to apoptosis. The electron microscopy images in the present study support this
speculation, as they showed significantly altered mitochondrial ultrastructure in cryAB-
silenced cardiomyocytes, suggestive of mitochondrial permeabilization and rupture (Fig. 5),
which would lead to apoptosis induction. Furthermore, given the ability of anti-oxidants to
protect against apoptosis in I/R injury by blocking the increased expression of p53, Bax, and
caspase 3 and by inhibiting caspase 3 activation (31), it may be that the oxidative stress
imposed by cryAB KD functions by the opposite mechanism, increasing the expression of
proapoptotic proteins and stimulating apoptosis.
Downstream of the interactions with mitochondrial proteins, cryAB may also protect from
apoptosis induced by oxidative stress by interacting with caspase 3 and caspase 12, further
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suggesting an involvement of cryAB in protection against not only the intrinsic pathway but also ER stress-induced apoptosis (41). In the present study and those of others (20, 21),cytosolic cryAB appears to interact with uncleaved caspase 3 to prevent its cleavage, and therefore its activation, thus preventing progression along the apoptotic cascade. In the absence of cryAB, caspase 3 activity increased, and it resulted in the higher levels of apoptosis observed in cryAB KD CMs. The apparent increased interactions of cryAB with cleaved caspase 3 observed in the present study have not been reported previously, to our knowledge. This may be indicative of the potential ability of cryAB to bind and inhibit active caspase 3, even after its cleavage, thus potentially inhibiting its proteolytic activity and cleavage of substrates in the cell and providing an additional point for intervention in preventing apoptosis in CMs. CryAB has been shown to bind caspase 12. Caspase 12 is activated by ER stress (30), suggesting that cytosolic cryAB is also potentially involved in preventing ER stress-induced apoptosis. ER stress also contributes to apoptosis, by leading to the release of Ca 2+ from the ER, causing depletion of ER Ca 2+ and activating Ca 2+ -dependent endonucleases involved in DNA fragmentation (14). Furthermore, Ca 2+ is also believed to modulate cytochrome c release directly by regulating the MPTP, leading to apoptosis (15). As a molecular chaperone, cryAB would be induced in response to ER stress,suggesting that in cryAB-silenced cells, ER stress would lead to rampant apoptosis. This comes in agreement with our previous findings showing an upregulation of cryAB in response to ER stress as well as decreased viability of cMs in the absence of cryAB under conditions of augmented ER stress (5). This study did not differentiate between apoptosis by the intrinsic pathway and apoptosis induced by ER stress, but determining the contribution of ER stress to apoptosis in the absence of cryAB would be of interest.Regulating the levels of apoptosis is an attractive target for ameliorating any ischemic injury in the heart. Our results contribute to delineating the involvement of cryAB in the intrinsic and ER stress pathway of apoptosis activated by ROS-induced oxidative damage. ROS are
known to play a major role in the pathogenesis of myocardial dysfunction in a variety of
conditions, including I/R injury (16), with H 2O 2 playing a significant role in oxidative stress
injury (39). Therefore, determining where in the apoptotic cascade cryAB intervenes to
prevent H 2O 2 injury can have therapeutic implications for cardiac disease. For instance, it
has been shown that administration of exogenous cryAB significantly improves murine
cardiac function after I/R injury and that it decreases caspase 3 activity and apoptosis in
hypoxic human endothelial cells but not in mouse atrial CMs (40). Consistent with the work
by Velotta et al. (40), who investigated cryAB in human endothelial cells, our study in
ventricular CMs showed that cryAB intervenes and modulates apoptosis by interacting with
VDAC, TOM 20, caspase 3, and caspase 12 in ventricular CMs. This suggests that
therapeutic administration of cryAB could target and inhibit the intrinsic and ER stress
pathways of apoptosis in ventricular CMs, thus potentially contributing to improved cardiac
function. Further studies focusing on the extrinsic pathway of apoptosis and alternate death
pathways, such as autophagy and necrosis, would therefore prove quite insightful.
Acknowledgments
The authors thank Dr. Andrea Jurisicova and Dr. Rudiger von Harsorf for valuable discussions and insight.
GRANTS
This work was funded by an Ontario Graduate Scholarship (to R. Chis), Heart and Stroke Foundation of Ontario
Grant T-6281 (to A. O. Gramolini), an Early Research Award from the Ontario Ministry of Research and
Innovation (to A. O. Gramolini), an unrestricted grant from Boehringer Ingelheim Canada (to A. O. Gramolini), and
a Heart and Stroke/Lewar Centre Fellowship (to N. Bousette). A. O. Gramolini was a New Investigator of Heart
and Stroke Foundation Canada and holds a Canada Research Chair in Cardiovascular Proteomics and Molecular
Therapeutics.
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Fig. 1.α-Crystallin B (cryAB) and phosphorylated cryAB (PcryAB) distribution in cardiac cells.
A : immunoblot of cryA
B and PcryAB in different subcellular fractions isolated from adult
mouse hearts. The distribution of GAPDH as a marker for the cytosolic fraction, histone H3
for the nuclear fraction, and Na +-K +-ATPase for the microsomal fraction was assessed. A
minimum of three experiments was performed for each fractionation. B : sub-cellular
fractionation of wild-type (WT) adult mouse heart lysates run on a continuous 20–60%
sucrose gradient. Fractions 1–12 contained the highest to lowest sucrose concentrations,
respectively. The mitochondrial marker voltage-dependent anion channel (VDAC), the
plasma membrane protein marker Na +-K +-ATPase, and GAPDH as a cytosol protein were
detected as markers to monitor the fractionation procedure. A minimum of 3 experiments
was performed for each gradient. C : electron microscopy images showing immunogold
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staining of cryAB and PcryAB under control conditions along with negative controls for
cryAB and PcryAB staining. *Presence of gold particles. Scale bar = 100 nm. Images are
representative of a minimum of 50 separate microscopy fields. PMC Canada Author Manuscript
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Fig. 2.
Viability in cryAB-silenced cardiomyocytes (CMs). A : immunoblot for cryAB and actin in
CMs either transduced with scrambled short hairpin (sh)RNAs (Scram shRNA) or cryAB
targeting shRNA virus [cryAB knockdown (KD)]. In cryAB KD cells, cryAB levels were
~28% of the expression levels in CMs transduced with the Scram shRNA control plasmid.
B : quantification of cryAB expression levels in CMs transduced with cryAB-targeting
shRNA (cryAB KD) compared with CMs transduced with Scram shRNA. C : quantification
of viability in CMs transduced with cryAB-targeting shRNA (cryAB KD) compared with
Scram shRNA at increasing H 2O 2 concentrations. *P < 0.05 vs. Scram shRNA control CMs
and **P < 0.05 vs. KD control; #P < 0.05, differences between groups (Scram shRNA vs.
KD). Values are reported as means ± SE; n = 3/treatment. D : quantification of caspase 3
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activity in Scram shRNA (*P < 0.05) and cryAB KD (**P < 0.05) CMs after exposure to 60
μM H2O2 compared with control conditions. #P < 0.05, differences between groups (Scram
shRNA vs. KD). Values are reported as means ± SE; n = 6/treatment. E: quantification of
TUNEL-positive nuclei in Scram shRNA (*P < 0.05) and KD (**P < 0.05) CMs after
exposure to 60 μM H2O2 compared with control conditions. #P < 0.05, differences between
groups (Scram shRNA vs. KD). Values are reported as means ± SE; n = ~1,000 cells/
treatment. PMC Canada Author Manuscript
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Fig. 3.
