短发夹结构RNA对人腹膜间皮细胞转化生长因子-β1表达和超微结构的影响

凌光辉; 刘伏友; 彭佑铭; 段绍斌; 李军; 陈星

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中国血液净化 ›› 2007, Vol. 6 ›› Issue (9) : 490-495.

基础研究

短发夹结构RNA对人腹膜间皮细胞转化生长因子-β1表达和超微结构的影响

凌光辉刘伏友彭佑铭段绍斌李军陈星

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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/105 cells和(16.32 2.71)pg/105 cells,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。

Abstract

Objectives Transforming growth factor- 1 (TGF- 1) 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- 1 on the expression of TGF- 1 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- 1 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- 1 mRNA was detected by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The TGF- 1 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- 1 was upregulated significantly in HPMCs stimulated with 4.25% PD solution (P＜0.01). TGF- 1 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- 1 shRNA vectors. Conclusions TGF- 1 specific shRNA can significantly inhibit the expresson of TGF- 1 and ameliorates the ultrastructural abnormalities in HPMCs stimulated with 4.25% PD solution. Our results of this in vitro study suggest that TGF- 1 specific shRNA expression vectors transfer might be of the therapeutic benefit in peritoneal fibrosis. However, further studies in vivo are needed to confirm this hypothesis.