缺氧共培养血管内皮细胞产生的fgf2对绒毛外滋养mmp9、timp1表达及其侵袭力影响

1、2,1华技大学同济医学院附属同济医院妇产科省咸宁学院妇产科教研室咸宁:在妊娠过程中，绒毛外滋养细胞侵润螺旋动脉并替代其内皮细胞，从而将螺旋动脉改建成高容量、低阻抗的血管以满足生长发育的，这（TIMP1）对绒毛外滋养细胞的侵袭力和限速步骤起重要作用。先兆子痫由于全身小动脉痉挛导致血管内皮受损并应激产生一系列的血管生成因子。FF是最强的血管生长因子之一，它对受损内皮细胞的修复起一定的保护作用，但对绒毛外滋养细胞分泌MMP9、TIMP1及其侵袭力是否有影响还不清楚。本实验用人脐静脉血管内皮细胞株EC304和绒毛外滋养细胞株TEV-1在体行二维缺氧共培养，用荧光定量、estern-BltEC304细胞中FF2与TEV1细胞中MMP9、TIMP1的变化，并用ranlEC304细胞中FF2mRN及其蛋白表达升高，而TEV1细胞中MMP9R降、TIMP1mRN及其蛋白表达升高，且滋养细胞侵袭力亦下降。结论：受损的螺旋动脉内皮细胞可阻碍绒毛外滋养细胞的侵入，其机制可能是内皮细胞应激产生的FF2调控绒毛外滋养细胞MMP9分泌下降、TIMP1升高所致。:Hypoxiaco-culture；FGF2；MMP9；TIMP1；Trophoblast；Invasion基质金属蛋白酶（Matrixmetalloproteinases，MMPs）是一组降解细胞外基质的中性蛋白酶，MMPs受其组织抑制剂(Tissueinhibitorofmetalloproteinases,TIMPs)侵，使螺旋动脉重铸受阻，即胎盘浅着床而先兆子痫。那么为了弄清先兆子VEGF、EGF、FGF4、FGF10可促进滋养细胞分泌MMP9、但FGF2则无此作用[4。而在先兆子痫患者中由于全身小动脉痉挛导致血管内皮受损并应激产生一系列的血管生成因子如VEGF5、6]、EGF7、8、8]、FGF等10。这些血管生成因子对受损内皮细胞的修复起一定的保护作用但对绒毛外滋养细胞的侵袭力是否有影响还未见有因此，本实验用人脐静脉血管内皮细胞株ECV304和绒毛外滋养细胞株TEV-1在体外行二维缺氧共培养可较好模拟先兆子痫螺旋动脉内皮细胞和滋养细胞在体内的缺氧环境，探讨先兆子痫螺旋动脉内皮细胞产生的碱性成纤维细胞生长因子(basicfibroblastgrowthfactor,FGF2)对绒毛外滋养细胞MMP9、TIMP1表达及其侵袭力的影响，目的说MaterialsandCelllinesandco-cultureconditions：TEV-111humanextravilloustrophoblastcellline,中文大学惠赠)是来源于正常早孕的人胎盘绒毛中分离的原代滋养细胞通过表达人HPV16E6/E7的逆转录载体（PLXSN—E6/E7）转染而构建成的永生化的人但无恶性转化能力。ECV304（Humanumbilicalveinendothelialcells，本产前诊断实验室冻存）。Transwell（孔径0.4um，膜直径24mm，corningcostar）上室滴加2mlTEV-/ml，下室滴加/ml，培养行荧光定量PCR、WesternBlot检测上室TEV-1细胞MMP9、TIMP1的表达量，下室PCR：1mlTrizol(Invitrogen)用酚-氯仿法抽提总RNA，去除组的DNA，按逆转录cDNA试剂盒说明（Ferments）合成cDNA，进FGF2、MMP9、TIMP1β-actinmRNA扩增。FGF2引物序列：上游为5’-CTAACCGTTACCTGGCTATG-3’，下游为5’-TTTGCCCAGTTCGTTT-3’，度166bp;MMP9引物序列:上游为5’-GCAGAGGACTGTACCGC-3’，下游引物5’-AGGTTTGGAATCTGCCCAGGT-3’196bp；TIMP1引物序列：上游为5’-CACCTTCAGCGTTATGA-3’，下游为5’-GAGAAACTCCTCGCTGCGGTT-3’157bp；β-actin5’-CATTAAGGAGAAGCTGTGCT-3’,下5’-GTTGAAGGTAGTTTCGTGGA3’,208bp(Invitrogen合成)。扩增体系25ul1ul100nM,SYBRGreenmix（ABI）12.5ul,dd2H2o9.5ul95℃5min，94℃20s，55℃20s，72℃延伸20s，循环45次。