Autophagy regulates vascular
Autophagy Regulates Vascular Endothelial Cell eNOS and ET-1Expression Induced by Laminar Shear Stress in an Ex Vivo Perfused
System
F ENGXIA
G UO ,1,3X IAOHONG L I ,1J UAN P ENG ,1Y ALING T ANG ,1Q IN Y ANG ,1L USHAN L IU ,1Z UO W ANG ,1Z HISHENG J IANG ,1M ING X IAO ,1C HUYU N I ,1R UIXING C HEN ,1D ANGHENG W EI ,1,2and G UI -XUE W ANG 2
1
Institute of Cardiovascular Disease,Key Laboratory for Arteriosclerology of Hunan Province,University of South China,Hengyang 421001,China;2Key Laboratory of Biorheological Science and Technology (Chongqing University),Ministry of Education,Bioengineering College of Chongqing University,Chongqing 400044,China;and 3The Second People Hospital of
Heze City,Heze 274000,Shandong,China
(Received 17December 2013;accepted 10May 2014;published online 17May 2014)
Associate Editor Estefan?a Pen a oversaw the review of this article.
Abstract —Vascular endothelial cell function responds to
steady laminar shear stress;however,the underlying mech-anisms are not fully elucidated.In the present study,we examined the effect of steady laminar shear stress on vascular endothelial cell autophagy and endothelial cell nitric oxide synthase (eNOS)and endothelin-1(ET-1)expression using an ex vivo perfusion system.Human vascular endothelial cells and common arteries of New Zealand rabbits were pretreated with or without rapamycin or 3-MA for 30min.These were then placed in an ex vivo cell perfusion system or an ex vivo organ perfusion system under static conditions (0dynes/cm 2)or steady laminar shear stress (5or 15dynes/cm 2)for 1h.In both ex vivo perfusion vascular endothelial cells and vascular vessel segment,steady laminar shear stress promoted autophagy and eNOS expression and inhibited https://www.wendangku.net/doc/a34316814.html,pared with steady laminar shear stress treatment alone,the pretreatment of autophagy inducer rapamycin obviously strengthened the expression of eNOS and decreased the expression of ET-1in both the 5and 15dynes/cm 2treatment groups.Moreover,when pretreated with the autophagy inhibitor 3-MA,the eNOS expression was obviously inhibited and the ET-1expression was reversed.These ?ndings demonstrate that autophagy is upregulated under steady laminar shear stress,improving endothelial cell maintenance of vascular tone function.Keywords —Autophagy,Steady laminar shear stress,Endo-thelial cells,Endothelial nitric oxide synthase,Endothelin-1.
INTRODUCTION
Atherosclerosis is the leading cause of death in the developed world and the second cause in the devel-oping world.The endothelium plays a crucial role in controlling vascular tone and homeostasis via the re-lease of vasoactive factors.Endothelium dysfunction is the initiation step in atherosclerotic lesion formation and progression.Vascular endothelium is directly ex-posed to blood ?ow and senses blood ?ow shear stress,the frictional force acting on the endothelial cell sur-face as a result of blood ?ow.Previous studies have shown that vascular endothelium responds not only to shear stress but also to ?ow pattern.6,14,19,28,29Nonla-minar or disturbed blood ?ow results in inhibition of endothelial nitric oxide synthase (eNOS)production,vasodilatation,and endothelial cell repair.7,8Laminar shear stress promotes endothelial cell survival and quiescence,alignment with the direction of ?ow,and secretion of vasodilation and anticoagulation sub-stances.10Thus,laminar shear stress is generally con-sidered to play an important role in resistance to atherogenesis.However,the underlying mechanisms are not well understood.Further research on the response of vascular endothelium to laminar shear stress may contribute to prevent atherosclerosis.
Autophagy is an evolutionarily conserved process involved in removing misfolded or aggregated proteins,clearing damaged organelles,and eliminating intracel-lular pathogens.Most atherosclerotic plaque cells have autophagic marks,and autophagy dysfunction increases with atherosclerotic plaque progression.
Address correspondence to Dangheng Wei,Institute of Cardio-vascular Disease,Key Laboratory for Arteriosclerology of Hunan Province,University of South China,Hengyang 421001,China and Gui-xue Wang,Key Laboratory of Biorheological Science and Tech-nology (Chongqing University),Ministry of Education,Bioengi-neering College of Chongqing University,Chongqing 400044,China.Electronic mail:weizhonghua99@https://www.wendangku.net/doc/a34316814.html,,guixue_wang@https://www.wendangku.net/doc/a34316814.html,
Fengxia Guo,Xiaohong Li,and Juan Peng contributed equally to this study.
Annals of Biomedical Engineering ,Vol.42,No.9,September 2014(ó2014)pp.1978–1988DOI:
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ó2014Biomedical Engineering Society
1978
Blocking macrophage autophagy promotes plaque necrosis in advanced atherosclerosis.17Kim et al.13 demonstrated that epigallocatechin gallate assists in prevention of cardiovascular disease by contributing to the regulation of ectopic lipid accumulation in aortic endothelial cells(ECs)via facilitated autophagic?ux. Our previous works showed that increased EC autoph-agy induced by oxidized low density lipoprotein(ox-LDL)antagonized EC apoptosis and partly neutralized the effect of increased EC monolayer permeability mediated by the downregulation of VE-cadherin.26 In the present study,we hypothesized that steady laminar shear stress upregulates vascular endothelial cell autophagy to improve its function.Thus,we investigated the e?ect of steady laminar shear stress on vascular endothelial cell autophagy and the e?ect of autophagy intervention on endothelial cell eNOS and ET-1expression under steady laminar shear stress.We observed that vascular endothelial cell autophagy was upregulated when exposed to steady laminar shear stress and endothelial cell maintenance of vascular tone function was strengthened by promotion of autophagy and attenuated by blocking of autophagy.
MATERIALS AND METHODS
Materials
New Zealand rabbits, 1.9–2.5kg in weight,were purchased from the animal center of the University of South China.Dulbecco’s modi?ed Eagle’s medium (DMEM)containing trypsin,bovine serum albumin (BSA),rapamycin,and3-Methyladenine(3-MA)was obtained from Sigma-Aldrich(St.Louis,MO).Reve-rAid TM First Strand cDNA Synthesis Kit was pur-chased from Invitrogen(Carlsbad,USA).Monoclonal rabbit antibodies against p62,beclin1,microtubule-associated protein1light chain3(LC3),eNOS,ET-1, and b-actin were obtained from Santa Cruz Biotech-nology(Santa Cruz,CA).
