羊膜培养液体外抑制兔角膜上皮细胞血管内皮生长因子的表达.doc

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1、羊膜培养液体外抑制兔角膜上皮细胞血管内皮生长因子的表达【摘要】目的：探讨羊膜培养液对角膜上皮细胞中血管内皮细胞生长因子(vascularendothelialgrowthfactor,VEGF)表达的影响。方法：刮除并收集新鲜兔角膜上皮细胞,传代培养接种于35mm培养皿及自制的羊膜培养皿上。实验分为4组,I组(对照组)：无血清的DMEM培养液，II组：去上皮的羊膜培养液,川组：未去上皮的羊膜培养液，IV组：将细胞直接接种于无上皮羊膜。作用48h后用Trizol法提取各组样本的总RNA,进行RTPCR一步法反应检测各组VEGFmRNA表达并与

2、3actin比较。结果

3、：正常角膜上皮细胞中有VEGF基因表达，在III,IV组中表达受到明显抑制(P<0.01,n=5)o结论：羊膜培养液明显抑制VEGFmRNA在角膜上皮细胞中的表达。【关键词】羊膜；角膜新生血管；血管内皮生长因子；角膜上皮细胞AbstractATM:Todetectthesuppressioneffectsofculturesupernatantofhumanamnioticmembraneontheexpressionofvascularendothelialgrowthfactor(VEGF)inrabbitcornealepithelial(RCE)cells

4、.METHODS:ThenormalRCEcellswereculturedfor7daysunti1therewereconfluencesonthecultures.Thecellsweresubculturedtoplastic(3groups,IIII,Iwascontrol)andnakedamnioticmembrane(IVgroup)culturesrespectively.Thenoneofplasticgroup(IIgroup)wasculturedbyculturesupernatantofhumanamnioticmembranewith

5、outepitheliallayerasitsmedium;anotherplasticgroup(IIIgroup)wasculturedbyculturesupernatantofhumanamnioticmembranewithepitheliallayerasitsmedium;theothertwogroups(TandTVgroup)werestillculturedbyDMEM(freefromFBScomponents)・After48hours,thetotalRNAsofthesecultureswereextractedanddetected

6、byRTPCR.RESULTS:TherewereexpressionsofVEGFmRNAfromIgroupandIIgroup,however,theexpressionsweresignificantlysuppressedinITTgroup(usingculturesupernatantofhumanamnioticmembranewithepitheliallayerasitsmedium)andIVgroup(RCEcultereddirectlyonnakedamnioticmembrane)(P<0.01,n=5).CONCLUSION:Amn

7、ioticmembranetransplantationispartlythroughdepressingthemRNAexpressionofVEGFincornealcellstoreducethecornealneovascularizationofinjuriedcornealsurface.Anditmaybeanefficientmethodofcuringcornealneovascularizationinclinic.KEYWORDS:amnioticmembrane;cornealneovascularization;vascularendot

8、helialgrowthfactor;cornealepithelialcello引言正常的角膜无血管，角膜新生血管(cornealneovascularization,CNV)常由损伤及各种炎症刺激诱导产生。严重的角膜新生血管化会导致视力下降甚至视力丧失；随着新生血管的长入，角膜的免疫赦免地位丧失。虽然目前有多种方法用于治疗角膜新生血管，但均未获得满意疗效。临床上羊膜角膜移植及羊膜移植眼表重建，均显示其有抑制角膜新生血管的作用[1],虽然已知羊膜的细胞外基质及可溶性细胞因子可能参与这一作用机制23],然而具体作用机制仍不明。最近研究表明，羊膜培养液可明显地抑制角

9、膜新生血管