获得目的与内参的比值(ΔCt)，以含量最低的一个样本为标准，其余各样本的含量均为相对该样本的倍数(ΔΔCt),目的mRNA为相对定量值（2-△△ct）12WesternAttheendofthecultureperiod,cellswerewashedwithice-coldPBSandlysedbysonicationinlysisbuffer(RIPA,，ProMab,USA).Insolublematerialwasremovedbycentrifugation(12000g,40C,10min)andtheproteincontentinthesupernatantwasdeterminedwiththeBradfordproteinkitassay(biyuntian,shanghai,).Aliquotsofprotein(10ug)wereresolvedbySDSandelectrotransferredtonitrocellulosemembranes.Themembraneswereblocked(2h,roomtemperature)inblotto(Tris-bufferedsalineatpH7.6with0.05%Tween20(TBS-T)and0.5%dehydratednon-fatmilk).AfterrinsingwithTBS-T,membraneswereimmunoblottedwithantibodiestoFGF2MMP9,TIMP1orGAPDH（BeijingSeajetScientificInc.）.ThebandswerevisualizedusingECLreagentsandtatedwithGelproImageSoftware(Gelpro4.0,MediaCybernetics,USA).Transwellinvasion：参考Sato costar，（100ul/孔，Sigma）37℃30min2mlTEV-1细胞悬液（0.2%BSA/ml/ml（300μmol/L5 yses：SPSS11.5softwarepackagewasusedforstatistical experimentswereperformedatthreetimes,andvaluesaregivenasthe±S.DatawereysedbyANOVAandt-testsandspearmancorrelation ysis.ThelevelofsignificancewassetatP<0.05.43210各组ECV304细胞FGF2mRNA表达的比较：与各对照组比较，缺氧组43210图 （P﹥0.05 对照 缺氧0图 缺氧组MMP9mRNA的表达量在24h、48h、72h均下降，两组比较均有统计学差异(t1=34.86,t2=16.05,t3=14.56,P<0.01)，而缺氧组TIMP1mRNA的表达量在24h、48h、72h765对照组缺氧组43210765对照组缺氧组43210图3MMP9、TIMP1mRNATEV-1MMP9、TIMP1ECV304FGF2蛋两组比较有统计学差异(t1=10.68,t2=17.82,t3=11.13,P<0.01)TIMP1蛋白的表达t3=11.12,P<0.01)MMP9/TIMP1ECV304FGF2蛋白呈负相关(r=0.978,p<0.01)；但二组各时间点均无显著差异（P﹥0.05）（4） 对照组MMP9MMP9TIMP1TIMP104MMP9、TIMP1细胞侵袭力测定：Transwell（50.8±4.52）个,缺氧组为（29.6±3.91）个，缺氧组TEV-1细胞matrigel基底膜的细图5缺氧 图6对照0图772h(extravilloustrophoblast,EVT)。EVT侵入螺旋动脉并对其改建的过程被称为螺中，EVT侵入螺旋动脉有两个期：第一个在妊娠早期10周内，此时EVT侵入血管壁中,并逆行扩展至蜕膜-肌层交界处，同时伴发血管中层肌纤维和弹力纤维破坏,以及纤维蛋白样物质沉积,即妊娠期螺旋动脉的生理性变化[15];第二次在孕14～20周间,血管的生理性变化扩展到螺旋动脉的肌段,有时甚至达放射动脉的末端[14]。这种重铸使螺旋动脉进行性扩张,呈漏斗状,以适应胎盘增加的需要。先兆子痫患者缺乏妊娠中期EVT侵入的第二次,故螺旋动脉的肌段缺乏性，因此动物模型在此方面提供的信息往往是有限的[17、18]；研究内螺旋动脉普遍依赖于对胎盘活检或切除的标本进行组织化学或免疫组化的分析[19-27]，但这种组脉血管内皮细胞株ECV304和绒毛外滋养细胞株TEV-1在体外行二维缺氧共培养，可较好模拟先兆子痫螺旋动脉内皮细胞和滋养细胞在体内的缺氧环境，为研究先兆先兆子痫一般发生在孕20周后，由于全身小动脉痉挛导致血管内皮受损并应激产生一系列的血管生成因子如VEGF5、6]、EGF7、8、8]、FGF等10。这些血管生成因结合型的形式在组织中。