Fluid Shear Stress Experiments
A human umbilical vascular endothelial cell line (HUVEC)was obtained from the China Center for Type Culture Collection and passages40–80were used in this study.Rabbit bilateral common carotids were dissected free from the surrounding connective tissue and placed in a chamber?lled with DMEM containing10%fetal bovine serum,100l g/mL streptomycin and100U/mL penicillin.HUVECs(3.09105cells/mL)were seeded on a precoated with?bronectin glass slide to grow con?uence.ECs monolayer or vessel segments were connected to a parallel plate?ow chamber system (Figs.1a and1b).The perfusion culture medium con-sisted of serum-free DMEM containing antibiotics (100IU/L penicillin,100mg/L streptomycin).In addi-tion,5%dextran(molecular weight,70,000)was added to the culture medium to confer physiological?uid vis-cosity.Steady laminar?ow was controlled by the?ow rate over the endothelial monolayer.The shear stress (s w)acting on the cultured endothelial cell monolayer was determined by s w=6l Q/bh2,where l is?uid vis-cosity,Q is the?ow rate,b is the chamber width,and h is the?ow path height.25In conditions of steady laminar ?ow,shear stress(s w)acting on vessel endothelium is determined by s w=32l Q/p d3,where l is?uid viscos-ity,Q is?ow rate,and d is vessel diameter.30After pre-treatment with rapamycin or3-MA for30min,cultured vascular endothelial cells and vessel segments were exposures to shear stress of0,5or15dynes/cm2for1h. Then,vessel segments were washed with cold PBS. Subsequently,both of the vessel segment ends were closed and0.1%collagenase was injected.After incu-bated at37°C for15min,the vessel was massaged gently and endothelial cells were collected by centrifu-gation.The animal experimental protocols were ap-proved by the Institutional Animal Care and Use Committee of the University of South China.
Real-Time Quantitative PCR
Total RNA was isolated according to the manufac-turer’s instructions(TRIzol reagent;Invitrogen).Total RNA(2l g)was reverse transcribed using a TaqMan Reverse Transcription Reagents Kit(Applied Biosys-tems).Subsequently,2l g of cDNA was added to the real-time quantitative PCR system(Applied Biosystems) for evaluation of relative mRNA levels of Beclin1,LC3, eNOS,and ET-1,with b-actin as the internal control.The primers were followed as Table1.The relative expression of Beclin1,LC3,eNOS,and ET-1mRNA was deter-mined by normalizing the expression of each gene to b-actin gene following the22DD Ct method.
Western Blot Analysis
Protein expression of beclin1,LC3,p62,eNOS,and ET-1was analyzed by Western blot analysis.Whole protein concentration for loading was estimated by Bradford assay.Proteins were separated by SDS–PAGE and transferred onto PVDF membranes.After blocking with BSA,the membranes were incubated overnight at 4°C with anti-beclin1(1:500diluted),anti-LC3(1:1000 diluted),anti-p62(1:1000diluted),anti-eNOS(1:1000 diluted),anti-ET-1(1:1000diluted),or anti-b-actin (1:500diluted)antibodies.The corresponding peroxi-dase-conjugated secondary antibody was applied for2h and then enhanced chemiluminescence reagents were
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used to detect the targeted antigen.The abundance of targeted protein was analyzed using Labwork image analysis software.All experiments were performed at least in triplicate.
Statistical Analysis
The data are presented as mean values ±SEM.The unpaired Student’s t test or ANOVA was used to evaluate the signi?cance of the differences.The p val-ues <0.05were considered to be statistically signi?-cant.RESULTS
Steady Laminar Shear Stress Promotes Cultured
Vascular Endothelial Cell Autophagy In the present study,we tested the hypothesis that steady laminar shear stress induces autophagy in ECs.Autophagosome markers beclin-1and LC3were measured to assess human vascular endothelial cell autophagy in response to steady laminar shear stress.After treatment with steady laminar shear stress,the expression of beclin-1and LC3was signi?cantly increased (p <0.05),particularly in the 15dynes/cm 2treatment group (p <0.01),in the form of upregula-tion of autophagy (Figs.2a–2f).We performed immunoblotting with anti-p62antibody.p62(also
known as SQSTM1)associates with LC3and ubiquitin and is degraded via the autophagic pathway.A de-crease in p62indicates increased autophagic degrada-tion,whereas an increase indicates inhibited autophagic degradation.Under exposure to steady laminar shear stress,protein levels of p62were decreased (p <0.05),particularly in the 15dynes/cm 2treatment group (p <0.01)(Figs.2g and 2h),indicat-ing that steady laminar shear stress increased auto-phagosomal turnover in endothelial cells,promoting the protective effect of autophagic recycling of cyto-plasmic contents.
E?ect of Autophagy on Cultured Vascular Endothelial Cells eNOS Expression Under Steady Laminar Shear
Stress Previous studies suggested that steady laminar shear stress stimulates vascular endothelial cell eNOS expression.1,23Consistent with those results,steady laminar shear stress treatment elicited a signi?cant upregulation of eNOS expression (Figs.3a–3c).Com-pared with steady laminar shear stress treatment alone,the autophagy inducer rapamycin pretreatment elicited an obvious increase in eNOS protein expression.When pretreated with autophagy inhibitor 3-MA,the expression of eNOS expression was obviously inhibited (p <0.01)in both the 5and 15dynes/cm 2treatment groups (Figs.3d–3
i).
FIGURE 1.Diagrams of vascular endothelial cells or vascular vessel segment perfused system ex vivo .De?ned ?uid shear stress was applied to human vascular endothelial cells or vascular vessel segments for 1h.(a)Ex vivo vascular endothelial cell perfused system;(b)ex vivo vascular vessel segment perfused system.
TABLE 1.Real-time quantitative PCR primer.
Gene Forward
Reverse
Beclin 15￠-CAAGATCCTGGACCGTGTCA-3￠5￠-TGGCACTTTCTGTGGACATCA-3￠LC35￠-GCCTTCTTCCTGCTGGTGAAC-3￠5￠-AGCCGTCCTCGTCTTTCTCC-3￠eNOS 5￠-GCCAGAGCAGCACAAGAGTT-3￠5￠-ACGAGCAAAGGCACAGAAGT-3￠ET-15￠-CACCTGTCTTCCTTGGCTTC-3￠5￠-AAAATCGTTGCGTGGACTCT-3￠
b -actin
5￠-GCCAACACAGTGCTGTCTGG-3￠
5￠-GCTCAGGAGGAGCAATGATCTTG-3￠
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E?ect of Autophagy on Cultured Vascular Endothelial Cells ET-1Expression Under Steady Laminar Shear
Stress ET-1is a potent endogenous vasoconstrictor and plays an important role in the progression of athero-sclerosis.4,16Consistent with previous reports,steady laminar shear stress inhibited vascular endothelial cell ET-1expression at both the mRNA (Fig.4a)and protein levels,particularly in the 15dynes/cm 2treat-ment group (Figs.4b and 4c).We then investigated whether autophagy intervention affected vascular endothelial cell https://www.wendangku.net/doc/a34316814.html,pared with steady laminar shear stress treatment alone,autophagy inducer rapamycin pretreatment exhibited a further
inhibition in ET-1protein expression.However,when pretreated with autophagy inhibitor 3-MA,the downregulation of ET-1was reversed in both the 5and 15dynes/cm 2treatment groups (Figs.4d–4i).