Upregulation and translocation of cryAB and PcryAB to the mitochondria under oxidative
stress conditions. A : immunoblots of cryAB and PcryAB along with actin standards in WT
mouse adult CM controls or CMs exposed to 100 μM H 2O 2 for 1 h. B : quantification of
cryAB and PcryAB levels in adult mouse CMs after exposure to 100 μM H
2O 2 relative to
control levels (*P < 0.05). C : immunoblots of cytosolic and organellar subcellular fractions
for cryAB and PcryAB in cultured neonatal CMs under control conditions and after
exposure to 60 μM H 2O 2. The distribution of VDAC, a mitochondrial marker, and GAPDH,
a cytosolic marker, was assessed to monitor the purity of the fractions. D : quantification of
the mitochondrial and cytosolic distribution of cryAB and PcryAB in cultured neonatal CMs
after exposure to 60 μM H 2O 2 relative to control conditions (#P < 0.05). The band
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细胞凋亡试验常用的方法
细胞凋亡试验常用的方法（MTT法、荧光法、DNA琼脂糖凝胶电泳法与流式细胞仪检测法） （一）药物对肿瘤细胞的抑制效应的MTT法： 用培养基将肿瘤细胞调整至2 X108个/L，在96孔板中每孔加入100ul细胞悬液于37℃、5% CO2下培养过夜。 次日每孔加入不同浓度的药物100mg/L作为试验组，设加完全培养基不加药物的阴性对照，并用功能明确的药物为阳性对照和0.5%的乙醇溶剂对照，每组均设4-6个复孔（平行孔）、37℃、5% CO2继续培养。 培养至12h、24h、48h、实验终止前4-6h加入10ulMTT（5g/L），培养4-6h后，阴性对照孔中已形成明显的蓝紫色颗粒结晶时加100ul/孔SDS-HCl终止反应，于37℃存放过夜。 用酶标仪在A570波长下测吸光度值，按下式计算抑制率 抑制率（%）=（1-试验组平均吸光度值/阴性对照组平均吸光度值）x 100%。 （二）荧光法： 选用上述最佳浓度作用于肿瘤细胞，培养细胞48h后，收货细胞用PBS洗2-3次后用0.4%多聚甲醛室温下固定30min。 弃去固定液，并用PBS洗2次后，用1%Triton X-100作用4min加入适量的0.5mg/L DAPI 荧光染色60min，用PBS冲洗3次，取10ul滴片，干燥后于荧光显微镜下检测断裂的颗粒和片状荧光。 （三）DNA琼脂糖凝胶电泳法： 1、DNA提取： 用大方瓶培养肿瘤细胞，每瓶10ml，细胞浓度为3 x 108个/ml，每隔药物浓度、作用时间均设2瓶，共分3个时间段，4个药物浓度。共培养26瓶细胞。 分别于细胞中加入不同浓度的药物，于37℃、5% CO2中分别培养12h、24h、48h，收货细胞，用PBS洗2-3次。 于-20℃将细胞冷却处理10min后将细胞收集至离心管中，加1ml细胞裂解液，再加蛋白酶K，轻轻振摇使悬液混匀，成黏糊状，50℃过夜。 冷却后加入等体积的饱和酚溶液，混合后10000r/min离心10min，吸出上层水相，移至另一离心管中，再加入等体积饱和酚溶液重复抽提一次，直到无蛋白为止。 吸上清加入氯仿/异戊醇（24：1）按上述方法再抽提一次。 吸取水相层加入1/10体积的3mol/L的醋酸钠溶液，混匀。 再加入2.5倍体积冷无水乙醇，混合置-20℃处理30min后，10000r/min离心10min，沉淀部分为提供的DNA，弃去无水乙醇后用70%乙醇漂洗2次，将离心管倒扣在吸水纸上，吸干乙醇。 加入200ulTE缓冲液融解DNA，再加入25ul的RNA酶，置37℃作用30min，置4℃冰箱保存。 2、琼脂糖凝胶电泳： TBE缓冲液配制1.8%琼脂糖凝胶。在微波炉内煮沸至琼脂糖融解，待冷却至60℃时，加入溴化乙锭，使其终浓度为0.5mg/ml，混匀后灌胶。 待凝胶固定后放入含TBE电泳液的电泳槽内，使TBE电泳液盖过凝胶。 取10-15ul提取的各组DNA样品液与上样缓冲液按4：1比例混匀后点样。 60V电泳1h，用紫外透射仪观察梯形条带。
秀丽线虫生殖细胞凋亡检测
题目：秀丽线虫生殖细胞凋亡检测 实验目的: 1. 掌握检测凋亡细胞的方法 2. 学习使用荧光染料活体染色的方法和步骤 ．实验原理 1. 秀丽隐杆线虫( Caenorhabditis elegans )：是一种无毒无害、可以独立生存的 线虫。其个体小，成体仅 1.5mm 长，为雌雄同体 ( hermaphrodites )，雄性个体仅占群体的 0.2%，可自体受精或双性生殖；在20℃下平均生活史为 3.5 天，平均繁殖力为 300-350 个；但若与雄虫交配，可产生多达 1400 个以上的后代。 1976 年， Sulston 和 Horvitz 利用秀丽隐杆线虫 ( Caenorhabditis elegans ) 研究发现，其约 13%的体细胞在胚胎发育中注定死亡，使得人们认识到细胞凋亡的遗传基础。 2. 荧光染料活体染色：本实验使用吖啶橙( Acridine orange )作为染色剂，该染 料对细胞具有慢性毒性，致癌性强，由于凋亡细胞因 DNA片段化可结合更多染料，荧光显微镜下呈亮绿色，可在荧光显微镜下快速方便的检测出，适用于多数品系。
