Differential and Synergistic Effects of Platelet

O RIGINAL A RTICLE

Differential and Synergistic Effects of Platelet-derived Growth Factor-BB and Transforming Growth Factor-b1 on Activated Pancreatic Stellate Cells

Claus Kordes,PhD,Stefanie Brookmann,Dieter Ha¨ussinger,MD,and Hanne Klonowski-Stumpe,PhD

Objective:The cytokines platelet-derived growth factor(PDGF) and transforming growth factor(TGF)-b1are major factors in?u-encing the transformation from the quiescent to the activated phenotype of pancreatic stellate cells(PSC),a process involved in the patho-genesis of chronic pancreatitis.Albeit much effort has been made to study the effects of PDGF and TGF-b1on PSCs,their interaction is still unclear,because these cytokines show both differential and synergistic effects as outlined by this study.

Methods:Culture-activated PSCs of rats were treated with PDGF-BB and TGF-b1.Subsequent changes of cell proliferation and migration were determined by cell counting,(+)-bromo-2#-deoxyur-idine enzyme-linked immunosarbant assay(ELISA),and migration assay.Gene expression,synthesis of proteins,and activation of ki-nases were further studied by reverse transcription-polymerase chain reaction,real-time polymerase chain reaction,ELISA,and Western blot.

Results:PDGF-BB increased PSC proliferation and migration,ac-companied by elevated expression of matrix metalloproteinases(MMP)-13and MMP-3.The mRNA amount of procollagen a2(I),a-smooth muscle actin(a-SMA),tissue inhibitor of metalloproteinase(TIMP)-1, and TGF-b1was also increased by PDGF-BB.In contrast,PDGF-BB reduced collagen type I in culture medium and synthesis of a-SMA. Treatment of PSC with TGF-b1decreased proliferation,had no signi-?cant effect on migration and MMP expression,but increased expres-sion and synthesis of procollagen a2(I)and a-SMA.Both cytokines induced phosphorylation of extracellular signal regulated kinase (ERK)-1/2and p38MAPK,but only PDGF-BB activated the protein kinase B signaling pathway.

Conclusion:PDGF-BB augments effects of TGF-b1on the mRNA level presumably because of up-regulation of TGF-b1synthesis and common signaling pathways of the2cytokines.However,at the protein level,PDGF-BB impairs typical TGF-b1effects such as in-creased synthesis of collagen(type I)and a-SMA.Moreover,PDGF-BB facilitates degradation of extracellular matrix proteins by enhance-ment of MMP synthesis,but MMP activity was probably limited because of elevated tissue inhibitor of metalloproteinase1expression.

Key Words:a-smooth muscle actin,collagen type I,pancreatic stellate cells,proliferation,matrix metalloproteinases,migration

(Pancreas2005;31:156–167)

P ancreatic?brosis in chronic pancreatitis and in pancreatic cancer seems to be the result of a dysregulation of cells producing extracellular matrix(ECM).Recent studies suggest that similar to the situation in liver,1activation of stellate cells plays an important role in pancreatic?brogenesis.2Haber et al3 showed that pancreatic stellate cells(PSCs)were activated during?brosis of pancreas in rats and humans.In the activated state,PSCs express a-smooth muscle actin(a-SMA)and,pro-liferate,migrate,and secrete ECM proteins,such as collagen type I.How the transition of PSCs from a quiescent state in the normal pancreas toward an activated stage is induced and regulated has only partly been studied.

Five years ago,Apte et al4showed that proliferation of pancreatic stellate cells was enhanced by platelet-derived growth factor(PDGF),and a-SMA expression and collagen syn-thesis was increased by transforming growth factor-b(TGF-b). Since then,there is growing evidence that PDGF and TGF-b are the most important factors for regulation of PSC proliferation and matrix deposition.PDGF was identi?ed as an effective mitogen for PSCs4–7and a stimulant of PSC migration.7–10There was1study reporting that the synthesis of?bronectin was in-creased by PDGF.5Regarding TGF-b,there is agreement that this cytokine stimulates the synthesis of extracellular matrix proteins such as collagen type I4–6,11,12and inhibits proliferation of PSCs.12To our knowledge,there are no reports concerning the effect of TGF-b on PSC migration.

Deposition of ECM in tissues is not only regulated by the synthesis of matrix proteins,but also by enzymatic deg-radation of ECM.Matrix metalloproteinases(MMPs)or matrixins are a family of Zn2+-containing peptidases capable of degrading ECM components such as collagens,elastin, gelatin,?bronectin,laminin,and proteoglycans.More than 23members of the MMP family have been characterized in vertebrates so far.13

Matrixins are divided into collagenases,gelatinases, stromelysins,membrane-type MMPs,and other subgroups.14,15 Members of the collagenase subgroup are collagenase-1

Received for publication September27,2004;accepted April6,2005. From the Clinic of Gastroenterology,Hepatology,and Infectiology, Heinrich-Heine-University,Du¨sseldorf,Germany.

Supported by the SFB575Experimental Hepatology and the Research Commission of the Medical Faculty of the Heinrich-Heine-University, Du¨sseldorf,Germany.

Reprints:Hanne Klonowski-Stumpe,Clinic of Gastroenterology,Hepatology, and Infectiology,Heinrich-Heine-University,Du¨sseldorf Moorenstra?e5, 40225Du¨sseldorf,Germany(e-mail:Hanne.Klonowski-Stumpe@ uni-duesseldorf.de).

Copyrightó2005by Lippincott Williams&Wilkins

(MMP-1),collagenase-2(MMP-8),and collagenase-3(MMP-13).They all cleave types I,II,and III collagens.The resulting protein fragments are further degraded by gelatinases such as MMP-2and MMP-9.14,15Rodents,however,apparently lack the MMP-1gene,and MMP-13(rat collagenase)seems to be an important interstitial collagenase.16

Recently it was reported that PSCs synthesize and secrete a number of MMPs including gelatinase-A(MMP-2), stromelysin-1(MMP-3),and gelatinase-B(MMP-9),as well as their inhibitors,tissue inhibitor of metalloproteinase(TIMP)-1 and TIMP-2.12,17Secretion of MMP-13is assumed by zymography for PSCs,17but de?nitive identi?cation of this enzyme by reverse transcription-polymerase chain reaction (RT-PCR)or Western blot analysis has not been performed thus far.