Steady Laminar Shear Stress Promotes Autophagy in
Rabbit Aorta Perfused Ex Vivo To ascertain the e?ect of steady laminar shear stress on endothelial cell autophagy,a rabbit common car-otid ex vivo perfused system was used.In agreement with the ex vivo cell perfusion results,steady laminar shear stress upregulated the expression of the vascular cell autophagic marker Beclin 1and LC at both
the
FIGURE 2.Shear stress promotes vascular endothelial cell autophagy.Steady laminar shear stress increased autophagosome markers beclin-1and LC3mRNA and protein levels (a–f),whereas the level of p62was decreased (g and h),n 54.
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mRNA (Figs.5a and 5d)and protein levels (Figs.5b,5c,5e,and 5f).
Autophagy A?ects Rabbit Aorta eNOS Expression
Under Steady Laminar Shear Stress Treatment with 15dynes/cm 2increased the levels of both eNOS mRNA (Fig.6a)and protein (Figs.6b and 6c).However,the 5dynes/cm 2treatment upregulated only eNOS protein levels and not the mRNA levels,suggesting that eNOS activation responds to steady laminar shear stress with a different pattern,such that low steady laminar shear stress affects the eNOS pro-tein level,whereas physiological steady laminar shear
stress regulates both mRNA and protein levels.Under steady laminar shear stress,the expression of eNOS was further strengthened by the pretreatment with rapamycin and obviously inhibited by the pretreatment with 3-MA (Figs.6d–6i).
E?ect of Autophagy on Rabbit Aorta ET-1Expression
Under Steady Laminar Shear Stress The e?ect of autophagy on ET-1suppression in-duced by steady laminar shear stress at an organism level was investigated.Administration of steady lami-nar shear stress at either 5or 15dynes/cm 2signi?-cantly suppressed the expression of ET-1,in
particular
FIGURE 3.Autophagy involvement of vascular endothelial cell eNOS expression induced by steady laminar shear stress.The expression levels of eNOS mRNA (a)and protein (b,c)was increased under steady laminar shear stress.When pretreated with autophagy inducer rapamycin or autophagy inhibitor 3-MA,the expression of eNOS expression in endothelial cells was further increased by rapamycin and was attenuated by 3-MA both in the 5and 15dyne/cm 2treatment groups (d–i).
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in the 15dynes/cm 2group (Figs.7a–7c).When pre-treated with rapamycin or 3-MA,the expression of ET-1was further inhibited by rapamycin and was re-versed by 3-MA both in the 5and 15dynes/cm 2treatment groups (Figs.7d–7i),con?rming that autophagy results in the inhibition of ET-1expression under steady laminar shear stress.
DISCUSSION
Dynamic interaction between shear stress and endothelial cells in?uences not only disease progression but also the clinical outcomes of vascular diseases,
including atherosclerosis and aneurysm.To provide more e?ective prevention approaches and therapeutic interventions for such diseases,the relationship between shear stress and vascular remodeling and adaptation should be explored in greater detail.Autophagy has been demonstrated as one of the most crucial cellular functions in the regulation of cell sur-vival and death,and altered autophagic activity is associated with atherosclerosis.5,22Further,it is well known that steady laminar shear stress counters the resistance of formation and progression.However,it remains to be elucidated whether autophagy is regu-lated by steady laminar shear stress.Further,its role in the bene?t to endothelial cells induced by
steady
FIGURE 4.Autophagy involvement of the inhibition of vascular endothelial cell ET-1expression induced by shear stress.Human vascular endothelial cells were exposed to shear stress (0,5or 15dynes/cm 2,1h),and then whole cell extracts were subjected to RT-PCR to detect changes in ET-1mRNA levels (a)and to western blot analysis to evaluate changes in ET-1protein levels (b,c).After pretreatment with rapamycin or 3-MA,endothelial cells were exposed to steady laminar shear stress for 1h.(d–f),5dynes/cm 2treatment group;(g–i),15dynes/cm 2treatment group.Data are mean 6SE;n 53.
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laminar shear stress is not fully understood.In the present study,we demonstrated that autophagic activity is upregulated by steady laminar shear stress,contributing to increased expression of eNOS and decreased expression of ET-1.
Autophagy,acting as a pro-survival mechanism that protects cells under stress or poor nutrient conditions,degrades damaged cell components or aged proteins into basic biomolecules and drives a degradation–regeneration cycle.A recent report described ATG5‘‘knockout’’mice exhibiting an advanced atheroscle-rotic lesion.24Statins enhance coronary arterial myo-cyte autophagy to exert their bene?cial effects in atherosclerosis.27We speculated that disruption of vascular endothelial cell autophagy is the main cause of endothelial cell dysfunction.In this study,we observed that the expression of beclin 1and LC3was increased and the levels of p62was decreased after treatment with steady laminar shear stress for 1h,which indicated that steady laminar shear stress pro-motes autophagy.Young et al.31showed that laminar shear stress increased phosphorylation of adenosine monophosphate-activated protein kinase (AMPK)and peaked at 1h.Activated AMPK might inhibit mTOR-dependent signaling and is a critic positive regulator of autophagy,11,12,18,20which means that AMPK path-way might play a critic role in steady laminar shear stress induced endothelial cell autophagy.Impaired production of nitric oxide (NO)is a hall-mark of endothelial dysfunction and promotes the development of cardiovascular disease.NO is gener-ated in endothelial cells by https://www.wendangku.net/doc/a34316814.html,Rocca et al.15showed that impaired vascular autophagy with aging plays an important role in impaired vascular tissues endothelium-dependent dilation and inhibition of autophagy increased oxidative stress and reduced NO production.In the present study,steady laminar shear stress upregulated eNOS mRNA and protein expres-sion in cultured endothelial cells and intact arteries.To investigate whether autophagy contributes to the upregulation of eNOS expression under steady laminar shear stress,we pretreated cultured endothelial cells and intact arteries with the autophagy inducer rapa-mycin or the autophagy inhibitor 3-MA.Rapamycin is an inhibitor of the Ser/Thr protein kinase ‘‘mammalian target of rapamycin’’(mTOR)and has been shown to exert both anti-proliferative and protective effects against the development of transplant vasculopathy and in-stent restenosis.3,32A previous study showed that rapamycin increased eNOS levels in low-shear regions,protecting these regions against atherosclero-sis.2In this study,we observed that steady laminar shear stress upregulated the expression of eNOS.When pretreated with autophagy inducer rapamycin,the expression of eNOS was obviously strengthened.However,when pretreated with autophagy
inhibitor
FIGURE 5.Steady laminar shear stress promotes autophagy in rabbit aorta perfused ex vivo .After treatment with steady laminar shear stress,mRNA and protein expressions of the autophagic markers Beclin 1(a–c)and LC3(d–e)in rabbit common carotids were increased.Data are mean 6SE;n 53.
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3-MA,the expression of eNOS was obviously inhibited under steady laminar shear stress.These results dem-onstrate that autophagy is involved in the regulation of eNOS expression under steady laminar shear stress,which supports the idea that the associations between autophagy and eNOS expression are mechanistically linked.Autophagy may,therefore,directly in?uence endothelial cell function by improving eNOS expres-sion.
