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

Suppression of VEGF expression in rabbit corneal epithelial cells cultured in human amniotic membrane

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(vascula rendothelial growth factor,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,Ⅲ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RT-PCR一步法反应检测各组VEGF mRNA表达并与β-actin比较。结果: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)。结论:羊膜培养液明显抑制VEGF mRNA在角膜上皮细胞中的表达。

Abstract:

AIM: To detect the suppression effects of culture super-natant of human amniotic membrane on the expression of vascular endothelial growth factor(VEGF) in rabbit corneal epithelial (RCE) cells. METHODS: The normal RCE cells were cultured for 7 days until there were confluences on the cultures. The cells were subcultured to plastic(3 groups,Ⅰ-Ⅲ, Ⅰ was control) and naked amniotic membrane (Ⅳ group) cultures respectively. Then one of plastic group (Ⅱ group) was cultured by culture supernatant of human amniotic membrane without epithelial layer as its medium; another plastic group (Ⅲ group) was cultured by culture supernatant of human amniotic membrane with epithelial layer as its medium; the other two groups (Ⅰand Ⅳgroup) were still cultured by DMEM (free from FBS components ).After 48 hours, the total RNAs of these cultures were extracted and detected by RT-PCR. RESULTS:There were expressions of VEGF mRNA from Ⅰ group and Ⅱ group, however, the expressions were significantly suppressed in Ⅲ group(using culture supernatant of human amniotic membrane with epithelial layer as its medium) and Ⅳ group (RCE cultered directly on naked amniotic membrane) (P<0.01, n=5). CONCLUSION: Amniotic membrane transplantation is partly through depressing the mRNA expression of VEGF in corneal cells to reduce the corneal neovascularization of injuried corneal surface. And it may be an efficient method of curing corneal neovascularization in clinic.