In this study,the effects of PDGF-BB and TGF-b1on culture-activated rat PSCs were investigated.We detected both differential and synergistic effects of the2cytokines.Incubation of PSC with PDGF-BB increased transcription of genes known to be regulated by TGF-b1such as procollagen a2(I),a-SMA, and TIMP-1.Moreover,we measured elevated expression and protein synthesis of TGF-b1after PDGF-BB treatment.Rises in procollagen a2(I)and a-SMA mRNA amounts caused by PDGF-BB were not accompanied by increased protein syn-thesis.PDGF-BB application reduced concentrations of these proteins in culture media or cell lysates.In addition,we found that MMP-13and MMP-3expression of PSCs was increased by PDGF-BB.Elevated production of MMP-13after PDGF-BB treatment of PSCs was veri?ed by Western blot analysis. Thus,PDGF-BB apparently facilitates degradation of ECM proteins to allow cell migration,but MMP activity is probably limited because of increased TIMP-1expression.

MATERIALS AND METHODS Isolation,Culture,and Cytokine

Treatment of PSCs

Pancreata of male Wistar rats(300–350g body weight) were prepared as described previously.18PSCs were isolated by a single Nycodenz gradient(Nycomed Pharma,Oslo,Norway) as described by Apte et al.19Cells were cultured in Dulbeccos modi?ed Eagle medium(Gibco,Invitrogen,Karlsruhe, Germany)containing10%fetal bovine serum(PAA Labora-tories,Coelbe,Germany)and0.01%gentamicin(Gibco). Con?uent cultures were trypsinized,washed with medium (centrifugation at747g),and adjusted to200,000cells per tissue culture dish(diameter6cm;Falcon,BD Bioscience Europe,Erembodegem,Belgium)to harvest proteins and RNA from cell lysates.Approximately100,000cells per well(6-well plate;Costar,Corning,Corning,NY)were used for collagen type I quanti?cation.All experiments were performed with serum-free medium containing0.01%gentamicin.In collagen type I quanti?cation experiments,medium was supplemented with50m g/mL ascorbic acid(Sigma,Deisenhofen,Germany). Cells were cultured for7and14days before treatment with PDGF-BB,TGF-b1,or interleukin(IL)-1a(Sigma).The cytokines were dissolved in phosphate-buffered saline(PBS)containing0.2%bovine serum albumin.Control cells were treated with solvent alone.

Assessment of Cell Proliferation

PSC proliferation was determined by microscopic counting of cells.After detachment of cells by trypsin-EDTA treatment,cell number was assessed in aliquots using a hemocytometer.Proliferation of cells was also measured using a colorimetric(+)-bromo-2#-deoxyuridine(BrdU)cell prolifera-tion assay[(+)-BrdU ELISA,Roche,Mannheim,Germany]. After6or13days of culture,PSCs(2500cells/well)were transferred to?at-bottomed96-well microtiter plates and again cultured for24hours.The culture medium was removed and replaced by serum-free medium containing10m mol/L BrdU. When indicated,PDGF-BB or TGF-b1was added for72 hours.BrdU incorporation was determined according to the manufacturer’s recommendations.

Collagen Type I Quanti?cation of

Culture Supernatant

The collagen type I concentration of culture medium was determined after48hours of cytokine treatment using an indirect ELISA.Culture supernatant was diluted in PBS(1:4), and50m L per well was coated on high adsorption microtiter plates(Maxisorp,96well;Nunc,Wiesbaden,Germany)over-night at4°C.Unbound protein was removed by washing with PBS-Tween(0.05%Tween20in PBS).Remaining binding sites of microtiter plates were blocked with5%milk powder in PBS-Tween(300m L/well)for1hour.The blocking solution was removed,and plates were washed with PBS-Tween.A biotin conjugated polyclonal rabbit anticollagen type I anti-body(0.5m g/mL in PBS-Tween;Abcam,Cambridge,UK) was added(50m L/well)and incubated for1hour.After removal of antibody solution,plates were washed with PBS-Tween.Streptavidin-horseradish peroxidase(1m g/mL in PBS-Tween;Dianova,Hamburg,Germany)was pipetted into the wells(50m L/well)and incubated for1hour.Finally,the enzyme solution was removed,plates were washed with PBS-Tween,and substrate solution containing the chromogen orthophenylene diamine(OPD tablets for ELISA;DakoCy-tomation Denmark A/S,Glostrup,Denmark)and H2O2 (30%,5m L/12mL)was added to allow enzyme reaction (50m L/well).After15minutes,the enzyme reaction was stopped by addition of0.5mol/L H2SO4(50m L/well).Optical density was measured at450nm using a microplate reader. Puri?ed rat collagen type I(Chondrex,Redmond,WA)served as a standard for the ELISA and could be quanti?ed down to a concentration of15ng/mL.The calibration curve of the assay was linear up to at least2000ng/mL collagen type I.The intra-assay coef?cient of variation was 6.2%(n=6)and the interassay coef?cient of variation was24.7%(n=12).The col-lagen type I concentration of culture supernatant was depen-dent on cell number.Antigen concentrations were calculated based on cell numbers determined by cell counting using a hemocytometer.

TGF-b1Quanti?cation of Culture Supernatant The TGF-b1concentration of culture medium was mea-sured after48hours of cytokine treatment using a sandwich

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ELISA according to the manufacturer’s recommendations (R&D Systems,Minneapolis,MN).Culture supernatants were acidi?ed with1N HCl to activate latent TGF-b1to immu-noreactive TGF-b1and subsequently were neutralized with 1.2N NaOH/0.5mol/L HEPES.

