bFGF小干扰RNA表达质粒抑制人晶状体上皮细胞增殖的实验研究  

bFGF siRNA expression plasmid inhibits growth of human LEC-B3 cells

作　　者：徐庆[1] 贾仁兵[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院眼科,200011

出　　处：《中华眼科杂志》2008年第12期1078-1082,共5页Chinese Journal of Ophthalmology

基　　金：上海市卫生局基金资助项目（034084）

摘　　要：目的探讨碱性成纤维细胞生长因子（bFGF）小干扰RNA（siRNA）表达质粒体外转染人晶状体上皮细胞LEC—B3对其增殖的影响，为防治晶状体后囊膜混浊（PCO）的临床研究提供思路。方法实验研究。以永生化的人晶状体上皮细胞LEC—B3为研究对象，首先用针对αB-晶状体蛋白的免疫组织化学方法鉴定LEC-B3细胞来源。在此基础上，用逆转录聚合酶链反应（RT—PCR）检测LEG—B3细胞中bFGF及其受体1（FGFRI）的mRNA表达。然后，构建bFGF siRNA表达质粒及其对照质粒，并在体外稳定转染LEC—B3细胞，干扰其bFGF基因表达，再用实时定量PCR和Western blot法检测转染后LEC-B3细胞中bFGF基因和蛋白表达，并以流式细胞术检测LEC-B3细胞中细胞增殖核抗原（PCNA）表达。将bFGFsiRNA表达质粒及其对照质粒转染后LEC-B3细胞中PCNA表达水平分别设为实验组和对照组，用成组设计的t检验比较两组PCNA表达差异。结果bFGF和FGFR1的mRNA在LEC—B3细胞中均有表达；bFGFsiRNA表达质粒稳定转染LEC—B3细胞后，其bFGF基因和蛋白表达降低，实验组PCNA表达明显降低，和对照组相比差异具有统计学意义（t=-5．0011，P=0．0005）。结论LEC-B3细胞组成性表达bFGF和FGFR1的mRNA；干扰bFGF基因表达可有效抑制LEC-B3细胞增殖。Objective Basic fibroblast growth factor （bFGF） has been thought to play an important role during the development of posterior capsule opacification （PCO） by promoting the growth of lens epithelial cells （LECs）. In the present study, we sought to explore the inhibition effect on LEC-B3 cells growth by plasmid-based RNA interference （RNAi） targeting bFGF. Method It is a prospective study. LEC-B3 cells, an immortal human lens capsule epithelial cell line, were identified as human lens epithelium by immunnhistochemiscal assay of orB-crystalline. Both bFGF and bFGF receptor 1 （ FGFR1 ） mRNA expressions of LEC-B3 cells were determined by reverse transcription polymerase chain reaction （ RT- PCR）. Based on the identification of bFGF expression, plasmid-based RNA interference （RNAi） was used to inhibit bFGF mRNA expression of LEC-B3 ceils by stable transfection. bFGF mRNA and protein levels were examined by real time PCR and western blot,respectively. Then the flow cytometry （FCM） was performed to evaluate proliferation cell nuclear antigen （PCNA） expression. PCNA level in bFGF-interfered LEC-B3 cells was compared to that of the control. P 〈 0. 05 was regarded as statistically different. Results bFGF and FGFR1 mRNAs were abundantly expressed in LEC-B3 cells, bFGF mRNA and protein levels were both downregulated by stable transfection of bFGF siRNA expression plasmid. PCNA expression was significantly decreased by inhibiting bFGF expression （ t = - 5. 0011, P = 0. 0005 ）. Conclusions bFGF and FGFR1 mRNA are extensively expressed in LEC-B3 cells. Plasmid-based RNAi targeting bFGF can lead to potent inhibition of LEC-B3 cells growth,which may play a part in dealing with the development of PCO.

关 键 词：成纤维细胞生长因子2 质粒 RNA 小分子干扰 白内障 上皮细胞 细胞增殖 

分 类 号：R686[医药卫生—骨科学]