实验材料及设备 1. 实验材料： a) 各品系秀丽隐杆线虫：N2(实验组) , ced-1::gfp (方法对照组)，ced- 3(阴性对照) b) OP50 c) M9培养基 d) NGM培养基 2. 实验设备： a) 普通光学显微镜 b) 载玻片若干，盖玻片若干，铂金丝 c) 暗箱 d) 吸水纸、滴管等 e) 荧光显微镜 四．实验方法及步骤 1. 线虫接种、同步化 2. 取样：在 12 孔板培养板上，每孔吸取 900μL 预先接入少量 OP50 的 M9 培养基，每孔用铂金丝挑取培养 20~30 条成体线虫 3. 染色：向 N2与 ced-3 品系中每孔加入 250μg/mL 吖啶橙 100μL, 混匀后 置于培养箱(避光)染色 45~60min。 4. 方法对照组观察：向 ced-1::GFP 品系中加入 1 滴盐酸左旋咪唑，麻痹线
细胞凋亡检测方法
细胞凋亡检测方法 一、细胞凋亡的形态学检测 1 光学显微镜和倒置显微镜 （1）未染色细胞：凋亡细胞的体积变小、变形，全面皱缩，细胞膜完整但出现发泡现象，细胞凋亡晚期可见凋亡小体，凋亡小体为数个圆形小体围绕在细胞周围。贴壁细胞出现皱缩、变圆、脱落。 （2）染色细胞： 姬姆萨（Giemsa）染色、瑞氏染色等：正常细胞核色泽均一；凋亡细胞染色质浓缩、边缘化，核膜裂解、染色质分割成块状和凋亡小体等典型的凋亡形态；坏死细胞染色浅或没染上颜色。 苏木素-伊红（HE）染色：细胞核固缩碎裂、呈蓝黑色、胞浆呈淡红色（凋亡细胞），正常细胞核呈均匀淡蓝色或蓝色，坏死细胞核呈很淡的蓝色或蓝色消失。 2 荧光显微镜和共聚焦激光扫描显微镜 一般以细胞核染色质的形态学改变为指标来评判细胞凋亡的进展情况。 常用的DNA特异性染料有：Hoechst 33342，Hoechst 33258，DAPI。三种染料与DNA 的结合是非嵌入式的，主要结合在DNA的A-T碱基区。紫外光激发时发射明亮的蓝色荧光。 Hoechst是与DNA特异结合的活性染料，能进入正常细胞膜而对细胞没有太大细胞毒作用。Hoechst 33342在凋亡细胞中的荧光强度要比正常细胞中要高。 DAPI为半通透性，用于常规固定细胞的染色。 PI和Hoechst33342双标：PI、Hoechst33342均可与细胞核DNA（或RNA）结合。但PI不能通过正常细胞膜，Hoechst则为膜通透性荧光染料，故细胞在处于坏死或晚期调
亡时细胞膜被破坏，这时可为PI着红色。正常细胞和中早期调亡细胞均可被Hoechst着色，但是正常细胞核的Hoechst着色的形态呈圆形，淡兰色，内有较深的兰色颗粒；而调亡细胞的核由于浓集而呈亮兰色，或核呈分叶，碎片状，边集。故PI着色为坏死细胞；亮兰色，或核呈分叶状，边集的Hoechst着色的为调亡细胞。 凋亡细胞体积变小，细胞质浓缩。细胞凋亡过程中细胞核染色质的形态学改变分为三期：Ⅰ期的细胞核呈波纹状（rippled）或呈折缝样（creased），部分染色质出现浓缩状态；Ⅱa期细胞核的染色质高度凝聚、边缘化；Ⅱb期的细胞核裂解为碎块，产生凋亡小体(图1)。 3 透射电子显微镜观察 凋亡细胞体积变小，细胞质浓缩。凋亡Ⅰ期（pro-apoptosis nuclei）的细胞核内染色质高度盘绕，出现许多称为气穴现象（cavitations）的空泡结构(图2)；Ⅱa期细胞核的染色质高度凝聚、边缘化；细胞凋亡的晚期，细胞核裂解为碎块，产生凋亡小体。 二、磷脂酰丝氨酸外翻分析（Annexin V法） 磷脂酰丝氨酸（Phosphatidylserine, PS）正常位于细胞膜内侧，但在细胞凋亡早期，PS可从细胞膜内侧翻转到细胞膜表面，暴露在细胞外环境中。磷脂酰丝氨酸的转位发生在凋亡早期阶段，先于细胞核的改变、DNA断裂、细胞膜起泡。体内的吞噬细胞可通过识别
细胞凋亡实验步骤及注意事项
细胞凋亡实验步骤及注意事项 一、实验目的 1、掌屋凋亡细胞的形态特征 2、学会用荧光探针对细胞进行双标记来检测正常活细胞、凋亡细胞与坏死 细胞的方法 二、实验原理 细胞死亡根据其性质、起源及生物学意义区分为凋亡与坏死两种不同类型。凋亡普遍存在于生命界,在生物个体与生存中起着非常重要的作用。它就是细胞在一 定生理条件下一系列顺序发生事件的组合,就是细胞遵循一定规律自己结束生命 的自主控制过程。细胞凋亡具有可鉴别的形态学与生物化学特征。 在形态上可见凋亡细胞与周围细胞脱离接触,细胞变园,细胞膜向内皱缩、胞浆浓缩、内质网扩张、细胞核固缩破裂呈团块状或新月状分布、内质网与细胞膜进一步融合将细胞分成多个完整包裹的凋亡小体,凋亡小体最后被吞噬细胞吞噬消化。在凋亡过程中细胞内容物并不释放到细胞外,不会影响其它细胞,因而不引起炎症反应。 在生物化学上,多数细胞凋亡的过程中,内源性核酸内切酶活化,活性增加。核DNA 随机地在核小体的连接部位被酶切断,降解为180-200bp或它的整倍数的各种片断。如果对核DNA进行琼脂糖电泳,可显示以180-200bp为基数的DNA ladder(梯状带纹)的特征。 相比之下,坏死就是细胞处于剧烈损伤条件下发生的细胞死亡。细胞在坏死早期 即丧失质膜完整性,各种细胞器膨胀,进而质膜崩解释放出其中的内容物,引起炎症反应,坏死过程中细胞核DNA虽也降解,但由于存在各种长度不等的DNA片断,不能形成梯状带纹,而呈弥散状。 一些温与的损伤刺激及一些抗肿瘤药物可诱导细胞凋亡,通常这些因素在诱导凋亡的同时,也可产生细胞坏死,这取决于损伤的剧烈程度与细胞本身对刺激的敏感 程度。 三尖杉酯碱(HT)就是我国自行研制的一种对急性粒细胞白血病,急性单核白血病等有良好疗效的抗肿瘤药物。研究表明HT在0、02~5μg/ml范围内作用2小时,即可诱导HL-60细胞凋亡,并表现出典型的凋亡特征。本实验用1μg/ml HT在体外诱导培养的HL-60细胞发生凋亡,同时也有少数细胞发生坏死。用 Hoechst33342与碘化丙啶(propidium iodide,PI)对细胞进行双重染色,可以区别凋亡、坏死及正常细胞。 细胞膜就是一选择性的生物膜,一般的生物染料如PI等不能穿过质膜。当细胞坏死时,质膜不完整,PI就进入细胞内部,它可嵌入到DNA或RNA中,使坏死细胞着
常用细胞凋亡检测方法(图)