Western Blot Analysis

At the end of the incubation period,the culture medium was removed,and the cells were immediately lysed on ice using a lysis buffer containing20mmol/L Tris-HCl(pH7.4), 140mmol/L NaCl,10mmol/L NaF,10mmol/L sodium pyrophosphate,1%Triton X-100,1mmol/L ethylenediami-netetraacetic acid,1mmol/L glycol ether diamine tetraacetic acid,1mmol/L sodium vanadate,20mmol/L b-glycerophos-phate,and protease inhibitor set(Roche,Mannheim,Germany). Proteins were quanti?ed according to Bradford,20subjected to SDS-polyacrylamide gel electrophoresis,21and transferred to nitrocellulose membranes22using a semidry transfer apparatus. Blots were blocked in5%bovine serum albumin containing 20mmol/L Tris-HCl(pH7.5),150mmol/L NaCl,and0.1% T ween20(TBS-T)and incubated at4°C overnight with the respective primary antibody.Primary antibodies used were polyclonal rabbit anti-phospho-p38MAPK antibody(Promega, Madison,WI),polyclonal rabbit anti-p38MAPK antibody(Santa Cruz,Santa Cruz,CA),monoclonal mouse anti-phospho p44/42 antibody,polyclonal rabbit anti-ERK1/2MAPK antibody (Upstate,Charlottesville,V A),polyclonal rabbit anti-phospho protein kinase B(PKB)antibody,monoclonal mouse anti-PKB antibody(Becton Dickinson,San Diego,CA),monoclonal mouse anti-a-SMA antibody(Sigma),monoclonal mouse anti-MMP-13antibody(Abcam),polyclonal anti-TGF-b1antibody conjugated to horseradish peroxidase(R&D Systems),mono-clonal mouse anti-GAPDH antibody(Chemicon,Hampshire, UK),and monoclonal mouse anti-b-actin antibody(Abcam). After incubation with the primary antibody,blots were washed with TBS-T and exposed to appropriate secondary antibody coupled with horseradish peroxidase(horseradish peroxi-dase conjugated anti-mouse or anti-rabbit antibodies;Bio-Rad, Mu¨nchen,Germany)for2hours.Blots were washed and in-cubated with enhanced chemiluminescence detection reagents (enhanced chemiluminescence Western blotting detection reagents;Amersham Biosciences Europe,Freiburg,Germany). Finally,blots were used to expose chemiluminescence?lms (Hyper?lm;Amersham Biosciences Europe).Densitometry of protein bands was performed using LabImage software(version 2.62).Molecular weights of protein bands were calculated by application of the biotinylated protein ladder detection pack (Cell Signaling Technology,Beverly,MA).

Reverse Transcription-Polymerase

Chain Reaction

Total RNAwas isolated from cultured PSCs after48hours of cytokine treatment using the Invisorb RNA Kit II(Invitek Gesellschaft fu¨r Biotechnik&Biodesign GmbH,Berlin, Germany).Puri?ed RNA was quanti?ed by spectrophotom-etry.The?rst-strand cDNA was made from1m g total RNA per 20-m L reaction volume with the RevertAid H Minus First Strand cDNA Synthesis Kit(Fermentas,St.Leon-Rot, Germany).PCR was performed using the23PCR Master Mix(Fermentas)and the following primers for MMP-3 (GenBank NM_133523),MMP-13(GenBank XM_343345), and b-actin(GenBank NM_031144)as controls—MMP-3 sense5#-CTGGAATGGTCTTGGCTCAT-3#,MMP-3antisense 5#-CTGACTGCATCGAAGGACAA-3#,MMP-13sense 5#-GCCATTACTAGTCTCCGAGGA-3#,MMP-13antisense 5#-GGAATTTGTTGGCATGACTCTCAC-3#,b-actin sense 5#-GCCCTAGACTTCGAGCAAGA-3#,and b-actin antisense 5#-CAGTGAGGCCAGGA TAGAGC-3#.All primer sets(MWG Biotech,Ebersberg,Germany)were used at a?nal concen-tration of200nmol/L and1or5m L template cDNA was added per25-m L reaction volume.PCR was conducted with a thermal cycler using standard protocols.Products of PCR were analyzed with2%agarose gels and ethidium bromide staining. Fragment sizes were calculated by application of the DNA ladder MassRuler(low range;Fermentas).

Quantitative Real-time RT-PCR

RNA was isolated using a total RNA extraction kit (RNeasy Kit;Qiagen,Hilden,Germany).The levels of gene expression were measured by quantitative,real-time,one-step RT-PCR with the help of QuantiTect SYBR Green RT-PCR kit (Qiagen)and the Gene Amp5700Sequence Detection System (Applied Biosystems,Foster City,CA)according to the man-ufacturer’s instructions.For ampli?cation of target sequences, 25ng of RNA was used per25-m L reaction volume.The speci?city of ampli?ed cDNA was checked by recording of dissociation curves.

Real-time PCR was conducted for procollagen a2(I) (GenBank NM_053356),a-SMA(GenBank X06801),MMP-13(GenBank XM_343345),MMP-3(GenBank NM_133523), TIMP-1(GenBank AA957593),and TGF-b1(GenBank NM_021578).Hypoxanthine guanine phosphoribosyl trans-ferase(HPRT;GenBank NM_012583)served as an internal standard.The sequences of primers used for real-time PCR were as follows:procollagen a2(I)sense5#-ACCCCAGCCA-AGAATGCATAC-3#,procollagen a2(I)antisense5#-CCA-GACATGCTTGTTGGCCT-3#,a-SMA sense5#-CTGCTGCT-TCCTCTTCTTCCC-3#,a-SMA antisense5#-GCCCA TCAG-GCAGTTCGTAG-3#,MMP-13sense5#-AGAGGTGAAAA-GGCTCAGTGCT-3#,MMP-13antisense5#-GTCTTCCCCG-TGTCCTCAAA-3#,MMP-3sense5#-CGGATCTTCACAG-TTGGAGTTTG-3#,MMP-3antisense5#-A TGTGGGTCAC-TTTCCCTGC-3#,TIMP-1sense5#-AGAACCGCAGCGAG-GAGTT-3#,TIMP-1antisense5#-AA TTTCCGTTCCTTAAA-CGGC-3#,TGF-b1sense5#-AGGACCTGGGTTGGAAGT-GG-3#,TGF-b1antisense5#-AGTTGGCATGGTAGCCCT-TG-3#,HPRT sense5#-TTGAATCATGTTTGTGTCATCA-GC-3#,and HPRT antisense5#-GGCTTTGTACTTGGCTTTT-CCAC-3#.

Cell Migration

The migration of PSCs was determined using the QCM-Chemotaxis96-Well Migration Assay(Chemicon,Temecula, CA).Brie?y,PSCs grown for6or13days were seeded in the migration chamber,which was separated by8-m m pore-sized membrane from a feeder tray,containing Dulbeccos modi?ed Eagle medium without or with PDGF-BB or TGF-b1.The amount of cells migrated through the membrane was

Kordes et al Pancreas Volume31,Number2,August2005

determined after an incubation period of 24hours by the use of ?uorescent dye CyQuant according to the manufacturer’s recommendations.

Statistics

Data were analyzed using the U test (Mann-Whitney)and analysis of variance.Values were considered signi?cant at P ,0.05.The program WinStat (version 1999.3)was used for statistical calculations.Results were expressed as mean values from at least 3independent experiments 6SEM.