ET-1,another speci?c marker of endothelial dys-function,acts as the natural counterpart of the vasodi-lator NO and is elevated in arteries with atherosclerotic lesions.A previous study showed that transgenic mice with endothelium-speci?c ET-1overexpression exhib-ited endothelial dysfunction,vascular remodeling,oxi-dative stress,and in?ammation.16Blocking ET-1has been proposed as a strategy for the prevention of ath-erosclerosis.21In this study,we observed that steady laminar shear stress inhibited endothelial cells ET-1expression.When pretreated with rapamycin,the expression of ET-1expression was further inhibited.However,when pretreated with 3-MA,the downregu-lation by steady laminar shear stress was reversed in both cultured endothelial cells and intact arteries.Studies showed that enhanced autophagy decreases the generation and accumulation of reactive oxygen species (ROS),9which may contributes to the downregulation of ET-1induced by steady laminar
stress.
FIGURE 6.Autophagy affects rabbit aorta eNOS expression induced by steady laminar shear stress.Steady laminar shear stress increased expression levels of both rabbit aorta eNOS mRNA (a)and protein (b,c).Rabbit aorta endothelial cells eNOS expression was increased by pretreatment with rapamycin and inhibited by pretreatment with 3-MA in both the 5and 15dynes/cm 2treatment group (d–i).
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Several limitations to our study need to be consid-ered.In this study,we only detected the relationship between laminar shear stress and endothelial cells autophagy.In vivo ,endothelial cells were not only exposed to laminar shear stress in ‘‘straight’’regions of vasculature,but also exposed to disturbed shear stress near arterial bifurcations,branch ostia and curvatures.Thus,more studies are required to determine the relationship between shear stress and autophagy and to elucidate the signaling pathways underlying the relationship.
The present study focused on autophagy and its role in vascular endothelial cell eNOS and ET-1expression under steady laminar shear stress.In this context,it appears that autophagy played a protective role in cul-tured vascular endothelial cells and intact arteries under an in vivo perfused system,promoting the expression of eNOS and inhibiting the expression of ET-1.These re-sults may shed light on the mechanisms by which shear stress forces modulate vascular homeostasis and pro-vide a strategy to ?ght against atherosclerosis via improving vascular endothelial cells autophagy.Future directions unraveling mechanisms that autophagy on endothelial cells function regulation is crucial to de?ne autophagy as a unique therapeutic target to ?ght against the development and progression of
atherosclerosis.
FIGURE 7.Autophagy affects inhibition of rabbit aorta ET-1expression induced by shear stress affects expression.Rabbit common carotids were treated with steady laminar shear stress for 1h with or without subsequent rapamycin or 3-MA for additional 30min.RT-PCR and Western blotting were used to detect mRNA and protein expression,respectively.a–c Rabbit aorta was treated rapamycin or 3-MA for 30min,followed by treatment with 5dynes/cm 2for 1h (d–f);rabbit aorta was exposed to rapamycin or 3-MA for 30min,followed by treatment with 15dynes/cm 2for 1h (g–i).
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ACKNOWLEDGMENTS