常用细胞凋亡检测方法（图） 转载请注明来自丁香园 发布日期：2012-02-16 13:41 文章来源：丁香通 关键词：丁香园生物专题义翘神州细胞培养点击次数：951 一、细胞凋亡的形态学检测 1、光学显微镜和倒置显微镜 ①未染色细胞：凋亡细胞的体积变小、变形，细胞膜完整但出现发泡现象，细胞凋亡晚期可见凋亡小体。贴壁细胞出现皱缩、变圆、脱落。 ②染色细胞：常用姬姆萨染色、瑞氏染色等。凋亡细胞的染色质浓缩、边缘化，核膜裂解、染色质分割成块状和凋亡小体等典型的凋亡形态。 2、荧光显微镜和共聚焦激光扫描显微镜 一般以细胞核染色质的形态学改变为指标来评判细胞凋亡的进展情况。常用的DNA 特异性染料有：HO 33342 (Hoechst 33342)，HO 33258 (Hoechst 33258), DAPI。三种种染料与DNA的结合是非嵌入式的，主要结合在DNA的A-T碱基区。紫外光激发时发射明亮的蓝色荧光。Hoechst是与DNA特异结合的活性染料，储存液用蒸馏水配成1mg/ml的浓度，使用时用PBS稀释，终浓度为10 ug/ml。DAPI为半通透性，用于常规固定细胞的染色。储存液用蒸馏水配成1mg/ml的浓度，使用终浓度一般为10 ug/ml。结果评判：细胞凋亡过程中细胞核染色质的形态学改变分为三期：Ⅰ期的细胞核呈波纹状（rippled）或呈折缝样（creased），部分染色质出现浓缩状态；Ⅱa期细胞核的染色质高度凝聚、边缘化；Ⅱb期的细胞核裂解为碎块，产生凋亡小体(图1)。 3、透射电子显微镜观察 结果评判：凋亡细胞体积变小，细胞质浓缩。凋亡Ⅰ期（pro-apoptosis nuclei）的细胞核内染色质高度盘绕，出现许多称为气穴现象（cavitations）的空泡结构(图2)；Ⅱa期细胞核的染色质高度凝聚、边缘化；细胞凋亡的晚期，细胞核裂解为碎块，产生凋亡小体。 二、磷脂酰丝氨酸外翻分析（Annexin V法） 磷脂酰丝氨酸（Phosphatidylserine, PS）正常位于细胞膜的内侧，但在细胞凋亡的早期，PS可从细胞膜的内侧翻转到细胞膜的表面，暴露在细胞外环境中(图3)。Annexin-V是一种分子量为35~36KD的Ca2+依赖性磷脂结合蛋白，能与PS高亲和力特异性结合。将Annexin-V进行荧光素（FITC、PE）或biotin标记，以标记了的Annexin-V作为荧光探针，利用流式细胞仪或荧光显微镜可检测细胞凋亡的发生。 碘化丙啶(propidine iodide, PI)是一种核酸染料，它不能透过完整的细胞膜，但在凋亡中晚期的细胞和死细胞，PI能够透过细胞膜而使细核红染。因此将Annexin-V 与PI匹配使用，就可以将凋亡早晚期的细胞以及死细胞区分开来。 方法
实验14-细胞凋亡的诱导和检测
实验14 细胞凋亡的诱导和检测 20世纪60年代人们注意到细胞存在着两种不同形式的死亡方式：凋亡（apoptosis）和坏死（necrosis）。细胞坏死指病理情况下细胞的意外死亡，坏死过程细胞膜通透性增高，细胞肿胀，核碎裂，继而溶酶体、细胞膜破坏，细胞容物溢出，细胞坏死常引起炎症反应。 细胞凋亡apoptosis一词来源于古希腊语，意思是花瓣或树叶凋落，意味着生命走到了尽头，细胞到了一定时期会像树叶那样自然死亡。凋亡是细胞在一定生理或病理条件下遵守自身程序的主动死亡过程。凋亡时细胞皱缩，表面微绒毛消失，染色质凝集并呈新月形或块状靠近核膜边缘，继而核裂解，由细胞膜包裹着核碎片或其他细胞器形成小球状凋亡小体凸出于细胞表面，最后凋亡小体脱落被吞噬细胞或邻周细胞吞噬。凋亡过程中溶酶体及细胞膜保持完整，不引起炎症反应。细胞凋亡时的生化变化特征是核酸切酶被激活，染色体DNA被降解，断裂为50～300 kb长的DNA片段，再进一步断裂成180～200bp整倍数的寡核苷酸片断，在琼脂糖凝胶电泳上呈现“梯状”电泳图谱(DNA Ladder)。细胞凋亡在个体正常发育、紫稳态维持、免疫耐受形成、肿瘤监控和抵御各种外界因素干扰等方面都起着关键性的作用。 1．细胞凋亡的检测方法 凋亡细胞具有一些列不同于坏死细胞的形态特征和生化特征，据此可以鉴别细胞的死亡形式。细胞凋亡的机制十分复杂，一般采用多种方法综合加以判断，同时不同类型细胞的凋亡分析方法有所不同，方法选择依赖于具体的研究体系和研究目的（表？）。
形态学观察方法：利用各种染色法可观察到凋亡细胞的各种形态学特征： （1）DAPI时常用的一种与DNA结合的荧光染料。借助于DAPI染色，可以观察细胞核的形态变化。 （2）Giemsa染色法可以观察到染色质固缩、趋边、凋亡小体形成等形态。 （3）吖啶橙(AO)染色，荧光显微镜观察，活细胞核呈黄绿色荧光，胞质呈红色荧光。凋亡细胞核染色质呈黄绿色浓聚在核膜侧，可见细胞膜呈泡状膨出及凋亡小体。 （4）吖啶橙(A())／溴化乙啶(EB)复染可以更可靠地确定凋亡细胞的变化，AO只进入活细胞，正常细胞及处于凋亡早期的细胞核呈现绿色；EB只进入死细胞，将死细胞及凋亡晚期的细胞的核染成橙红色。 （5）台盼蓝染色对反映细胞膜的完整性，区别坏死细胞有一定的帮助，如果细胞膜不完整、破裂，台盼蓝染料进入细胞，细胞变蓝，即为坏死。如果细胞膜完整，细胞不为台盼蓝染色，则为正常细胞或凋亡细胞。使用透射电镜观察，可见凋亡细胞表面微绒毛消失，核染色质固缩、边集，常呈新月形，核膜皱褶，胞质紧实，细胞器集中，胞膜起泡或出“芽”及凋亡小体和凋亡小体被临近巨噬细胞吞噬现象。 （6）木精-伊红（HE）染色是经典的显示细胞核、细胞质的染色方法，染色结果清晰。发生凋亡的细胞经HE染色后，其细胞大小的变化及特征性细胞核的变化：染色质凝集、呈新月形或块状靠近核膜边缘，晚期核裂解、细胞膜包裹着核碎片“出芽”凸出于细胞表面形成凋亡小体等均可明显显示出来。 DNA凝胶电泳：细胞发生凋亡或坏死，其细胞DNA均发生断裂，细胞小分子 质量DNA片段增加，高分子DNA减少，胞质出现DNA片段。但凋亡细胞DNA断裂点均有规律的发生在核小体之间，出现180～200 bp DNA片段，而坏死细胞的DNA断裂点为无特征的杂乱片段，利用此特征可以确定群体细胞的死亡，并可与坏死细胞区别。
细胞凋亡主要发生机制及相关作用研究
细胞凋亡主要发生机制及相关作用研究 摘要 细胞凋亡是一种有序的或程序性的细胞死亡方式，是细胞接受某些特定信号刺激后在基因调控下所发生的一系列细胞主动死亡过程，通常来说是一种正常生理应答反应。目前认为细胞凋亡信号传导通路主要包括三种：内源性途径、外源性途径以及内质网途径。细胞凋亡的研究已成为当前生命科学研究热点之一。研究细胞凋亡的信号传导通路及其调控对进一步认识和治疗凋亡相关疾病有重要意义。 关键词：细胞凋亡信号传导通路疾病治疗