RESULTS

Effect of PDGF-BB and TGF-b 1on Cell Proliferation

Incubation of PSCs with 20ng/mL PDGF-BB for 48hours resulted in a signi?cant increase of cell number compared with controls (PSCs day 7:210%67%;Fig.1A).

In contrast,application of 2ng/mL TGF-b 1for the same time decreased cell number signi?cantly (PSCs day 7:85%63%;Fig.1A).Similar results were obtained when the incubation period of PDGF-BB or TGF-b 1was prolonged (ie,72hours;data not shown).The opposing effects of PDGF-BB and TGF-b 1on cell proliferation were used for determination of optimal cytokine quantity.Cytokine concentrations of 2,10,and 20ng/mL were tested.The highest cell proliferation was detected at 20ng/mL PDGF-BB,whereas 2ng/mL TGF-b 1was suf?cient to reduce cell number.

Similar to the results obtained by cell counting,incor-poration of BrdU was increased in PSCs treated with PDGF-BB for 72hours (PSCs day 7:133%62%;Fig.1B).TGF-b 1induced a signi?cant decrease in BrdU incorporation (PSCs day 7:79%63%;Fig.1B),indicating reduced DNA synthesis in response to TGF-b 1.Determination of BrdU incorporation and cell counting displayed no major difference of PSC grown for 7or 14days before stimulation with PDGF-BB or TGF-b

1.

FIGURE 1.Effect of PDGF-BB and TGF-b 1on proliferation and BrdU incorporation of PSCs.PSCs were either left untreated (C)or treated with PDGF-BB (P;20ng/mL)or TGF-b 1(T;2ng/mL).A,Determination of cell number after 48hours of cytokine treatment by microscopic counting.Results were means 6SEM of 5to 11independent experiments (*,P ,0.01versus control).B,Determination of BrdU incorporation of PSCs after 72hours of cytokine treatment.Results were means 6SEM of 23to 32independent experiments (*,P ,0.001versus

control).

FIGURE 2.Effect of PDGF-BB and TGF-b 1on mRNA concentration of procollagen a 2(I)and collagen type I amount of culture medium.PSCs were either left untreated (C)or treated with PDGF-BB (P;20ng/mL)or TGF-b 1(T;2ng/mL)for 48hours.A,Quantitative determination of procollagen a 2(I)mRNA by real-time PCR.Results were means 6SEM of 4independent experi-ments (*,P ,0.01versus control).B,Determination of collagen type I concen-trations in culture supernatants against cell number.Results were means 6SEM of 5to 13independent experiments (*,P ,0.001versus control).

Pancreas Volume 31,Number 2,August 2005Effects of PDGF-BB and TGF-b 1on PSC

Effect of PDGF-BB and TGF-b 1on Collagen Type I and a -SMA

The mRNA concentration of the collagen precursor pro-tein procollagen a 2(I)was elevated by PDGF-BB (PSCs day 7:264%617%)and TGF-b 1(PSCs day 7:181%616%)as determined by real-time PCR (Fig.2A).In contrast,PDGF-BB caused reduction of collagen type I concentration of culture medium of PSCs cultured for 7days before cytokine treatment (61%63%;Fig.2B)compared with control cells.TGF-b 1increased collagen type I release of cells to culture medium (155%66%;Fig.2B),which was in good agreement with the results for mRNA amounts of procollagen a 2(I)obtained by real-time PCR.Both prolongation of the incubation period of cytokines (ie,72hours)and use of PSCs cultured for 14days before stimulation displayed the same effects of PDGF-BB and TGF-b 1on collagen type I concentration of culture media (data not shown).

The amount of a -SMA mRNA was increased after PDGF-BB (310%654%)and TGF-b 1(2105%6498%)treatment of PSCs cultured for 7days before stimulation as revealed by real-time PCR (Fig.3A).Protein synthesis of a -SMA was evaluated by Western blot analysis after addition of PDGF-BB or TGF-b 1.Similar to the effect on collagen type I concentration of culture medium,a -SMA levels were reduced by PDGF-BB (PSCs day 7:66%65%)and elevated by TGF-b 1(PSCs day 7:179%628%;Fig.3B and C).

The

FIGURE 3.Effect of PDGF-BB and TGF-b 1on mRNA and protein amount of a -SMA.PSCs were either left untreated (C)or treated with PDGF-BB (P;20ng/mL)or TGF-b 1(T;2ng/mL)for 48hours.A,Quantitative determination of a -SMA mRNA by real-time PCR.Results were means 6SEM of 4independent experiments (*,P ,0.05versus control).B,Semiquantitative determination of a -SMA of cell lysates by Western blotting.Results were means 6SEM of 4independent experiments (*,P ,0.01versus control).C,Representative Western blot of a -SMA of cell lysates (5m g protein per lane;glyceraldehyde-3-phosphate dehydrogenase served as

control).

FIGURE 4.Effect of PDGF-BB and TGF-b 1on cell migration.PSCs were either left untreated (C)or treated with PDGF-BB (P;20ng/mL)or TGF-b 1(T;2ng/mL).The amount of cells migrated through an 8-m m porous membrane was determined after 24hours of cytokine treatment.Results were means 6SEM of 3independent experiments (*,P ,0.05versus control).

Kordes et al Pancreas Volume 31,Number 2,August 2005

same effects were observed after prolongation of the incu-bation period (ie,72hours;data not shown).The a -SMA levels of PSC cultured for 14days before cytokine incubation were less altered by PDGF-BB (PSCs day 14:78%61%)and TGF-b 1(PSCs day 14:121%62%)compared with PSCs cultured for 7days.

Effect of PDGF-BB and TGF-b 1on Cell Migration

Incubation of PSCs with PDGF-BB resulted in highly increased cell migration through an 8-m m pore-sized mem-brane (PSCs day 7:164%621%;Fig.4)compared with control cells.In contrast,incubation with TGF-b 1did not in?uence migration of PSCs signi?cantly (PSCs day 7:90%64%;Fig.4).There was no major difference in the reaction of PSCs grown for 7or 14days before treatment with PDGF-BB or TGF-b 1(data not shown).

