靶向结缔组织生长因子shRNA慢病毒表达载体的构建及病毒包装

目的构建靶向结缔组织生长因子（CTGF）的shRNA慢病毒载体并检测其介导的RNA干扰（RNAi）对人视网膜色素上皮细胞（ARPE-19）CTGF表达的影响，为进一步研究CTGF在增殖性玻璃体视网膜病变（PVR）中的功能奠定基础。方法设计并合成三对针对CTGF的siRNA，通过WesternBlot筛选有效靶点，合成有效靶点的Oligo DNA，退火形成双链DNA，与线性化的pGC-LV-GFP载体连接，通过PCR筛选阳性克隆并进行DNA测序鉴定；将重组CTGFshRNA的质粒与病毒包装的辅助质粒共转染295T细胞，将包装产生的慢病毒感染ARPE-19细胞，WesternBbt检测CTGF的表达。结果通过双酶切和基因测序证实靶向CT-GF的shRNA慢病毒载体构建成功，Western Blot证实经慢病毒感染后的ARPE-19细胞中的CTGF的蛋白水平明显减低。结论成功构建了靶向CTGF的shRNA慢病毒表达载体，体外感染ARPE-19细胞可有效降低CTGF蛋白的表达，为深人研究CTGF在PVR中的功能提供了有力的工具。

Study on the mechanism of themolecular biology of hepatocyte growth factor and connective tissue factor in proliferative vitreoretinopathy

Objective To determine the role of CTGF in PVR, we successfully down - regulated the expression of CTGF by RNA interference( RNAi) technology in ARPE-19 cell line. Methods Design and synthesis three CTGF siRNA, screen effective targeted points in Western blot, combine Oligo DNA of effective targeted point, anneal to form double chain DNA, connect with linearing pGC-LV-GFP carrier, sequence DNA in PCR screening positive clones. Results The shRNA-expression vector of CTGF was constructed successfully. And RT-PCR and Western Blot results showed that CTGF expression was significantly inhibited by siRNA transfectants in ARPE-19 cells at mRNA and protein levels. Conclusion We successfully construct shRNA-expression vector of CTGF which paves a way for CTGF-targeted gene therapy of proliferative vitreoretinopathy.