The present research is supported by the National Natural Science Foundation of China(81370378, 30800449),the construct program of the key discipline in Hunan province and key grant from the National Natural Science Foundation of China(No.11332003).
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帕金森病中的自噬途径与关键药物靶点_欧阳亮
帕金森病中的自噬途径与关键药物靶点 欧阳亮, 张岚, 刘博* (四川大学华西医院, 生物治疗国家重点实验室, 生物治疗协同创新中心, 四川成都 610041) 摘要: 帕金森病 (PD) 是一种常见的神经退行性疾病。在过去几十年中, 对PD的发病机制的探索已有了较大的进步, 环境因素和遗传因素都会导致PD的发生, 然而它的具体发病机制仍然未知。最近的研究表明自噬过程或许与PD密切相关, 在许多PD患者和动物模型中都观察到了异常的自噬水平。此外, 一些PD相关蛋白, 如α-synuclein、Parkin和PINK1等都被发现参与自噬的调控, 被认为与PD的发病机制相关。本文综述了几种重要PD相关蛋白在自噬途径中的作用, 同时概述了通过调节自噬过程来治疗PD的潜在策略。 关键词: 自噬; 帕金森病; α-突触核蛋白; 线粒体自噬; 帕金森病治疗 中图分类号: R966 文献标识码:A 文章编号: 0513-4870 (2016) 01-0009-09 Autophagy pathways and key drug targets in Parkinson’s disease OUYANG Liang, ZHANG Lan, LIU Bo* (State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Chengdu 610041, China) Abstract: Parkinson’s disease (PD) is a common neurodegenerative disorder associated with aging. Great progresses have been made toward understanding the pathogenesis over the past decades. It seems that both genetic factors and environmental factors contribute to PD, while the precise pathogenesis still remains unknown. Recently, increasing evidence has suggested that autophagy dysregulation is closely related to PD. Dysregula-tion of the autophagic pathways has been observed in the brains of PD patients or in animal models of PD, and a number of PD-associated proteins, such as α-synuclein, Parkin and PINK1, were found to involve in autophagy, suggesting a link between autophagy and pathogenesis of PD. In this review, we summarized the role of PD-associated proteins in autophagy pathways. In addition, we described the efficacy of autophagy-modulating compounds in PD models and discussed promising strategies for PD therapy. Key words: autophagy; Parkinson’s disease; α-synuclein; mitophagy; Parkinson’s disease therapy 1 帕金森病概述 帕金森病 (Parkinson’s disease, PD) 是居于阿兹海默病后的第2位最常见的神经退行性疾病, 其主要病理学特征是在黑质中多巴胺能神经元细胞死亡, 黑质纹状体通路退化[1]。此外, 受损的多巴胺能神经元胞浆内存在着路易小体 (Lewy body, LB), 收稿日期: 2015-08-11; 修回日期: 2015-10-14. 基金项目: 国家自然科学基金资助项目 (81473091, 81260628). *通讯作者 Tel / Fax: 86-28-85503817, E-mail: liubo2400@https://www.wendangku.net/doc/a34316814.html, DOI: 10.16438/j.0513-4870.2015-0706 其内主要包含异常的或者聚集体形式的α-突触核蛋白(α-synuclein)[2]。脑内产生多巴胺的细胞逐渐丧失了影响神经系统的功能, 使患者控制肌肉的能力受限。在临床上, PD具有一些核心运动症状, 统称为震颤性麻痹, 包括静止性震颤、运动迟缓、肌强直、姿势不稳和步态障碍。此外, 临床描述的PD还包括几种非运动性症状, 如执行功能障碍、自主神经系统功能障碍、睡眠障碍、行为和精神方面的改变以及嗅觉障碍等[2?4]。据2013年美国国立PD基金会统计, 全世界有400万～600万名PD患者, 在工业化国家中流
细胞自噬与肿瘤治疗的研究进展
中国组织化学与细胞化学杂志 CHINESE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY 第29卷第2期2020年4月 V ol.29.No.2April.2020 〔收稿日期〕2020-01-06 〔修回日期〕2020-04-09 〔基金项目〕国家重点研究计划“重大慢性非传染性 疾病防控研究”重点专项2018年度定向项目子课题（2018YFC1311300）；湖北省第二届医学领军人才工程第二层次基金（[2019]47） 〔作者简介〕高利昆，女（1983年），汉族，在读博士 *通讯作者(To whom correspondence should be addressed)：dr_hongli@https://www.wendangku.net/doc/a34316814.html, 细胞自噬与肿瘤治疗的研究进展 高利昆1，袁静萍2，洪莉1* （武汉大学人民医院1妇产科，2病理科，武汉430060） 〔摘要〕自噬是一种高度保守的、存在于真核细胞内的自我降解机制，自噬水平异常会破坏细胞内稳态。自噬在肿瘤中既能抑制早期肿瘤发生又可促进肿瘤发展，尤其是其对多种肿瘤治疗有细胞毒性、细胞保护性的双重影响，如何调控自噬用于肿瘤治疗及化疗增敏已成为肿瘤防治领域的研究热点。在本综述中对自噬在肿瘤致病及进展过程中的具体作用展开了总结，重点讨论了自噬对肿瘤治疗的影响及当前靶向自噬改善肿瘤治疗的各种新策略。 〔关键词〕自噬；肿瘤治疗；化疗耐药 〔中图分类号〕R736.1 〔文献标识码〕A DOI ：10.16705/ j. cnki. 1004-1850. 2020. 02. 