大鼠结膜囊成纤维细胞TGFβ_2特异性siRNA的筛选及其载体构建的研究  

Screen of specific siRNA for transforming growth factor β_2 of Tenon’s fibroblast and construction of its vector

作　　者：马建民 赵家良[2] 卞爱琳[2] 程钢炜[2] 张文宝[2] 张华[2] 

机构地区：[1]首都医科大学附属北京同仁医院,北京100730 [2]中国医学科学院眼科研究中心北京协和医院,北京100730

出　　处：《眼科研究》2006年第6期577-581,共5页Chinese Ophthalmic Research

基　　金：国家自然科学基金资助(30371509)

摘　　要：目的筛选结膜囊成纤维细胞转化生长因子β2(TGFβ2)特异性siRNAs,并构建其载体。方法采用体外转录法合成4个TGFβ2siRNAs(L1、L2、L3、L4),分别转染体外培养的大鼠Tenon’s囊成纤维细胞,以未转染细胞作为对照,转染后48h采用RT-PCR技术检测所合成的siRNAs对TGFβ2mRNA表达的干扰作用;在此基础上,再构建有干扰作用siRNA的载体。结果与对照组相比,转染48h后,仅L4能够使大鼠Tenon’s囊成纤维细胞TGFβ2的mRNA表达水平明显下调,其余3条TGFβ2siRNAs未产生明显的干扰效果。经过测序检测证明成功构建了有干扰效果TGFβ2siRNA的载体。结论不同的TGFβ2siRNA对大鼠Tenon’s囊成纤维细胞TGFβ2mRNA表达具有不同的干扰效能;能够构建出有干扰效能的TGFβsiRNA特异性载体。Objective As the expression of transfornfing growth factor β （TGF2 β） of tenon＇ s fibroblasts is important for filtering bleb scarring formation after glaucoma surgery, it is benefit for preventing filtering bleb scarring through inhibiting its expression. This paper was to screen the effects of different TGF2β siRNAs on the expression of TGF1β mRNA and to construct the specific TGF2β siRNAs plasmid vector of rat Tenon＇ s fibroblasts. Methods Four TGF1β siRNAs （ L1 , L2 , L3 , L4 ） were transcribed in vitro and then transferred to cultured Tenon＇ s capsule fibroblasts in 30 SD rats. RT-PCR was performed to evaluate the levels of TGF1β mRNA in both transferred cells, and untransferred cells were as the controls. The specific TGF2 β siRNAs plasmid vector was constructed according to the screened TGF1β siRNAs. Results The expression of TGF1β mRNA of fibroblasts transferred with only L4 （0. 163 ± 0. 009 ） was reduced significantly in comparison with the control group（0. 433 ± 0. 011 ）, hut the interference function of others siRNAs seemed to be inactive. The plasmid vector of siRNA specific for TGFβ2 was proved to be constructed successfully by detecting its sequence. Conclusion Different siRNAs is designed to target the relevant TGFβ2 mRNA of rats Tenon＇ s fibroblast. The vector of siRNA specific for TGFβ2 is of potent interference ability for target tissue.

关 键 词：大鼠 结膜囊成纤维细胞 转化生长因子β2mRNA 转化生长因子β2siRNA载体 

分 类 号：R775[医药卫生—眼科] R988.1[医药卫生—临床医学]