当组织缺血、缺氧时，细胞外基质受破坏，结合的FGF2察先兆子痫患者的FGF2升高的结果相一致[31]。MMP9是一类基质蛋白水解酶,主要降解细胞外基质。TIMP1是MMP9的特异性抑制物,可1∶1以非共价键形式与MMP9结合。MMP9与TIMP1间的平衡对于细胞的浸润总之,本实验的意义在于:(一)该共培养模型弥补了既往用单细胞培养来模拟先兆子痫体内环境而可能致实验结果较片面的缺陷。（二）有助于了解和细胞的相互作用，为细胞有可能控制血管内绒毛外滋养细胞的侵袭提供一定的理论依据（三）说明血管内皮细胞因缺氧受损应激产生的FGF2，虽然其对血管的修复起一VandenSteenP,DuboisB,NelissenI,RuddP,DwekR&OpdenakkerG.BiochemistryandmolecularbiologyofgelatinaseBormatrixmetalloproteinase-9(MMP-9).CriticalReviewsinBiochemistryandMolecularBiology.2002,37:375–536.BischofP,MeisserA&CampanaA.ControlofMMP-9expressionatthematernal-fetalinterface.JournalofReproductiveImmunology.2002,55:3–10.CampbellS,RoweJ,JacksonC&GalleryE.Invitromigrationofcytotrophoblaststhroughadecidualendothelialcellmonolayer:theroleofmatrixmetalloproteinases.centa.2003,24E.Y.Anteby,C.Green®eld,S.Natanson-Yaron,etal.Vascularendothelialgrowthfactor,epidermalgrowthfactorandfibroblastgrowthfactor-4and-10stimulatetrophoblastsminogenactivatorsystemandmetalloproteinase-9.MolecularHumanReproduction.2004,10(4):LashGE,TaylorCM,TrewAJ,CooperS,etal.Vascularendothelialgrowthfactorandcentalgrowthfactorreleaseinculturedtrophoblastcellsunderdifferentoxygentensions.GrowthFactors.2002,20(4):197-210.WathenKA,TuuttiE,StenmanUH,etal.Maternalserum-solublevascularendothelialgrowthfactorreceptor-1inearlypregnancyendinginpreeclampsiaorintrauterinegrowthretardation.JClinEndocrinolMetab.2006,91(1):180-184.LevyR,SmithSD,ChandlerK,SadovskyY,NelsonDM.Apoptosisinhumanculturedtrophoblastsisenhancedbyhypoxiaanddiminishedbyepidermalgrowthfactor.AmJPhysiolCellPhysiol.2000,278(5):C982-C988.FaxenM,NasiellJ,BlanckA,NisellH,LunellNO.AlteredmRNAexpressionpatternofcentalepidermalgrowthfactorreceptor(EGFR)inpregnanciescomplicatedbypreeclampsiaand/orintrauterinegrowthretardation.AmJPerinatol.1998,15(1):9-13.PerkinsJ；StJohnJ；AhmedA；ModulationoftrophoblastcelldeathbyoxygenEGF.MolMed.2002,8(120:847-M.C.LYGNOS,K.LPAPPA,H.A.PAPADAKL.etal.ChangsinMaternalsmaLevelsofVEGF,bFGF,TGF-β1,ET-1andsKLDuring 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