015 Recent progress in autophagy and tumor therapy Gao Likun 1, Yuan Jingping 2, Hong Li 1* (1Department of Obstetrics and Gynecology, 2Department of Pathology, Renmin Hospital of Wuhan University, Wuhan 430060, China) 〔Abstract 〕 Autophagy is a highly conservative self-degradation mechanism that exists in eukaryotic cells. Abnormal levels of autophagy will destroy intracellular homeostasis. Autophagy can not only suppress early tumorigenesis but also promote tumor devel-opment, especially it has dual effects of cytotoxicity and cytoprotection on various tumor treatments. How to regulate autophagy for tumor treatment and chemotherapy sensitization has become research hotspots in the field of cancer prevention and treatment. In this review, the specific role of autophagy in the pathogenesis and progression of tumors is summarized, focusing on the impact of autoph -agy on tumor treatment and the current various new strategies targeted on autophagy to improve tumor treatment. 〔Keywords 〕Autophagy; tumor therapy; chemoresistance 自噬(autophagy)是真核细胞内发生的一种高度保守的自我降解机制[1]。生理状态下，基础活性下的自噬会降解并清除受损或死亡细胞器、错配蛋白质等物质，维持细胞内稳态[2]，自噬异常则会打破细胞原有平衡而导致肿瘤发生。自噬可分为3种类型：巨自噬（macroautophagy ）、微自噬（microau-tophagy ）及分子伴侣介导的自噬(chaperone-mediated autophagy ，CMA)。巨自噬涉及到特有的双层膜囊泡即自噬体的形成，可捕获自噬相关货物并进一步将货物传送至溶酶体形成自噬溶酶体[1]。微自噬不涉及胞浆内双层膜囊泡的形成，而是由溶酶体内陷或 突出直接吞噬细胞内物质[3]。分子伴侣介导的自噬的特征则是通过热休克蛋白家族与位于溶酶体膜上的溶酶体相关膜蛋白2（LAMP-2A ）共同作用于胞浆内物质后将其转运至溶酶体内发生降解[4]。本文中所讨论的自噬即为巨自噬，目前了解最多。1 自噬发生过程与调控 自噬的发生是一个动态发展的连续过程，主要包括双层膜结构的起始阶段、延伸阶段、成熟降解阶段从而完成自噬体形成融合到溶酶体，进一步形成自噬溶酶体，降解其内包裹的物质并释放得以重新利用[1]。迄今为止发现，自噬全过程由超过30个自噬相关基因（autophagy related gene, ATG ）及相应蛋白严格调控[5]。依据功能和生理上的相互作用机制， ATG 蛋白参与形成五个分子复合体[5]：①ULK1复合体：由ULK1/ULK2、ATG13/101、FIP200组成，该复合体的形成及活化启动自噬的发生。②ATG9复合体：唯一的整体跨膜核心蛋白，促进磷酸的转运。③PI3KC3复合体：包括将PI 转化为PI-3磷酸盐（PI3P ）的催化亚单位真空蛋白34（VPS34）、beclin1和囊泡运输因子p115与ATG14联结一起组
细胞自噬和肿瘤
细胞自噬和肿瘤 摘要：近年来，细胞自噬与肿瘤的关系是研究热点。研究表明，自噬是肿瘤的双刃剑；自噬调节剂与细胞毒性药物联用在临床应用上具有很大潜力。对于细胞自噬和癌症发生之间关系还需要更加深入的研究，这将会有助于人类更好地认识并最终攻克癌症。本文将针对细胞自噬在肿瘤发生过程的作用、主要的信号调节通路以及其在治疗中的作用进行简单介绍。 关键词：细胞自噬、肿瘤、信号调节通路、治疗 一、细胞自噬和肿瘤的关系 细胞自噬(autophagy)是指真核生物中的一些受损或衰老的蛋白质以及细胞器被双层膜结构的自噬小泡包裹后，送入溶酶体中进行降解并得以循环利用的过程。细胞自噬过程大致分为4个阶段:首先，细胞浆中出现游离前自噬泡，诱导起始与成核；前自噬泡包裹受损或衰老的蛋白质以及细胞器，逐渐发展成为由双层膜结构形成的自噬泡；自噬泡与溶酶体膜融合后，内膜及其包裹的物质进入溶酶体，被其中的酶水解；最后，溶酶体的物质分解为细胞生存所必需的组成成分，底物降解再循环。 肿瘤发生的机制很复杂，主要表现为细胞过度增殖，具有高代谢的特点，对营养和能量的需求很高；另外肿瘤细胞在恶劣的细胞外环境下（如代谢压力、缺氧、缺乏血供、养分及生长因子不足、抗肿瘤治疗等）具有很强的凋亡抵抗能力，逆境生存的能力极强。这些都有赖于肿瘤细胞的自噬活性。 正常生理情况下，细胞自噬能够及时清除细胞中产生的受损或衰老的蛋白质以及细胞器等，有利于细胞保持自稳状态。细胞自噬可以抑制细胞发生癌变，首先，细胞自噬可以清除坏死的细胞器，调整内源性的压力，从而稳定基因组，减少细胞向癌细胞的转变；其次，细胞自噬既可以加强细胞检验点的检查作用，又起到了稳定基因组的作用，从而减少了细胞的癌变。然而肿瘤一旦形成，细胞自噬又会为癌细胞提供更丰富的营养，促进肿瘤生长。不过，目前大多数研究者还是认为，细胞自噬是一种对抗细胞癌变的机制。(见图1)。[1] 图1、细胞自噬在肿瘤发生和发展中的角色 二、检测细胞自噬与细胞癌变的一般方法
自噬与肿瘤的关系及抗肿瘤药物
自噬与肿瘤的关系及抗肿瘤药物 张庆余任皓刘洁朱润芝 广东医学院附属医院肝胆外科研究室 湛江市肝胆相关疾病重点实验室 摘要：自噬是将细胞内受损、变性或衰老的蛋白质以及细胞器运输到溶酶体进行消化降解的过程.正常生理情况下，细胞自噬利于细胞保持自稳状态;在发生应激时，细胞自噬防止有毒或致癌的损伤蛋白质和细胞器的累积，抑制细胞癌变;然而肿瘤一旦形成，细胞自噬为癌细胞提供更丰富的营养，促进肿瘤生长.因此，在肿瘤发生发展的过程中，细胞自噬的作用具有两面性.对于细胞自噬和肿瘤发生之间的关系有待深入的研究，这将会有助于人类更好地认识并最终攻克肿瘤。 关键词：肿瘤，自噬，抗肿瘤药物 Abstract:Autophagy is the major intracellular degradation system by which cytoplasmic materials (denatured protein, damaged organelles) are delivered to and degraded in the lysosome to maintain homeostasis. Once carcinogenesis rising up, autophagy would be also employed by cancer cells. Autophagy plays an important role in cancer cells– both in protecting against cancer as well as potentially contributing to the growth of cancer. However, autophagy can also contribute to cancer by promoting survival of tumor cells that have been starved. The relationship between autophagy and tumorigenesis need to be further researched, which will help humanity better understand and ultimately overcome the cancers. Keywords: Tumor, Autophagy, Anti-tumor drug 1 细胞自噬的概念 1.1 自噬的基本概念 传统的细胞死亡方式分类包括细胞坏死和细胞凋亡2种，后者又称为程序性细胞死亡，而新近的研究发现，除坏死和凋亡之外还存在其他细胞死亡方式，例如自噬[1] 。 