ABSTRACT Apoptosis is an orderly or programmed cell death way, is a series of cells active death process under gene regulation that after cell accepted certain specific signal stimulation, it is a normal physiological response. At presently, the cell apoptosis signaling pathways mainly includes three types: intrinsic pathway, extrinsic pathway, and the way of endoplasmic reticulum. The research of apoptosis has become the life science research hotspot. Researching cell apoptosis signaling pathways and regulation can get further understanding and also have the important meaning to treatment of apoptosis related diseases. Key words: A poptosis Signal conduct pathway Treatment of diseases
细胞凋亡的几种检测方法
细胞凋亡的几种检测方法 1、形态学观察方法 （1）HE（苏木精—伊红染色法）染色、光镜观察：凋亡细胞呈圆形，胞核深染，胞质浓缩，染色质成团块状，细胞表面有“出芽”现象。 （2）丫啶橙（AO)染色，荧光显微镜观察：活细胞核呈黄绿色荧光，胞质呈红色荧光。凋亡细胞核染色质呈黄绿色浓聚在核膜内侧，可见细胞膜呈泡状膨出及凋亡小体。 （3）台盼蓝染色：如果细胞膜不完整、破裂，台盼蓝染料进入细胞，细胞变蓝，即为坏死。如果细胞膜完整，细胞不为台盼蓝染色，则为正常细胞或凋亡细胞。此方法对反映细胞膜的完整性，区别坏死细胞有一定的帮助。 （4）透射电镜观察：可见凋亡细胞表面微绒毛消失，核染色质固缩、边集，常呈新月形，核膜皱褶，胞质紧实，细胞器集中，胞膜起泡或出“芽”及凋亡小体和凋亡小体被临近巨噬细胞吞噬现象。 2、DNA凝胶电泳 细胞发生凋亡或坏死，其细胞DNA均发生断裂，细胞内小分子量DNA片断增加，高分子DNA减少，胞质内出现DNA片断。但凋亡细胞DNA断裂点均有规律的发
生在核小体之间，出现180－200bpDNA片断，而坏死细胞的DNA断裂点为无特征的杂乱片断，利用此特征可以确定群体细胞的死亡，并可与坏死细胞区别。正常活细胞DNA 电泳出现阶梯状（LADDER)条带；坏死细胞DNA电泳类似血抹片时的连续性条带 3、酶联免疫吸附法（ELISA)核小体测定 凋亡细胞的DNA断裂使细胞质内出现核小体。核小体由组蛋白及其伴随的DNA片断组成，可由ELISA法检测。 检测步骤 1、将凋亡细胞裂解后高速离心，其上清液中含有核小体； 2、在微定量板上吸附组蛋白体’ 3、加上清夜使抗组蛋白抗体与核小体上的组蛋白结合‘ 4、加辣过氧化物酶标记的抗DNA抗体使之与核小体上的DNA结合’ 4、加酶的底物，测光吸收制。 用途 该法敏感性高，可检测5*100/ml个凋亡细胞。可用于人、大鼠、小鼠的凋亡检测。该法不需要特殊仪器，
细胞凋亡的检测(含图片) 陈英玉
细胞凋亡的检测 细胞凋亡与坏死是两种完全不同的细胞凋亡形式，根据死亡细胞在形态学、生物化学和分子生物学上的差别，可以将二者区别开来。细胞凋亡的检测方法有很多，下面介绍几种常用的测定方法。 一、细胞凋亡的形态学检测 根据凋亡细胞固有的形态特征，人们已经设计了许多不同的细胞凋亡形态学检测方法。 1 光学显微镜和倒置显微镜 （1）未染色细胞：凋亡细胞的体积变小、变形，细胞膜完整但出现发泡现象，细胞凋亡晚期可见凋亡小体。 贴壁细胞出现皱缩、变圆、脱落。 （2）染色细胞：常用姬姆萨染色、瑞氏染色等。凋亡细胞的染色质浓缩、边缘化，核膜裂解、染色质分割 成块状和凋亡小体等典型的凋亡形态。 2 荧光显微镜和共聚焦激光扫描显微镜 一般以细胞核染色质的形态学改变为指标来评判细胞凋亡的进展情况。 常用的DNA特异性染料有：HO 33342 (Hoechst 33342)，HO 33258 (Hoechst 33258), DAPI。三种染料与DNA的结合是非嵌入式的，主要结合在DNA的A-T碱基区。紫外光激发时发射明亮的蓝色荧光。 Hoechst是与DNA特异结合的活性染料，储存液用蒸馏水配成1mg/ml的浓度，使用时用PBS稀释成终浓度为2~5mg/ml。 DAPI为半通透性，用于常规固定细胞的染色。储存液用蒸馏水配成1mg/ml的浓度，使用终浓度一般为0.5 ~1mg/ml。 结果评判：细胞凋亡过程中细胞核染色质的形态学改变分为三期：Ⅰ期的细胞核呈波纹状（rippled）或呈折缝样（creased），部分染色质出现浓缩状态；Ⅱa期细胞核的染色质高度凝聚、边缘化；Ⅱb期的细胞核裂解为碎块，产生凋亡小体(图1)。
细胞凋亡机制的研究及其意义
细胞凋亡机制的研究及其意义 摘要: 细胞凋亡是维持神经系统正常发育, 维持其免疫系统正常功能所必需过程。目前, 对细胞凋亡的研究已经成为生命科学领域研究的热点。本文就细胞凋亡的发生机制、基因调节机制等方面作一综述。 关键词: 细胞凋亡; 机制；意义 引言：细胞凋亡对机体的健康发育甚为重要，在生理条件下，它作为机体正常细胞群生长与死亡相协调的重要方式，有利于清除多余的细胞、无用细胞、发育不正常细胞、有害细胞、完成正常使命的衰老细胞；有利于维持机体细胞群的自身稳定，从而维持器官组织的正常发育。细胞凋亡过少时，机体易患肿瘤性疾病、自身免疫性疾病；细胞凋亡过多时，机体易患神经系统方面的疾病。人的艾滋病等疾病之所以发生，主要是由于机体细胞凋亡发生异常的结果。 正文： 1、细胞凋亡机制 1.1 信号传递机制 凋亡一般由细胞外的调节因素与其在细胞表面的受体结合而启动。经活化的受体又启动胞内第二信号系统，激活核酸内切酶，引起DNA裂解，进而引发细胞凋亡。细胞外的调节因素包括生理活性因子：如肿瘤坏死因子、转化生长因子及表皮生长因子等；非生理因素：如X射线、紫外线、一氧化氮、毒素及化疗药物等；感染因素：如EB病毒、腺病毒及HIV病毒等。有学者认为，细胞凋亡的信号传导能使用或部分利用细胞增殖和分化过程中的传统信号途径。传统信号途径包括G 结合蛋白信号途径和酶蛋白信号途径，前者可以调节第二信使cAMP和钙离子的生成，细胞内cAMP和钙离子浓度的变化可以对细胞凋亡产生影响；后者可通过酪氨酸蛋白激酶（PTK）、Ras-MAPK或JaK-STAT等途径参与凋亡信号的传导。但众多研究表明可直接启动细胞凋亡的信号途径或死亡信号途径是两种死亡因子，即肿瘤坏死因子和Fas配体与细胞膜表面的相应受体TNF受体和37? 结合以后所发生的凋亡反应。目前对TNF和FasL与相应受体结合所介导的细胞凋亡信号途径及其机制已取得了突破性进展 1.2 酶学机制 1.2.1 caspases蛋白酶 胱冬蛋白酶（caspases）是近几年研究的热点之一，属于ICE/CED3蛋白酶家族成员，目前发现至少有14种之多,分别命名为caspases1-caspases14。与细胞凋亡密切相关，它是通过级联反应，最终激活核酸内切酶来实现的。也有人认为凋亡并不总是引起caspases的释放，而caspases的释放也并不总是引起凋亡，很可能还与细胞的迁移和分化有关.。蛋白酶前体可在天冬氨酸位点上被切断成3部分，H2N端是抑制区域被移去，另一端COOH端断裂成一大一小亚单位
