磷酸钙纳米颗粒介导shRNA抑制腹膜间皮细胞TGF-β_1的表达  

Transfection of shRNA expressing plasmid by calcium phosphate nanoparticles inhibits the high glucose-induced TGF-β1 expression in human peritoneal mesothelial cells

作　　者：凌光辉[1] 刘伏友[1] 彭佑铭[1] 段绍斌[1] 刘虹[1] 李瑛[1] 李军[1] 刘霆[2] 

机构地区：[1]中南大学肾脏病研究所,中南大学湘雅二医院肾内科,长沙410011 [2]中南大学湘雅医院消化科,长沙410011

出　　处：《中国血液净化》2008年第3期149-152,共4页Chinese Journal of Blood Purification

摘　　要：目的转化生长因子-β1(TGF-β1)是腹膜纤维化的关键介质之一,在此前的实验中已经制备了靶向TGF-β1的shRNA表达质粒。本研究使用化学修饰的方法制备了一种新型的非病毒载体-磷酸钙纳米颗粒(CPNP),透射电镜和Zeta电位显示其直径23.5～34.5nm,表面电荷为+16.8mV。在本研究中探讨使用CPNP介导转染shRNA质粒,研究其对高糖诱导的人腹膜间皮细胞(HPMC)表达TGF-β1和纤维连接蛋白(FN)的影响。方法shRNA表达质粒或作为对照的空白质粒在磷酸钙纳米颗粒介导下转染体外培育的HPMC,转染后的细胞被高浓度D-glucose(50mmol/L)刺激48h,然后监测TGF-β1和FN的表达。结果HPMC被高浓度葡萄糖刺激48h后其TGF-β1和FN的表达均上调,而使用磷酸钙纳米颗粒介导转染shRNA表达质粒则可明显抑制高糖对TGF-β1和FN的诱导效应。结论鉴于shRNA表达质粒对腹膜间皮细胞TGF-β1的有效抑制和CPNP的载体作用,我们认为联合shRNA表达质粒和CPNP可能成为腹膜纤维化基因治疗极具前景的新方法。Objective Transforming growth factor-β1 （TGF-β1） is one of the key mediators in peritoneal fibrosis. Short hairpin RNAs （shRNA） transcribed under the control of U6 or H1 promoter in plasmid can trigger silence of the target gene in mammalian cells. We have developed TGF-β1 shRNA expression plasmids in a previous study. A novel nonviral vector calcium phosphate nanoparticle （CPNP） was also developed by means of a chemistry method. Transmission electron microscopy and Zeta potential demonstrated that CPNP was of 23.5～34.5 nm in diameter and had positive surface charges of ＋16.8 mV. In this study, we investigated the transfection of shRNA expressing plasmid mediated by CPNP on high glucose-induced TGF-β 1 expression in human peritoneal mesothelial cells （HPMCs）. Methods The TGF-β1 shRNA expression plasmid （pcDU6-A1-B1） was transfected into HPMCs mediated by CPNP. Transfected cells were then stimulated with 50mmol/L D-glucose for 48 hours, and the expression of TGF-β 1 and fibronectin were evaluated Results TGF-β1 expression was upregulated significantly in control HPMCs stimulated with 50mmol/L D-glucose for 48 hours. Such induction was inhibited by the transfection of shRNA expression plasmid （pcDU6-A1-B1） into the cells mediated by CPNP. Fibronectin expression was also upregulated significantly by the stimulation of 50mmol/L D-glucose for 48 hours. The 50mmol/L D-glucose-induced fibronectin expression became insignificant in HPMCs transfected with the shRNA expression plasmid, as measured by enzyme-linked immunosorbent assay （ELISA）. Conclusion The introduction of shRNA expression plasmid into HPMCs effectively inhibited high glucose-induced TGF-β1 expression in these cells. Calcium phosphate nanoparticles were useful for the introduction of this plasmid into HPMCs. shRNA expression plasmid introduced by calcium phosphate nanoparticles may be a promising approach for the gene therapy of peritoneal fibrosis.

关 键 词：腹膜纤维化 转化生长因子-Β1 RNA干扰 纳米颗粒 

分 类 号：R459.5[医药卫生—治疗学]