细胞自噬是真核生物中进化保守的对细胞内物质进行周转的重要过程。该过程中一些损坏的蛋白或细胞器被双层膜结构的自噬小泡包裹后，送入溶酶体(动物）或液泡（酵母和植物）中进行降解并得以循环利用。细胞自噬最早由Ashford和Ponen于1962年用电子显微镜在人的肝细胞中观察到，是细胞中初级溶酶体处理内源性底物的重要过程，同时参与维持蛋白代谢平衡及细胞内环境的稳定，其在清除废物、结构重建以及细胞生长发育中起重要作用[2]。自噬存在于真核细胞的病理生理过程中，近年随着对自噬研究的不断深入，其在肿瘤中的作用日渐引起广泛关注。目前研究表明自噬功能异常在肿瘤发生、发展中均扮演重要角色[3] 。在生物进化中，细胞自噬是一种保守的过程，从酵母到植物细胞再到哺乳动物，都存在这样的过程，并且其中的很多调节因子在多个生物种中都能找到其同源体[4]。 1.2 与自噬相关的重要细胞器——溶酶体 损坏的蛋白或细胞器在体内降解主要有泛素-蛋自酶体途径（UPS）和溶酶体途径（细胞自噬）。UPS和自噬在降解过程、机制、亚细胞定位、降解底物、系统活性等方面均有区别，细胞自噬是UPS受损时的代偿途径，细胞自噬可以在UPS抑制时激活；细胞自噬的长期抑制可导致UPS功能受损，但UPS不能代偿细胞自噬被抑制的功能[5] 。本文主要讨论溶酶体途径。 自噬是细胞对持续性内外刺激的非损伤性应答反应，以维持细胞结构、代谢和功能的平
肿瘤的自噬机制及自噬相关治疗
Vol.5 No.1Feb. 2019生物化工Biological Chemical Engineering 第 5 卷 第 1 期 2019 年 2 月肿瘤的自噬机制及自噬相关治疗 司新红１，高睿１，商雨婷１，李若谨１，王澈２＊ （１．辽宁师范大学化学化工学院，辽宁大连 １１６０２９；２．辽宁省生物技术与分子药物研发重点实验室，辽宁大连 １１６０８１） 摘?要： 自噬是一种细胞自我降解的过程。当细胞处于应激状态下受损时，细胞启动自噬来清除受损的蛋白质和细胞器，细胞依赖自噬促进细胞内成分的再利用，维持代谢稳态与细胞生长。越来越多的研究表明，自噬在肿瘤细胞中也具有非常重要的作用，一方面适度的自噬可以使受损的肿瘤细胞存活，另一方面过度的自噬则会加速肿瘤细胞的死亡。阐明肿瘤细胞中的自噬机制有助于发现新的抗肿瘤分子靶点，进而开发出有效的基于自噬机制的抗肿瘤药物。本文通过阐述自噬在肿瘤细胞中的发生机制来探讨自噬对肿瘤细胞生存的影响，并进一步探讨自噬在肿瘤治疗中的意义。 关键词： 自噬；肿瘤细胞；肿瘤治疗中图分类号：Ｒ７３０．５ 文献标志码：Ａ Ｔｕｍｏｒ Ａｕｔｏｐｈａｇｙ Ｍｅｃｈａｎｉｓｍ ａｎｄ Ａｕｔｏｐｈａｇｙ Ｒｅｌａｔｅｄ Ｔｒｅａｔｍｅｎｔ Ｓｉ Ｘｉｎ－ｈｏｎｇ１，Ｇａｏ Ｒｕｉ１，ＳｈａｎｇＹｕ－ｔｉｎｇ１，Ｌｉ Ｒｕｏ－ｊｉｎ１，Ｗａｎｇ Ｃｈｅ２＊ （１．Ｓｃｈｏｏｌ ｏｆ Ｃｈｅｍｉｓｔｒｙ ａｎｄ Ｃｈｅｍｉｃａｌ Ｅｎｇｉｎｅｅｒｉｎｇ， Ｌｉａｏｎｉｎｇ Ｎｏｒｍａｌ Ｕｎｉｖｅｒｓｉｔｙ，Ｌｉａｏｎｉｎｇ Ｄａｌｉａｎ １１６０２９； 2.Liaonin ｇ Ｋｅｙ Ｌａｂｏｒａｔｏｒｙ ｏｆ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ ａｎｄ Ｍｏｌｅｃｕｌａｒ Ｄｒｕｇ Ｄｅｖｅｌｏｐｍｅｎｔ， Ｌｉａｏｎｉｎｇ Ｄａｌｉａｎ １１６０８１） Abstract: Ａｕｔｏｐｈａｇｙ ｉｓ ａ ｐｒｏｃｅｓｓ ｏｆ ｃｅｌｌ ｓｅｌｆ－ｄｅｇｒａｄａｔｉｏｎ． Ｗｈｅｎ ｃｅｌｌｓ ａｒｅ ｄａｍａｇｅｄ， ａｕｔｏｐｈａｇｙ ｉｓ ｉｎｉｔｉａｔｅｄ ｔｏ ｒｅｍｏｖｅ ｄａｍａｇｅｄ ｐｒｏｔｅｉｎｓ ａｎｄ ｏｒｇａｎｅｌｌｅｓ． Ｃｅｌｌｓ ｒｅｌｙ ｏｎ ａｕｔｏｐｈａｇｙ ｔｏ ｐｒｏｍｏｔｅ ｔｈｅ ｒｅｕｓｅ ｏｆ ｉｎｔｒａｃｅｌｌｕｌａｒ ｃｏｍｐｏｎｅｎｔｓ， ｍａｉｎｔａｉｎｉｎｇ ｍｅｔａｂｏｌｉｃ ｈｏｍｅｏｓｔａｓｉｓ ａｎｄ ｓｕｒｖｉｖａｌ ｏｆ ｃｅｌｌｓ； Ｉｎｃｒｅａｓｉｎｇ ｌｉｎｅｓ ｏｆ ｅｖｉｄｅｎｃｅ ｈａｖｅ ｓｈｏｗｎ ｔｈａｔ ａｕｔｏｐｈａｇｙ ａｌｓｏ ｐｌａｙｓ ｉｍｐｏｒｔａｎｔ ｒｏｌｅｓ ｉｎ ｃａｎｃｅｒ ｃｅｌｌｓ． Ｏｎ ｔｈｅ ｏｎｅ ｈａｎｄ， ｍｏｄｅｒａｔｅ ａｕｔｏｐｈａｇｙ ｃａｎ ｒｅｓｃｕｅ ｔｈｅ ｄａｍａｇｅｄ ｃａｎｃｅｒ ｃｅｌｌｓ ｔｏ ｓｕｒｖｉｖｅ．Ｏｎ ｔｈｅ ｏｔｈｅｒ ｈａｎｄ， ｅｘｃｅｓｓｉｖｅ ａｕｔｏｐｈａｇｙ ａｃｃｅｌｅｒａｔｅｓ ｃａｎｃｅｒ ｃｅｌｌｓ ｄｅａｔｈ． Ｅｌｕｃｉｄａｔｉｎｇ ｔｈｅ ｍｏｌｅｃｕｌａｒ ｍｅｃｈａｎｉｓｍｓ ｏｆ ａｕｔｏｐｈａｇｙ ｉｎ ｃａｎｃｅｒ ｃｅｌｌｓ ｈｅｌｐｓ ｕｓ ｄｉｓｃｏｖｅｒ ｎｅｗ ｍｏｌｅｃｕｌａｒ ｔａｒｇｅｔｓ ａｎｄ ｄｅｖｅｌｏｐ ｅｆｆｅｃｔｉｖｅ ａｕｔｏｐｈａｇｙ－ｂａｓｅｄ ａｎｔｉ－ｃａｎｃｅｒ ｄｒｕｇｓ． Ｉｎ ｔｈｉｓ ｒｅｖｉｅｗ， ｗｅ ｓｕｍｍａｒｉｚｅｄ ｔｈｅ ｍｏｌｅｃｕｌａｒ ｍｅｃｈａｎｉｓｍｓ ｏｆ ａｕｔｏｐｈａｇｙ ｉｎ ｃａｎｃｅｒ ｃｅｌｌｓ， ａｎｄ ｆｕｒｔｈｅｒ ｄｉｓｃｕｓｓｅｄ ｔｈｅ ｓｉｇｎｉｆｉｃａｎｃｅ ｏｆ ａｕｔｏｐｈａｇｙ ｉｎ ｃａｎｃｅｒ ｔｈｅｒａｐｙ． Keywords: Autophagy; Cancer cells; Cancer therapy １ 细胞自噬的分子机制 细胞自噬是进化过程中高度保守的自我调节机 制，可分为巨自噬（或大自噬，Macroautophagy）、微 自噬（或小自噬，Microautophagy）和分子伴侣介导 的自噬（Chaperone-mediated autophagy）。巨自噬通 过形成具有双层膜结构的自噬体（autophagosome）， 包裹细胞质中的物质并与溶酶体结合形成自噬溶酶 体，继而在自噬溶酶体中将物质降解，最终将降解后的物质用于细胞生存和功能维持；微自噬是通过溶酶体或液泡表面直接吞噬特定的细胞器将其降解；分子伴侣介导的自噬则是通过分子伴侣将胞浆蛋白导入溶酶体内腔进行降解的过程[1-2]。巨自噬与微自噬通常是非选择性的过程，而分子伴侣自噬为选择性降解过程。在分子伴侣自噬过程中，热休克蛋白（HSC70）特异性识别具有KFERQ 基序的底物，只有被识别之后的这一类底物才可以被运送至溶酶体进行降解[3]。 司新红（1993—），女，河南郑州人，硕士，研究方向：有机化学。 王澈（1977—），女，吉林长春人，博士，教授，研究方向：生物技术与分子药物研发。E-mail: wangche@https://www.wendangku.net/doc/a34316814.html,。文章编号：2096-0387（2019）01-0153-03
自噬与肿瘤的关系及抗肿瘤药物.doc