TUNEL法检测细胞凋亡
细胞在发生凋亡时，会激活一些DNA内切酶，这些内切酶会切断核小体间的基因组DNA。基因组DNA 断裂时，暴露的3'-0H 可以在末端脱氧核苷酸转移酶（Terminal Deox yn ucleotidyl Tran sferase,TdT）的催化下加上荧光素（FITC）标记的dUTP（fluorescein-dUTP），从而可以通过荧光显微镜或流式细胞仪进行检测，这就是TUNEL 法检测细胞凋亡的原理。 TUNEL法特异性检测细胞凋亡时产生的DNA断裂，但不会检测出射线等诱导的DNA断裂（和细胞凋亡时的断裂方式不同）。这样一方面可以把凋亡和坏死区分开，另一方面也不会把 射线等诱导发生DNA断裂的非凋亡细胞判断为凋亡细胞。 针对问题2（TUNEL法的实验原理是什么？）： 基本原理：对不同组织切片先增加细胞膜通透性，然后让rTDT和bio标记的dUTP进入细 胞内，在rTDT的辅助下dUTP与核断裂的DNA 3 -0H结合，再用HRP标记的链霉亲和素与dUTP 上的biot in 结合（每个链霉亲和素至少可以再结合3个biot in 分子），最后用DAB 过氧化氢与SP上的辣根过氧化物酶HRP发生氧化、环化反应，形成苯乙肼聚合物而呈现棕褐色，最终通过计数每张切片上不同视野中TUNEL阳性细胞的比例来判断细胞凋亡发生情 况。■ 1. TUNEL工作原理：简单说就是一一TUNEL细胞凋亡检测试剂盒是用来检测细胞在凋亡过程中细胞核DNA的断裂情况。 其原理是；生物素（biot in ）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdT En zyme）的 作用下，可以连接到凋亡细胞中断裂的DNA的3' - 0H末端，并可与连接了的辣根过氧化酶的 链霉亲和素（Streptavidin-HRP ）特异结合，在辣根过氧化酶底物二氨基联苯胺（DAB的存在下，产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在普通 显微镜下即可观察和计数凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA的断裂，因而没有3'-0H形成，很少能够被染色。 针对问题3 （TUNEL实验中几个关键步骤是什么？）： 1. 充分脱蜡和水化。脱蜡可以先60度20min，再用二甲苯两次5~10min ;而水化用梯度乙 醇从高浓度到低浓度浸洗，这些以便后面的结合反应充分、均匀； 2. 把握好细胞通透的时间。一般根据切片的厚薄，选择蛋白酶k的孵育时间，常用10~30min, 几um切片用短时间；几十um切片用长时间，通过摸索达到既不脱片，有能够使后面的酶和 抗体进入胞内。 3. 适当延长TUNEL反应液的时间。一般是37度1h,你也可以根据你的凋亡损伤程度，选择更长的时间，可长至2h,但要结合你最终的背景着色。 4. DAB显色条件的选择。一般DAB反应10分钟左右，结合镜下控制背景颜色，最长不超过 30min;我不喜欢用promega公司提供的DAB液（桃红色），不利于辨认棕褐色。 5. PBS的充分清洗。我个人认为，在TUNEL反应后和酶标反应后的清洗应十分严格，可增加 次数达5次，因为这些清洗直接决定最后切片的非特异性着色。 6. 此外，内源性POD的封闭也十分关键。对于肝脏、肾脏等血细胞含量多的组织，我的经 验是适当延长封闭时间和升高过氧化氢的浓度，可以达到很好的封闭效果，且不影响最终的 特异性染色。 针对问题5.细胞通透的原理、通透剂的浓度、孵育时间及其配制方法？ 1. 蛋白酶K是消化膜蛋白，从而起打孔作用，增加
(完整)常见细胞凋亡检测的方法与注意事项
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常见细胞凋亡检测的方法与注意事项 大家常把细胞凋亡和细胞坏死混淆,其实两者是不同的细胞死亡形式，大家可以在死亡细胞的形态、生化和分子指标上将二者区分开来，细胞凋亡检测的方法不少，这里就总结下几种常用的检测方法. 细胞凋亡检测更多详情，点击查看不可不知的细胞检测方法——MTT 一、细胞凋亡的形态学检测 根据凋亡细胞固有的形态特征，人们已经设计了许多不同的细胞凋亡形态学检测方法。 1 光学显微镜和倒置显微镜 （1) 未染色细胞：凋亡细胞的体积变小、变形，细胞膜完整但出现发泡现象，细胞凋亡晚期可见凋亡小体。 贴壁细胞出现皱缩、变圆、脱落. (2）染色细胞：常用姬姆萨染色、瑞氏染色等.凋亡细胞的染色质浓缩、边缘化，核膜裂解、染色质分割 成块状和凋亡小体等典型的凋亡形态。 2 荧光显微镜和共聚焦激光扫描显微镜 一般以细胞核染色质的形态学改变为指标来评判细胞凋亡的进展情况。 常用的DNA特异性染料有:HO 33342 (Hoechst 33342），HO 33258 (Hoechst 33258), DAPI。三种染料与DNA的结合是非嵌入式的，主要结合在DNA的A-T碱基区。紫外光激发时发射明亮的蓝色荧光。 Hoechst是与DNA特异结合的活性染料，储存液用蒸馏水配成1mg/ml的浓度，使用时用PBS稀释成终浓度为2~5mg/ml。 DAPI为半通透性，用于常规固定细胞的染色。储存液用蒸馏水配成1mg/ml的浓度，使用终浓度一般为0.5 ～1mg/ml。 结果评判：细胞凋亡过程中细胞核染色质的形态学改变分为三期：Ⅰ期的细胞核呈波纹状（rippled）或呈折缝样(creased)，部分染色质出现浓缩状态；Ⅱa期细胞核的染色质高度凝聚、边缘化;Ⅱb期的细胞核裂解为碎块,产生凋亡小体(图1）。 3 透射电子显微镜观察 结果评判：凋亡细胞体积变小,细胞质浓缩。凋亡Ⅰ期（pro—apoptosis nuclei)的细胞核内染色质高度盘绕，出现许多称为气穴现象（cavitations)的空泡结构(图2）；Ⅱa期细胞核的染色质高度凝聚、边缘化;细胞凋亡的晚期，细胞核裂解为碎块,产生凋亡小体。 图2
秀丽线虫生殖细胞凋亡检测