Effect of PDGF-BB and TGF-b 1on MMP-13,MMP-3,and TIMP-1

Treatment of PSCs with PDGF-BB for 48hours elevated the mRNA amount of MMP-13(PSCs day 7:187%633%;Fig.5A)compared with control cells as determined by real-time PCR.After TGF-b 1application,the expression rate of MMP-13tended to decrease (PSCs day 7:57%68%;Fig.5A),but this effect was not signi?cant.Increased expression of MMP-13by PDGF-BB incubation was veri?ed by RT -PCR (Fig.5B).IL-1a at a concentration of 5ng/mL also led to a strong increase of MMP-13expression as revealed by RT -PCR (Fig.5C).Elevated synthesis of MMP-13after PDGF-BB treatment of PSCs was detected by Western blot analysis (PSCs day 7:139%69%),whereas TGF-b 1did not sig-ni?cantly change protein levels of MMP-13(PSCs day 7:87%616%;Fig.5D and E).

The amount of MMP-3mRNA was also elevated after PDGF-BB treatment (PSCs day 7:255%6109%)as deter-mined by real-time PCR (Fig.6A)and RT -PCR (Fig.6B).

The

FIGURE 5.Effect of PDGF-BB and TGF-b 1on expression and protein synthe-sis of MMP-13.PSCs were either left untreated (C)or treated with PDGF-BB (P;20ng/mL),TGF-b 1(T;2ng/mL),or IL-1a (IL-1;5ng/mL)for 48hours.A,Quantitative determination of MMP-13mRNA by real-time PCR.Results were means 6SEM of 4independent experiments (*,P ,0.05versus control).B,Representative RT-PCR of MMP-13after PDGF-BB and TGF-b 1treatment (b -actin served as control).C,Representative RT-PCR of MMP-13after IL-1a treatment (b -actin served as control).D:,Semiquantitative de-termination of MMP-13protein of cell lysates by Western blotting.Results were means 6SEM of 3independent experiments (*,P ,0.05versus control).E,Representative Western blot of MMP-13after PDGF-BB and TGF-b 1treatment (MMP-13:25m g protein per lane;b -actin:5m g protein per lane;b -actin served as control).Pancreas Volume 31,Number 2,August 2005Effects of PDGF-BB and TGF-b 1on PSC

MMP-3expression was not signi?cantly changed by TGF-b 1(PSCs day 7:82%641%;Fig.6A).

Elevated expression rates of MMP-13and MMP-3were accompanied by increased mRNA amounts of TIMP-1when PDGF-BB was applied (PSCs day 7:791%6258%).Treat-ment of PSC with TGF-b 1also led to increased mRNA levels of TIMP-1(PSCs day 7:177%637%),but this effect was less pronounced compared with PDGF-BB application (Fig.7).

Effect of PDGF-BB and TGF-b 1on TGF-b 1

To clarify the partially strong effects of PDGF-BB on genes known to be regulated by TGF-b 1,the mRNA of TGF-b 1was quanti?ed by real-time PCR.Treatment of PSCs with PDGF-BB elevated the mRNA amount of TGF-b 1(PSCs day 7:252%638%),whereas rising mRNA levels of TGF-b 1after TGF-b 1application were not signi?cant (PSCs day 7:132%620%;Fig.8A).The concentrations of total TGF-b 1of culture supernatants were increased by PDGF-BB treatment (42.4631pg/mL;data not shown)as determined by a sandwich ELISA,whereas TGF-b 1was not detectable in culture media of control cells.Elevated TGF-b 1synthesis after addition of PDGF-BB was further con?rmed by Western blot analysis of cell lysates.The levels of the precursor protein of TGF-b 1were increased by PDGF-BB (327%663%)com-pared with control cells.Also,treatment of PSCs with TGF-b 1seemed to enhance the synthesis of the TGF-b 1precursor protein (195%661%),but this effect was not signi?cant (Fig.8B and C).

Effect of PDGF-BB and TGF-b 1on Protein Kinases

Incubation of PSCs with 20ng/mL PDGF-BB resulted in a rapid and sustained increase of PKB phosphorylation (PSCs day 7:925%6184%after 15minutes),which was more pronounced in PSCs cultured for 14days before PDGF-BB treatment (Fig.9A and B).In contrast,2ng/mL TGF-b 1did not in?uence the amount of activated PKB (Fig.10A and B).Both cytokines induced an increase in phosphorylation of

ERK-1/2.After 15minutes of incubation,ERK-1phosphor-ylation was elevated up to 988%6107%by PDGF-BB and up to 1050%6387%by TGF-b 1compared with control cells (PSCs day 7).TGF-b 1induced a sustained increase of p38

MAPK

FIGURE 6.Effect of PDGF-BB and TGF-b 1on MMP-3expression.PSCs were either left untreated (C)or treated with PDGF-BB (P;20ng/mL)or TGF-b 1(T;2ng/mL)for 48hours.A,Quantitative determination of MMP-3mRNA by real-time PCR.Results were means 6SEM of 4independent experiments (*,P ,0.05versus control).B,Representative RT-PCR of MMP-3(b -actin served as

control).

FIGURE 7.Effect of PDGF-BB and TGF-b 1on TIMP-1expres-sion.PSCs were either left untreated (C)or treated with PDGF-BB (P;20ng/mL)or TGF-b 1(T;2ng/mL)for 48hours.Quantitative determination of TIMP-1mRNA by real-time PCR.Results were means 6SEM of 5independent experiments (*,P ,0.05versus control).

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phosphorylation,whereas incubation of PSCs with PDGF-BB caused only transient activation of p38MAPK (Figs.9and 10).

DISCUSSION

Modi?cation of Cell Proliferation,Cell Migration,and Protein Synthesis by PDGF-BB and TGF-b 1

Incubation of culture-activated PSCs with PDGF-BB stimulated cell proliferation and migration.These effects of PDGF-BB on proliferation and migration of PSCs support the ?ndings of others.4–10Migration of cells in tissues is augmented by ECM-degrading enzymes such as MMP .Therefore,cell migration should be accompanied by up-regulation of MMP synthesis.In fact,the expression and production of the inter-stitial collagenase MMP-13as well as the expression of MMP-3was increased by PDGF-BB in our study.There are only a few reports showing a rise of MMP-13synthesis after PDGF treatment of osteoblasts and chondrocytes,23,24whereas several studies exist regarding the up-regulation of MMP-3expression by PDGF .25MMP activity is not only regulated by gene tran-scription and translation,but also by proteolytic activation of MMP and binding of MMP to TIMP .In our study,a strong elevation of TIMP-1gene expression was measured after PDGF-BB treatment of PSCs,which suggests that the activity of secreted MMP was probably limited.A similar effect of PDGF on TIMP-1expression rate was recently observed in hepatic stellate cells (HSCs).26However,this is the ?rst study regarding the up-regulation of MMP-13and MMP-3expression of PSC after PDGF-BB treatment.Increased amounts of these 2matrixins most likely facilitate migration and dispersion of PSCs through the pancreas,suggesting an important function of PDGF-BB for the formation of ?brosis in vivo.

