短发夹结构RNA对人腹膜间皮细胞转化生长因子-β_1表达和超微结构的影响  

Effects of short hairpin RNAs on TGF-β1 expression and ultrastructure in HPMCs induced by peritoneal dialysis solution

作　　者：凌光辉[1] 刘伏友[1] 彭佑铭[1] 段绍斌[1] 李军[1] 陈星[1] 

机构地区：[1]中南大学湘雅二医院肾内科,长沙410011

出　　处：《中国血液净化》2007年第9期490-495,共6页Chinese Journal of Blood Purification

基　　金：国家自然科学基金资助项目(30370811)

摘　　要：目的研究靶向转化生长因子-β1(TGF-β1)的短发夹RNA(shRNA)表达载体对腹膜透析液诱导的人腹膜间皮细胞(HPMC)表达TGF-β1的影响,并观察其对HPMC超微结构的影响。方法利用pcDU6质粒构建两个靶向TGF-β1的shRNA真核表达载体pcDU6-A1-A2和pcDU6-B1-B2,使用脂质体介导转染腹膜透析液刺激下的HPMC,采用半定量逆转录多聚酶链式反应(RT-PCR)和酶联免疫吸附(ELISA)检测shRNA表达载体对于TGF-β1基因和蛋白质表达水平的抑制效果,使用透射电镜观察HPMC超微结构的改变。结果HPMC暴露于4.25%腹膜透析液中48小时后,其上清中TGF-β1的浓度与对照组相比显著增高分别为(39.71?6.60)pg/105cells和(16.32?2.71)pg/105cells,P<0.01),1.5%腹膜透析液对于TGF-β1蛋白合成无明显影响;而预先转染了shRNA载体的两组与转染pcDU6载体(空载体)的组相比,其由4.25%腹膜透析液诱导的TGF-β1蛋白增高幅度明显减少,分别下降了39.2%和47.2%(P<0.01);两组转染不同的shRNA载体的HPMC之间相比TGF-β1浓度无显著差异,pcDU6载体(空载体)转染组与4.25%腹膜透析液刺激组TGF-β1浓度亦无显著差异。透射电镜显示HPMC暴露于4.25%腹膜透析液可导致细胞扁平、微绒毛减少和线粒体水肿等,而转染shRNA载体组细胞的改变相对较轻。结论pcDU6质粒载体介导的短发夹结构RNA(shRNA)能够明显抑制腹膜透析液刺激下的人腹膜间皮细胞TGF-β1的高表达,并减轻其超微结构的异常,提示shRNA表达载体可能有益于腹膜纤维化的防治。Objectives Transforming growth factor-β1 （TGF-β1） is one of the key mediators of the peritoneal fibrosis. Short hairpin RNAs （shRNA） transcribed by vectors contained U6 or H1 promoter can trigger sequence-selective gene silencing in mammalian cells. This study investigated the effects of shRNA targeting TGF-β1 on the expression of TGF-β1 and ultrastructure in human peritoneal mesohelial cells （HPMCs） induced by peritoneal dialysis solution （PD solution）. Methods All tests were performed on the human peritoneal mesothelial cell （HMrSV5）. TGF-β1 specific shRNA expression vectors were constructed and introduced to HPMCs stimulated with two commercially available PD solution （1.50% and 4.25% Dianeal） or DMEM to 48h. The dialysis solutions were diluted twofold with DMEM. Expression of TGF-β1 mRNA was detected by semiquantitative reverse transcription polymerase chain reaction （RT-PCR）. The TGF-β1 protein level in the culture supernatant was analyzed by enzyme-linked immunosorbent assay. Ultrastructure changes of HPMCs were observed by transmission electron microscopy. Results The expression of TGF-β1 was upregulated significantly in HPMCs stimulated with 4.25% PD solution （P 〈 0.01）. TGF-β1 expression in pcDU6 plasmid vector-mediated shRNA group were obviously downregulated when compared to the 4.25% PD solution group and the pcDU6 void vector group （P 〈 0.01）, with no significant difference among pcDU6 plasmid vector-mediated shRNA groups （P 〈 0.01）. Ultrastructural studies of HPMCs exposed to 4.25% PD solution showed cell flattening, reduced microvilli, and intracellular organelles compatible with dysfunctional mitochondria. In contrast, the ultrastructural morphology of HPMCs was relatively preserved after introduction with TGF-β1 shRNA vectors. Conclusions TGF-β1 specific shRNA can significantly inhibit the expresson of TGF-β1 and ameliorates the ultrastructural abnormalities in HPMCs stimulated with 4.25% PD solution. Our results of this in vitro study sug

关 键 词：转化生长因子-Β1 RNA干扰 腹膜透析液 人腹膜间皮细胞 

分 类 号：R446.6[医药卫生—诊断学]