题目：秀丽线虫生殖细胞凋亡检测 一．实验目的: 1.掌握检测凋亡细胞的方法 2.学习使用荧光染料活体染色的方法和步骤 二．实验原理 1.秀丽隐杆线虫（Caenorhabditis elegans）：是一种无毒无害、可 以独立生存的线虫。其个体小，成体仅 1.5mm长，为雌雄同体（hermaphrodites），雄性个体仅占群体的0.2%，可自体受精或双性生殖；在20℃下平均生活史为3.5天，平均繁殖力为300-350个；但若与雄虫交配，可产生多达1400个以上的后代。1976年，Sulston和Horvitz利用秀丽隐杆线虫（Caenorhabditis elegans）研究发现，其约13%的体细胞在胚胎发育中注定死亡，使得人们认识到细胞凋亡的遗传基础。 2.荧光染料活体染色：本实验使用吖啶橙（Acridine orange）作为 染色剂，该染料对细胞具有慢性毒性，致癌性强，由于凋亡细胞因DNA片段化可结合更多染料，荧光显微镜下呈亮绿色，可在荧光显微镜下快速方便的检测出，适用于多数品系。 三．实验材料及设备
1.实验材料： a)各品系秀丽隐杆线虫：N2（实验组）, ced-1::gfp（方法对照组），ced- 3（阴性对照） b)OP50 c)M9培养基 d)NGM培养基 2.实验设备： a)普通光学显微镜 b)载玻片若干，盖玻片若干，铂金丝 c)暗箱 d)吸水纸、滴管等 e)荧光显微镜 四．实验方法及步骤 1.线虫接种、同步化 2.取样：在12孔板培养板上，每孔吸取900μL预先接入少量OP50 的M9培养基，每孔用铂金丝挑取培养20~30条成体线虫 3.染色：向N2与ced-3品系中每孔加入250μg/mL吖啶橙100μL, 混匀后置于培养箱（避光）染色45~60min。 4.方法对照组观察：向ced-1::GFP品系中加入1滴盐酸左旋咪唑， 麻痹线虫后在荧光显微镜下观察。
常用的细胞凋亡检测方法
细胞凋亡与坏死是两种完全不同的细胞凋亡形式，根据死亡细胞在形态学、生物化学和分子生物学上的差别，可以将二者区别开来。细胞凋亡的检测方法有很多，下面介绍几种常用的测定方法。 一、细胞凋亡的形态学检测 根据凋亡细胞固有的形态特征，人们已经设计了许多不同的细胞凋亡形态学检测方法。 1 光学显微镜和倒置显微镜 （1）未染色细胞：凋亡细胞的体积变小、变形，细胞膜完整但出现发泡现象，细胞凋亡晚期可见凋亡小体。 贴壁细胞出现皱缩、变圆、脱落。 （2）染色细胞：常用姬姆萨染色、瑞氏染色等。凋亡细胞的染色质浓缩、边缘化，核膜裂解、染色质分割 成块状和凋亡小体等典型的凋亡形态。 2 荧光显微镜和共聚焦激光扫描显微镜 一般以细胞核染色质的形态学改变为指标来评判细胞凋亡的进展情况。 常用的DNA特异性染料有：HO 33342 (Hoechst 33342)，HO 33258 (Hoechst 33258), DAPI。三种染料与DNA的结合是非嵌入式的，主要结合在DNA的A-T碱基区。紫外光激发时发射明亮的蓝色荧光。 Hoechst是与DNA特异结合的活性染料，储存液用蒸馏水配成1mg/ml的浓度，使用时用PBS稀释成终浓度为2~5mg/ml。 DAPI为半通透性，用于常规固定细胞的染色。储存液用蒸馏水配成1mg/ml的浓度，使用终浓度一般为0.5 ~1mg/ml。 结果评判：细胞凋亡过程中细胞核染色质的形态学改变分为三期：Ⅰ期的细胞核呈波纹状（rippled）或呈折缝样（creased），部分染色质出现浓缩状态；Ⅱa期细胞核的染色质高度凝聚、边缘化；Ⅱb期的细胞核裂解为碎块，产生凋亡小体(图1)。 3 透射电子显微镜观察 结果评判：凋亡细胞体积变小，细胞质浓缩。凋亡Ⅰ期（pro-apoptosis nuclei）的细胞核内染色质高度盘绕，出现许多称为气穴现象（cavitations）的空泡结构(图2)；Ⅱa期细胞核的染色质高度凝聚、边缘化；细胞凋亡的晚期，细胞核裂解为碎块，产生凋亡小体 二、磷脂酰丝氨酸外翻分析（Annexin V法） 磷脂酰丝氨酸（Phosphatidylserine, PS）正常位于细胞膜的内侧，但在细胞凋亡的早期，PS可从细胞膜的内侧翻转到细胞膜的表面，暴露在细胞外环境中(图3)。Annexin-V是一种分子量为35~36KD的Ca2+依赖性磷脂结合蛋白，能与PS高亲和力特异性结合。将Annexin-V 进行荧光素（FITC、PE）或biotin标记，以标记了的Annexin-V作为荧光探针，利用流式细胞仪或荧光显微镜可检测细胞凋亡的发生。 碘化丙啶(propidine iodide, PI)是一种核酸染料，它不能透过完整的细胞膜，但在凋亡中晚期的细胞和死细胞，PI能够透过细胞膜而使细胞核红染。因此将Annexin-V与PI匹配使用，就可以将凋亡早晚期的细胞以及死细胞区分开来。
细胞凋亡检测,细胞凋亡实验步骤,检测方法
细胞凋亡检测，细胞凋亡实验步骤，检测方法 一、定性和定量研究 只定性的研究方法：常规琼脂糖凝胶电泳、脉冲场倒转琼脂糖凝胶电泳、形态学观察（普通光学显微镜、透射电镜、荧光显微镜） 进行定量或半定量的研究方法：各种流式细胞仪方法、原位末端标记法、ELISA 定量琼脂糖凝胶电泳。 二、区分凋亡和坏死 可将二者区分开的方法：琼脂糖凝胶电泳，形态学观察（透射电镜是是区分凋亡和坏死最可靠的方法），Hoechst33342/PI双染色法流式细胞仪检测，AnnexinV/PI双染色法流式细胞仪检测等。 不能将二者区分开的方法：原位末端标记法、PI单染色法流式细胞仪检测等。 三、样品来源不同选择 组织：主要用形态学方法(HE染色，透射电镜、石蜡包埋组织切片进行原位末端标记，ELISA或将组织碾碎消化做琼脂糖凝胶电泳)。 四、细胞凋亡检测 1、早期检测: 1) PS(磷脂酰丝氨酸)在细胞外膜上的检测 2)细胞内氧化还原状态改变的检测 3)细胞色素C的定位检测 4) 线粒体膜电位变化的检测 2、晚期检测： 细胞凋亡晚期中，核酸内切酶在核小体之间剪切核DNA，产生大量长度在 180-200 bp 的DNA片段。 对于晚期检测通常有以下方法： 1) TUNEL(末端脱氧核苷酸转移酶介导的dUTP缺口末端标记) 2) LM-PCR Ladder (连接介导的PCR检测) 3) T elemerase Detection (端粒酶检测) 3、生化检测： 1）典型的生化特征：DNA 片段化 2）检测方法主要有：琼脂糖凝胶电泳、原位末端标记（TUNEL）等 3）TUNEL（末端脱氧核苷酸转移酶介导的dUTP缺口末端标记） 4）通过DNA末端转移酶将带标记的dNTP （多为dUTP）间接或直接接到DNA 片段的3’-OH端，再通过酶联显色或荧光检测定量分析结果。可做细胞悬液、福尔马林固定或石蜡处理的组织、细胞培养物等多种样本的检测。 4、LM-PCR Ladder (连接介导的PCR检测) 当凋亡细胞比例较小以及检测样品量很少（如活体组织切片）时，直接琼脂糖电泳可能观察不到核DNA的变化。通过LM-PCR,连上特异性接头，专一性地扩增梯度片段，从而灵敏地检测凋亡时产生梯度片段。此外，LM-PCR 检测是半定量的，因此相同凋亡程度的不同样品可进行比较。如果细胞量很少，还可在分离提纯DNA后，用32P-ATP和脱氧核糖核苷酸末端转移酶（TdT）使DNA标记，