Synthesis of MMP-13has been detected in various invading carcinomas.27–29In vitro MMP-13expression in tumor cell lines was enhanced by tumor necrosis factor-a and TGF-b 1.30–32IL-1a was shown to elevate expression of MMP-13in the myo?broblast cell line MG2,which derives from HSCs.33Therefore,IL-1a was used as positive control to induce increased MMP-13mRNA synthesis in our study.In contrast to PDGF-BB,TGF-b 1had no signi?cant effect on expression and synthesis of this matrixin in PSCs.This was in agreement with the ?ndings of Cao et al,34who observed no changes of MMP-13expression or concentration of this enzyme in culture medium after TGF-b 1treatment of HSCs.Like MMP-13,MMP-3expression of PSCs was not sig-ni?cantly altered by TGF-b 1in our experiments,whereas Shek et al 12found decreased MMP-3secretion of PSC after TGF-b 1application.Our ?ndings strengthen the view that TGF-b 1has no major stimulating effects on ECM degrada-tion.In addition,incubation of PSCs with TGF-b 1caused increased collagen type I concentrations of culture medium,reduced cell proliferation,and did not stimulate cell migration.In contrast to TGF-b 1,application of PDGF-BB reduced the secretion of collagen type I per cell,but increased mRNA concentrations of procollagen a 2(I).Elevated mRNA amounts of type I and type III procollagens were also detected

after

FIGURE 8.Effect of PDGF-BB and TGF-b 1on expression and protein synthesis of TGF-b 1.PSCs were either left untreated (C)or treated with PDGF-BB (P;20ng/mL)or TGF-b 1(T;2ng/mL)for 48hours.A,Quantitative determination of TGF-b 1mRNA by real-time PCR.Results were means 6SEM of 4independent experiments (*,P ,0.01versus control).B,Semiquantitative determination of TGF-b 1precursor protein of cell lysates by Western blotting.Results were means 6SEM of 4independent experiments (*,P ,0.01versus control).C,Representative Western blot of TGF-b 1precursor protein of cell lysates (TGF-b 1precursor:25m g protein per lane;b -actin:5m g protein per lane;b -actin served as control).

Pancreas Volume 31,Number 2,August 2005Effects of PDGF-BB and TGF-b 1on PSC

PDGF treatment of HSCs.35The same effect was observed for a -SMA in our study.PDGF-BB elevated mRNA concen-trations of a -SMA,but decreased a -SMA protein levels.

It is well known that the transcription of procollagen a 2(I)and a -SMA is increased by TGF-b 1in PSCs.Albeit Shek et al 12observed no signi?cant effect of TGF-b 1on TIMP-1expression in PSCs,several studies using HSCs detected up-regulation of TIMP-1expression after TGF-b 1application.34,36To clarify the partially strong expression of genes known to be regulated by TGF-b 1after PDGF-BB treatment as revealed by our experiments,we measured the mRNA and protein amounts of TGF-b 1and found increased levels of this cytokine.Increased transcription of TGF-b 1in response to PDGF application was also described for HK-2cells,37which are derived from immortalized human proximal tubular epithelial cells.This effect was further observed in HSCs indirectly,because PDGF-BB up-regulated the expres-sion of connective tissue growth factor (CTGF),and the induction of CTGF transcription by PDGF-BB can be reduced in the presence of neutralizing TGF-b 1antibody.38PDGF-BB apparently promotes auto-stimulation of PSCs with TGF-b 1,which might explain increased transcription of procolla-gen a 2(I),a -SMA,and TIMP-1.At the protein level,however,decrease of collagen type I and a -SMA was detected after addition of PDGF-BB in our experiments,probably because of impaired synthesis of these proteins.Increased MMP-13re-lease of PSCs accompanied by enhanced degradation of collagen type I might contribute to reduced levels of this ECM protein in culture medium.Secreted MMP-3is most likely involved in this process,because MMP-13is activated by proteolytic activity of MMP-3.39

TGF-b 1increased the mRNA amount of procollagen a 2(I),TIMP-1,and a -SMA as well as enhanced protein syn-thesis of collagen type I and a -SMA,although the

expression

FIGURE 9.PDGF-BB–mediated kinase acti-vation.PSCs were cultured for 7and 14days,and growth was inhibited by serum deprivation and exposed to PDGF-BB (20ng/mL)for the time periods indicated.Activation of PKB,ERK-1/2,and p38MAPK was studied by Western blotting using phospho-speci?c antibodies (total PKB,ERK-1/2,and p38MAPK served as controls).A,Representa-tive Western blots of phospho-PKB,phos-pho-ERK-1/2,and phospho p38MAPK after PDGF-BB treatment as well as total PKB,ERK-1/2,and p38MAPK (20m g protein per lane).B,Activation of PKB (:),ERK-1(j ),and p38MAPK (

)after PDGF-BB treatment.Results were means 6SEM of 5independent experiments and calculated as a relative in-crease of activation compared with the un-treated control (*,P ,0.001versus control).

Kordes et al Pancreas Volume 31,Number 2,August 2005

rate of procollagen a 2(I)was lower compared with PDGF-BB treatment.This discrepancy can be explained at least in part with increased half-life time of procollagen mRNA by TGF-b 1as published for HSCs.40

Signal Transduction of PDGF-BB and TGF-b 1

PDGF consists of 2polypeptide chains that are connected with disul?de bonds.The 2different polypeptide chains A and B form functional PDGF-AA,-AB,or -BB isoforms.41These isoforms bind to the tyrosine kinase receptors a (PDGF-R a )and b (PDGF-R b )of PDGF at the cell membrane.PDGF-AA is the exclusive ligand of PDGF-R a ,whereas PDGF isoforms containing the B chain activate both receptors.42Ligand-coupled PDGF receptors undergo auto-phosphorylation and activate the phosphatidylinositol-3-kinase pathway followed

by phosphorylation of PKB (Akt).43PDGF receptors can also activate the Ras-Raf-ERK1/2signal transduction pathway.8

Several studies were performed regarding the activation of kinases after PDGF treatment of PSCs.It was shown that PDGF induces phosphorylation of extracellular signal regu-lated kinases (ERK1/2),7,8,10mitogen-activated protein kinase p38(p38MAPK ),44and PKB.7,10This was con?rmed by our experi-ments.PDGF-BB induced sustained activation of ERK1/2and PKB,whereas elevated phosphorylation of p38MAPK in response to PDGF-BB was moderate and transient.Interestingly,PKB activation was more pronounced in PSCs cultured for 14days before cytokine treatment compared with those cultured for 7days.This suggests an elevated sensitivity of fully activated PSC for PDGF-BB.Further studies are needed to clarify this

phenomenon.

