PRS—CTGF—siRNA对TGF—β1诱导的人腹膜间皮细胞细胞外基质及VEGF表达的影响  

PRS-CTGF-siRNA attenuates matrix production and the expression of vascular endothelial growth factors induced by transforming growth factor β1 in cultured human peritoneal mesothelial cells

作　　者：肖力[1] 刘伏友[1] 彭佑铭[1] 段绍斌[1] 刘虹[1] 刘映红[1] 凌光辉[1] 

机构地区：[1]中南大学湘雅二医院肾内科中南大学肾脏病研究所,长沙410011

出　　处：《中华肾脏病杂志》2008年第8期575-580,共6页Chinese Journal of Nephrology

基　　金：国家自然科学基金（30370811）

摘　　要：目的观察pRetw—Super（PRS）反转录病毒载体介导的表达人结缔组织生长因子（CTGF）小分子干扰RNA（siRNA）对体外培养的人腹膜间皮细胞（HPMC）细胞外基质和血管内皮生长因子（VEGF）表达的影响。方法根据siRNA靶序列要求及PRS反转录病毒载体特点分别设计4对寡核苷酸，构建表达人CTGF基因siRNA的PRS—CTGF—siRNA1-4重组反转录病毒载体。以脂质体2000将重组反转录病毒载体转染PT67包装细胞，继而感染HPMC。采用RT—PCR法检测mRNA表达及Western印迹法检测蛋白质表达。结果5μg／L外源性转化生长因子β1（TGF—β1）刺激可诱导HPMC表达CTGF、纤连蛋白（FN）、Ⅰ型胶原（ColⅠ）、层粘连蛋白（LN）和VEGF明显增高；而PRS—CTGF—siRNA 1～4组与TGF—β1刺激组比较，HPMC细胞内CTGF、FN、ColⅠ、LNmRNA和蛋白表达和VEGF mRNA表达明显较低（P〈0．01），各干扰组对CTGF mRNA抑制率分别为69．3％、22．2％、27．4％和38-8％，其中以PRS-CTGF—siRNA 1组最为明显；同时PRS-CTGF-siRNA 1组相对于TGF—β1刺激组，VEGF蛋白表达也明显较低（P〈0．01）；而PRS空载体组与TGF-β1刺激组比较，差异无统计学意义（P〉0．05）。结论PRS—CTGF—siRNA重组反转录病毒载体可明显抑制TGF-β1诱导的细胞外基质及VEGF表达的增加。Objective To investigate the effects of connective tissue growth factor （CTGF） siRNA delivered by pRetro-Super （PRS） retrovirus vector on extracellular matrix and VEGF expression in human peritoneal mesothelial cells （HPMC）. Methods Four pairs of oligonucleotides including 64 bp DNA were designed and synthesized in vitro according to siRNA target sequence and PRS retrovirus desire. PRS-CTGF-siRNA1-4 recombinant retrovirus vectors were constructed. The recombinant retrovirus vectors containing CTGF-siRNA were transferred into PT67 packaging cell lines with lipofectamine 2000, then infected HPMC. mRNA expression was determined by semi-quantitative RT-PCR and protein expression was determined by Western blot. Results Both mRNA and protein expressions of CTGF, FN, Col Ⅰ, laminin （LN） and VEGF were significantly increased in HPMC with 5 μg/L TGF-β1 stimulation （P〈0.01, respectively）. CTGF, FN, Col Ⅰ, LN mRNA and protein and VEGF mRNA expression stimulated by TGF-β1 were significantly decreased in HPMC infected with PRS-CTGF-siRNA 1-4 retrovirus vectors （P〈0.01, respectively）. The inhibitory rates on CTGF were 69.3%, 22.2%, 27.4% and 38.8%, respectively （P〈0.01）. At the same time, there was also a significant reduction of VEGF protein expression in HPMC infected with PRS-CTGF-siRNA1 vector （P〈0.01）. There was no significant difference in HPMC infected with PRS void vector. Conclusion CTGF siRNA delivered by PRS retrovirus vector can effectively inhibit the enhancement of extracellular matrix and VEGF expression stimulated by TGF-β1 in HPMC.

关 键 词：RNA 小分子干扰 转化生长因子β 血管内皮生长因子类 结缔组织生长因子 腹膜间皮细胞 

分 类 号：R346[医药卫生—基础医学]