细胞凋亡检测方法
细胞凋亡的检测方法 一、细胞凋亡概念： 细胞凋亡是指为维持内环境的稳定，有基因控制的细胞自主的程序性死亡。 细胞凋亡不是一件被动的过程，而是主动过程，它涉及一系列基因的激活、表达以及调控等的作用；它并不是病理条件下，自体损伤的一种现象，而是为更好地适应生存环境而主动争取的一种死亡过程。 细胞凋亡与胚胎发育、自身免疫耐受、肿瘤发生、病毒感染等生理、病理过程密切相关,近年来一直是生物医学领域各专业的研究热点。选择合适的凋亡检测方法是研究细胞凋亡研究的关键。 二、细胞凋亡的检测方法： 1. 磷酯酰丝氨酸(PS)外翻法(Annexin V 法) 在凋亡细胞中，磷酯酰丝氨酸 (PS) 从质膜内侧转移到外侧，暴露在细胞外环境中。 荧光基团或荧光蛋白标记的膜联蛋白V 可与暴露在质膜外侧的PS 结合，用于识别凋亡细胞。碘化丙啶(propidine iodide, PI)是一种核酸染料，它不能透过完整的细胞膜，但在凋亡中晚期的细胞和死细胞，PI 能够透过细胞膜而使细胞核红染。因此将Annexin-V 与PI 匹配使用，就可以将凋亡早晚期的细胞以及死细胞区分开来。 应用实例：以FITC Annexin V/ PI Apoptosis Kit 为例子 2. Caspase-3活性的检测: 半胱氨酸蛋白酶caspase 家族蛋白的激活是凋亡进程中的一个必要的决定性事件。其中caspase-3的激活在凋亡信号传导的许多途径中发挥着关键的作用。Caspase-3正常以酶原（32KDa ）的形式存在于胞浆中，在凋亡的早期阶段，它被激活，活化的Caspase-3由两个大亚基（17KDa ）和两个小亚基（12KDa ）组成， 图1. 使用10 μM 喜树碱处理Jurkat 细胞4 小时作为阳性实验组（右图），同时设置未处理组做阴性对照（左图）。使用FITC Annexin V/ PI Apoptosis Kit 对以上两组细胞进行相应的实验处理，流式细胞仪进行观察。
细胞凋亡的几种检测方法
细胞凋亡的几种检测方 法 Company number：【WTUT-WT88Y-W8BBGB-BWYTT-19998】
细胞凋亡的几种检测方法 1、形态学观察方法 （1）HE（苏木精—伊红染色法）染色、光镜观察：凋亡细胞呈圆形，胞核深染，胞质浓缩，染色质成团块状，细胞表面有“出芽”现象。 （2）丫啶橙（AO)染色，荧光显微镜观察：活细胞核呈黄绿色荧光，胞质呈红色荧光。凋亡细胞核染色质呈黄绿色浓聚在核膜内侧，可见细胞膜呈泡状膨出及凋亡小体。 （3）台盼蓝染色：如果细胞膜不完整、破裂，台盼蓝染料进入细胞，细胞变蓝，即为坏死。如果细胞膜完整，细胞不为台盼蓝染色，则为正常细胞或凋亡细胞。此方法对反映细胞膜的完整性，区别坏死细胞有一定的帮助。 （4）透射电镜观察：可见凋亡细胞表面微绒毛消失，核染色质固缩、边集，常呈新月形，核膜皱褶，胞质紧实，细胞器集中，胞膜起泡或出“芽”及凋亡小体和凋亡小体被临近巨噬细胞吞噬现象。 2、 DNA凝胶电泳 细胞发生凋亡或坏死，其细胞DNA均发生断裂，细胞内小分子量DNA片断增加，高分子DNA减少，胞质内出现DNA片断。但凋亡细胞DNA断裂点均有规
律的发生在核小体之间，出现180－200bpDNA片断，而坏死细胞的DNA断裂点为无特征的杂乱片断，利用此特征可以确定群体细胞的死亡，并可与坏死细胞区别。 正常活细胞DNA 电泳出现阶梯状（LADDER)条带；坏死细胞DNA电泳类似血抹片时的连续性条带 3、酶联免疫吸附法（ELISA)核小体测定 凋亡细胞的DNA断裂使细胞质内出现核小体。核小体由组蛋白及其伴随的DNA片断组成，可由ELISA法检测。 检测步骤 1、将凋亡细胞裂解后高速离心，其上清液中含有核小体； 2、在微定量板上吸附组蛋白体’ 3、加上清夜使抗组蛋白抗体与核小体上的组蛋白结合‘ 4、加辣过氧化物酶标记的抗DNA抗体使之与核小体上的DNA结合’ 4、加酶的底物，测光吸收制。 用途 该法敏感性高，可检测5*100/ml个凋亡细胞。可用于人、大鼠、小鼠的凋亡检测。该法不需要特殊仪器，
秀丽线虫生殖细胞凋亡检测 细胞学实验报告
生命科学学院专业生物技术 2016级生技班612组 姓名同实验者 2018年 4 月 23日 题目：秀丽线虫生殖细胞凋亡检测 一．实验目的: 1.掌握检测凋亡细胞的方法 2.学习使用荧光染料活体染色的方法和步骤 二．实验原理 1.秀丽隐杆线虫（Caenorhabditis elegans）：是一种无毒无害、可 以独立生存的线虫。其个体小，成体仅 1.5mm长，为雌雄同体（hermaphrodites），雄性个体仅占群体的0.2%，可自体受精或双性生殖；在20℃下平均生活史为3.5天，平均繁殖力为300-350个；但若与雄虫交配，可产生多达1400个以上的后代。1976年，Sulston和Horvitz利用秀丽隐杆线虫（Caenorhabditis elegans）研究发现，其约13%的体细胞在胚胎发育中注定死亡，使得人们认识到细胞凋亡的遗传基础。 2.荧光染料活体染色：本实验使用吖啶橙（Acridine orange）作为 染色剂，该染料对细胞具有慢性毒性，致癌性强，由于凋亡细胞因DNA片段化可结合更多染料，荧光显微镜下呈亮绿色，可在荧光显微镜下快速方便的检测出，适用于多数品系。
生命科学学院专业生物技术 2016级生技班612组 姓名同实验者 2018年 4 月 23日 三．实验材料及设备 1.实验材料： a)各品系秀丽隐杆线虫：N2（实验组）, ced-1::gfp（方法对照组），ced- 3（阴性对照） b)OP50 c)M9培养基 d)NGM培养基 2.实验设备： a)普通光学显微镜 b)载玻片若干，盖玻片若干，铂金丝 c)暗箱 d)吸水纸、滴管等 e)荧光显微镜 四．实验方法及步骤 1.线虫接种、同步化 2.取样：在12孔板培养板上，每孔吸取900μL预先接入少量OP50 的M9培养基，每孔用铂金丝挑取培养20~30条成体线虫 3.染色：向N2与ced-3品系中每孔加入250μg/mL吖啶橙100μL, 混匀后置于培养箱（避光）染色45~60min。