FIGURE 10.TGF-b 1–mediated kinase activa-tion.PSCs were cultured for 7and 14days,and growth was inhibited by serum depri-vation and exposed to TGF-b 1(2ng/mL)for the time periods indicated.Activation of PKB,ERK-1/2,and p38MAPK was studied by West-ern blotting using phospho-speci?c anti-bodies (total PKB,ERK-1/2,and p38MAPK served as controls).A,Representative West-ern blots of phospho-PKB,phospho-ERK-1/2,and phospho p38MAPK after TGF-b 1treat-ment as well as total PKB,ERK-1/2,and p38MAPK (20m g protein per lane).B,Activa-tion of PKB (:),ERK-1(j ),and p38MAPK (

)after TGF-b 1treatment.Results were means 6SEM of 5independent experiments and cal-culated as a relative increase of activation compared with the untreated control (*,P ,0.001versus control).

Pancreas Volume 31,Number 2,August 2005Effects of PDGF-BB and TGF-b 1on PSC

Activation of the ERK1/2signal transduction pathway by PDGF-BB was shown to regulate proliferation8,44and mig-ration7of PSCs.PDGF-induced migration of PSCs involves the PI3-kinase pathway7,10and interaction of both signal pathways was assumed.10In fact,Treinies et al45showed for NIH3T3cells that the signal transduction pathways of ERK1/2and PI3-kinase interact to stimulate DNA synthesis and cell proliferation.Therefore,the failure of TGF-b1to stimulate PSC proliferation,despite activation of ERK1/2,can be explained by the lacking activation of PKB,the downstream effector of PI3-kinase.

It is well known that TGF-b initiates signaling by binding to and bringing together TGF-b serine/threonine kinase receptors type I and type II,located at the cell membrane. Binding of TGF-b1to receptor II induces phosphorylation of the receptor I kinase domain,which propagates the signal through phosphorylation of R-Smad proteins(Smad2and3). The activated Smads dimerize with Co-Smad(Smad4)and translocate into the nucleus,where they regulate the tran-scription of target genes in conjunction with other nuclear transcription factors.46One of these target genes of TGF-b1is procollagen a2(I).47

Growth arrest of PSCs after TGF-b1treatment may involve the signal transduction by Smads.Ohnishi et al48 reported that activation of Smad3mediated inhibition of PSC proliferation,whereas ERK1/2was involved in regulation of synthesis and secretion of TGF-b1.The outcome of activated ERK1/2after PDGF-BB and TGF-b1treatment seems to depend on the signaling context.

Treatment of PSCs with TGF-b1also resulted in sus-tained phosphorylation of p38MAPK.Furukawa et al49sug-gested that,in HSCs,TGF-b1activated p38MAPK phosphorylated a linker region of Smad3and facilitated binding of Smad3to Co-Smad as well as translocation of this complex into the nucleus and gene transcription of procollagen a2(I).Involvement of p38MAPK in signal transduction of PSCs in response to TGF-b1 was also studied by our group.The inhibitor of p38MAPK SB203580reduced TGF-b1–induced increase of collagen type I concentration of culture medium of about60%com-pared with PSCs that were treated with TGF-b1alone(control: 100%;2ng/mL TGF-b1:173%617%;25m mol/L SB203580:90%620%;25m mol/L SB203580and2ng/mL TGF-b1:113%621%).This indicated a connection of the 2signal transduction pathways through p38MAPK and Smads.

However,PDGF-BB also activated p38MAPK,a process that might be associated with proliferation of PSCs7or up-regulation of MMP-13expression as shown for IL-1a in MG2 cells,33but this has not been elucidated for PDGF-BB thus far.

In conclusion,PSCs are activated by both TGF-b1and

and p38MAPK may be involved in elevated transcription of target genes of TGF-b1after PDGF-BB application.In contrast,syn-thesis of typical proteins of activated stellate cells like collagen type I and a-SMA,which are controlled by TGF-b1,is impaired by PDGF-BB treatment.TGF-b1clearly increases deposition of ECM proteins and promotes?brotic processes. The effects of PDGF-BB predominantly favor abundance and dispersion of PSCs through the pancreas.Up-regulation of MMP expression and synthesis is most likely a prerequisite for migration of PSCs.The meaning of increased expression of TIMP-1along with MMP-13and MMP-3as detected in our experiments must be investigated in future studies.

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2017山香教育理论基础整理笔记(教育学、心理学、教育心理学)