---------------------------------------------------------------最新资料推荐------------------------------------------------------ 自噬与肿瘤的关系及抗肿瘤药物.doc 自噬与肿瘤的关系及抗肿瘤药物摘要： 自噬是将细胞内受损、变性或衰老的蛋白质以及细胞器运输到溶酶体进行消化降解的过程.正常生理情况下，细胞自噬利于细胞保持自稳状态；在发生应激时，细胞自噬防止有毒或致癌的损伤蛋白质和细胞器的累积，抑制细胞癌变；然而肿瘤一旦形成，细胞自噬为癌细胞提供更丰富的营养，促进肿瘤生长.因此，在肿瘤发生发展的过程中，细胞自噬的作用具有两面性.对于细胞自噬和肿瘤发生之间的关系有待深入的研究，这将会有助于人类更好地认识并最终攻克肿瘤。 关键词： 肿瘤，自噬，抗肿瘤药物 Abstract： Autophagy is the major intracellular degradation system by which cytoplasmic materials （denatured protein， damaged organelles） are delivered to and degraded in the lysosome to maintain homeostasis. Once carcinogenesis rising up，autophagy would be also employed by cancer cells. Autophagy plays an important role in cancer cells? both in protecting against cancer as well as potentially contributing to the growth of cancer. However ， autophagy can also contribute to cancer by promoting survival of tumor cells that have been starved. The relationship between autophagy and tumorigenesis 1 / 16
自噬与肿瘤
收稿日期:2006205226 基金项目:国家自然科学基金资助项目(30400531)作者简介:温莹浩(19772),女,江西萍乡人,第二军医大学东方肝胆外科医院硕士生,从事肿瘤基因治疗研究. 自噬与肿瘤 温莹浩,殷正丰 (第二军医大学东方肝胆外科医院分子肿瘤实验室,上海200438) 关键词:肿瘤;自噬;癌基因;基因,抑制,肿瘤 中图分类号:R 730 文献标识码:A 文章编号:100121692(2006)0520482205 细胞稳态依赖于大分子物质生物合成和分解代谢间的平衡。自噬(au tophagy )即是细胞用来达到这种动态平衡的重要机制之一,它是普遍存在于大部分真核细胞中的一种现象,从酵母到人类存在着共同的自噬分子调控机制。 真核细胞的降解主要有两条途径,即蛋白酶降解和自噬,但只有自噬具有降解整个细胞器的效力。自噬是大分子物质和细胞器在溶酶体腔发生的降解途径。自噬的作用主要是清除降解细胞内受损伤的细胞结构、衰老的细胞器,以及不再需要的生物大分子等,同时也为细胞内细胞器的构建提供原料,即细胞结构的再循环。因此,自噬是细胞成分更新、发育、分化及组织重塑的重要调控机制。自噬也与包括肿瘤在内的多种人类疾病有关。有趣的是,自噬能够保护细胞,也能造成细胞损伤。1 自噬的作用及意义 “au tophagy ”源于希腊词语,其语源是“phagy ”=吃与“au to ”=自我,因此自噬是一动态过程。自噬始于包裹膜包裹胞内大分子物质和细胞器后形成具有双层或多层膜的液泡即自噬体(au top hago som e )或自噬液泡(au top hagic vacuo le )。包裹膜可能源于粗面内质网的无核糖区、反面高尔基体网络或其它有膜囊泡体。自噬体膜相关蛋白的缺乏是从酵母到哺乳细胞的共同特征,故难以确定自噬体膜的起源。对大鼠纯化自噬体的分析显示,自噬体膜富集三种也存在于胞液中的蛋白:精氨琥珀酸合酶、32磷酸甘油醛脱氢酶N 2端截断变异体和短链22烯酰CoA 水合酶变异体,但其在自噬体形成中的作用尚未明确[1]。自噬体形成后,通过与溶酶体融合形成自噬性溶酶体(au 2top hago lyso som e ),完成对包裹物质的降解和再循环 利用。 自噬主要有三种作用[2]:(1)自噬是对营养缺乏的一种适应性反应,如氨基酸缺乏在肝脏和培养细胞中可以诱导自噬。自噬对大分子物质的降解能够产生调节新陈代谢和生物合成的氨基酸及其它因子,为细胞提供能量,维持细胞的生命活动。氨基酸是自噬的重要调节因子,缺失氨基酸在肝脏中迅速诱导自噬,最初可观察到自噬体,经历7～8分钟后降解液泡出现。从自噬体的形成到转化成残余体这一完整自噬过程约需20分钟,营养缺乏的肝细胞中自噬途径对胞质蛋白这种非选择性的降解率为3%～4% 小时。(2)自噬能够调节过氧化物酶体、线粒体、内质网的更新,故自噬被认为是一种参与细胞质稳态的看家机制,在许多病理生理和应激刺激状况 下可观察到对这些细胞器选择性的分隔。 (3)自噬参与特定的组织特异性功能。自噬与肺泡表面活性剂 生物合成有关。黑质多巴胺能神经元神经黑色素的生物合成应归于胞质多巴胺2苯醌被分隔入自噬液泡。在红细胞成熟过程中,细胞核排出后需要自噬清除核糖体、线粒体等细胞器。2 肿瘤细胞中自噬的作用 自噬是肿瘤细胞对外源性应激具有不同结果的反应,多项证据表明自噬在肿瘤发生中同时具有促进和抑制作用。 首先,自噬是保护肿瘤细胞避免受到低营养、电离辐射和治疗损伤所诱导的应激的一种保护机制。在肿瘤进展过程中,由于血供减少,肿瘤细胞特别是肿瘤内部细胞在乏氧和营养受限的状况下生存[3]。有趣的是,乏氧和营养缺失均能刺激自噬启动,通过自噬对胞内物质的降解和循环利用而改变新陈代谢。肿瘤细胞对应激作出适应性反应得以生存,同时,自噬对线粒体的分隔可防止促凋亡因子如细胞 色素和凋亡诱导因子(A IF )的扩散。自噬的这种作用能够帮助细胞逃逸凋亡。乳腺、结肠和前列腺癌细
细胞自噬与肿瘤治疗综述
细胞自噬与肿瘤治疗 施晓莹 摘要：细胞自噬是真核生物的一种进化保守的新陈代谢的过程，双层膜结构的自噬小泡包裹损坏的蛋白或细胞器，将其送入动物体的溶酶体或植物体和酵母的液泡中进行降解，使之得以循环利用，来维持细胞内的稳态。现在有研究表明，细胞自噬对肿瘤发生的不同阶段具有促进和抑制的双重作用，可通过对自噬的深入研究研制出治疗肿瘤的药物。 关键字：自噬肿瘤治疗 前言：细胞自噬是高度保守的自我消化和细胞存活维持稳态的过程。本文将对细胞自噬的过程、种类及功能进行简单介绍，并概述目前细胞自噬对治疗肿瘤的研究进展。随着环境的不断恶化和人类对自身健康的忽视，患癌率在不断上升，研制出有效治疗肿瘤的药物已迫在眉睫，探究细胞自噬如何抑制肿瘤的发生是目前科学界最感兴趣的项目之一，对人类今后的生命发展有着重要意义。 细胞自噬（autophagy）是真核生物的一种进化保守的新陈代谢的过程，双层膜结构的自噬小泡包裹损坏的蛋白或细胞器，将其送入动物体的溶酶体或植物体和酵母的液泡中进行降解，使降解产物氨基酸、核苷酸、游离脂肪酸等可得以循环利用，来维持细胞内环境的动态平衡。通常，寿命较短的蛋白质如调控蛋白等通过泛素-蛋白酶体系统进行降解；而寿命较长的蛋白质及细胞结构则通过细胞自噬途径，由溶酶体进行降解。 1.细胞自噬的过程 1.1膜泡的形成 当外界有辐射、低氧、饥饿、高温等因素或细胞内部发生应激反应如遇细胞器损坏或突变蛋白等均会引诱细胞发生自噬，此时来自内质网或细胞质中的双层膜形成膜泡，即前自噬体。 1.2自噬体的形成 膜泡募集自噬蛋白进入到吞噬泡成核中心，泛素化耦联系统对其进行辅助, 募集 Atg3、Atg7、Atg10、Atg8/LC3Atg4到前自噬体结构，使囊泡膜逐渐扩展，包裹细胞内待降解的细胞器或其他细胞内含物，然后闭合形成自噬体。 1.3自噬溶酶体的融合 自噬体酸化与细胞骨架微管系统相互作用，并与溶酶体融合形成自噬溶酶体。 1.4自噬体的降解 溶酶体内的一些酶参与水解自噬体，自噬体膜的受体蛋白能够降解错误折叠的蛋白质多聚体，还能够降解功能失常的整个线粒体、过氧化物体、高尔基体等细胞器，甚至可以清除细胞内的病原体，并且还能提供营养物质和 ATP给细胞再利用[1]。（见图一）