第一章教育与教育学 1、《学记》——“教也者，长善而救其失者也” 2、战国时荀子——“以善人者谓之教” 3、许慎在《说文解字》中认为“教，上所施，下所效也。”“育，养子使作善也。” 4、最早将“教育”一词连用的则是战国时期的孟子：“得天下英才而教育之，三乐也。” 5、分析教育哲学的代表人物谢弗勒在《教育的语言》中把教育定义区分为三种： 规定性定义：作者自己认为的定义，即不管他人使用的“教育”的定义是什么，我认为“教育”就是这个意思。运用规定性定义虽然有一定的自由度，但是，要求作业在后面的论述和讨论中，前后一贯地遵守自己的规定。 描述性定义：回答“教育实际上是什么”的定义。尽量不夹杂自己的主观看法，适当地对术语或者使用该术语的方法进行界定。 纲领性定义：回答“教育应该是什么”的定义。即通过明确或隐含的方式告诉人们教育应该是什么或者教育应该怎么样。 6、教育是一种活动。“教育”是以一种“事”的状态存在，而不是以一种“物”的状态出现。因而。我们就把“活动”作为界定教育的起点。 7、教育活动是人类社会独有的活动。 8、“生物起源论”代表人物： 利托尔诺在《各人种的教育演变》中指出教育是超出人类社会以外的，在动物界中就存在的。 沛西·能在《教育原理》中也认为教育是一个生物学过程，扎根于本能的不可避免的行为。 9、“终身教育”概念的提出，指明人在生理成熟后仍继续接受教育。 10、社会性是人的教育活动与动物所谓“教育”活动的本质区别。 11、教育的本质：教育活动是培养人的社会实践活动。 12、教育是人类通过有意识地影响人的身心发展从而影响自身发展的社会实践活动。 13、学校教育是一种专门的培养人的社会实践活动。 14、学校教育自出现以来就一直处于教育活动的核心。 15、学校教育是由专业人员承担的，在专门机构——学校中进行的目的明确、组织严密、系统完善、计划性强的以影响学生身心发展为直接目标的社会实践活动。 16、学校教育的特征：①可控性②专门性③稳定性 17、教育概念的扩展——大教育观的形成 18、1965年，法国教育家保罗·朗格朗在《终身教育引论》中指出，教科文组织应赞同“终身教育”的原则。 19、1972年，埃德加·富尔在《学会生存》中对“终身教育”加以确定，并提出未来社会是“学习化社会”。 20、“终身教育”概念以“生活、终身、教育”三个基本术语为基础。 从时间上看，终身教育要求保证每个人“从摇篮到坟墓”的一生连续性的教育过程； 从空间上看，终身教育要求利用学校、家庭、社会机构等一切可用于教育和学习的场所； 从方式上看，终身教育要求灵活运用集体教育、个别教育、面授或远距离教育； 从教育性质上看，终身教育即要求有正规的教育与训练，也要求有非正规的学习和提高，既要求人人当先生，也要求人人当学生。 21、教育的形态，是指教育的存在特征或组织形式。 22、在教育发展史上，教育的形态经历了从非形式化到形式化，再到制度化教育的演变。

教育学教育心理学理论及代表人物

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教育心理学第三章 学习的基本理论

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教育学心理学主要理论及代表人物

昆体良古罗 马 1.《雄辩术原理》世上第一部研究系统的教学方法论著，被公认为是西方教育史上的伟大 教育家，是第一位教学理论家和教学法专家。 2. 最早提出分班教学的思想 杜威美国1.提出实用主义教育学，杜威出版《民主主义与教育》《经验与教育》，克伯屈出版《设计教学法》，提倡活动课。 思想：强调儿童的主体地位：①教育即生活，教育即生长②教育社会化③做中学④教育即经验的不断改造。以儿童为中心，反对教师中心论 2.现代教育代言人现代教育的主要特点是民主 3.教育无目的论“教育是一个社会过程。” 4.问题的解决杜威的五步模式①困惑②诊断③假设④推断⑤验证 5.问题解决步骤的五步模式⑴疑难⑵分析⑶假设⑷检验和评价⑸结论 桑代克 美 国 1.1903年出版西方第一本《教育心理学》，是教育心理学体系的创始，标志着教育心理学 称为一门独立的学科。 2.学习理论之联结派的学习理论——联结学习：尝试－错误说（小猫“迷箱”试验） 试误成功条件：练习律、准备律、效果律 3.教育心理学体系（现代教育心理学）和联结主义学习心理学创始人，被誉为教育心理学 之父 4. 学习迁移理论之联结主义的相同要素说（代表人物：桑代克、伍德沃斯） 桑代克：相同要素说，即学习上的迁移是相同联结的转移。 伍德沃斯：共同成分说，即两种学习活动含有共同成分，则发生迁移，学习也就更容易。 以刺激——反应联结理论为基础。只有当学习情景和迁移情景存在共同成分时，才能产生迁移。即材料相似性是决定迁移的条件 5.现代教育测验之父 6.智力水平越高，迁移越大。 7.问题解决理论之试误说，又称联结说——（猫“迷箱”实验） 问题的解决过程是刺激情境与适当反应之间的联结完成的，联结的建立是通过尝试错误完成的。 贾德美国1.学习迁移理论之机能心理学的经验泛化说—“水下击靶”实验 他认为一个人对他的经验进行了概括，就可以完成从一个情境到另一个情境的迁移。概括就等于迁移，原理、法则等概括化的理论知识对迁移作用很大。 沃尔夫德国 1.学习迁移理论之官能心理学的形式训练说 他把迁移的实质理解为新的官能经训练而发展，认为促进迁移的条件与学习内容无大关系而偏重于形式。 魏特海默 苛勒德国 1.学习理论之认知派学习理论——格式塔的顿悟学习理论（黑猩猩取香蕉实验）：学习是 一个顿悟的过程，是突然察觉到解决问题的办法。主要代表人物：魏特海墨、科夫卡和克勒 2.学习迁移理论之格式塔学派的关系转换说（代表人物：苛勒）—“小鸡啄米实验” 强调“顿悟”是迁移的一个决定因素。强调个体的作用，愈能加以概括化，愈易产生迁移。 3.问题解决理论之顿悟说（苛勒）——黑猩猩取香蕉实验 4.格式塔心理学（完形心理学）创始人魏特海墨、科夫卡和克勒研究内容是意识体验， 论点“整体大于部分之和” 解决问题时从整体把握全部问题情境和认知结构的豁然改组，而不是一次次经验的积累。 反对元素分析认为每一个心理现象都是一个整体是一个格式塔是一个完形 学习的实质在于构造完型，刺激与反应之间的联系而需要意识作为中介 布鲁美国1.结构化教材和发现学习模式（明确结构，掌握课题，提供资料→建立假说，推测答案→验证→做出结论） 2.领导美国20C60y的结构主义课程改革，主张突出学科基本结构，让学生通过发现法学习，重视智力发展（动机原则、结构原则、程序原则、反馈原则） 3.学习理论之现代认知学习理论——认知发现理论 强调认知学习和认知发展，提倡发现学习。学习的核心内容是各门学科的基本知识结构。3教学方法：发现学习，新课标中也叫“探究学习”。即教师提出课题和一定的材料，引导学生自己进行分析、综合、抽象、概括等一系列活动，最后得到学习结果。 4.提出假设考验说，研究人工概念的形成（人需要利用已有的知识主动提出一些可能的假设，即猜想这个概念是什么）——人工概念是认为的、在程序上模拟的概念，这种方法最早是赫尔于1920年首创的。 5.强调非特殊成分的迁移，也叫普遍迁移。即学习了基本的普遍的概念或原理，可作为学

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