表达过氧化物酶增殖物激活受体γ1全长基因质粒的构建及其在转染的系膜细胞中功能的鉴定  

The establishment of plasmid over-expressing mouse peroxisome proliferator-activated receptor γ1 and functional identification in mesangial cells

作　　者：孙晶[1] 马骥[1] 郝传明[1] 顾勇[1] 林善锬[1] 

机构地区：[1]复旦大学附属华山医院肾脏科,上海200040

出　　处：《中华医学杂志》2005年第33期2338-2343,共6页National Medical Journal of China

基　　金：国家自然科学基金资助项目(30200125)

摘　　要：目的构建过氧化物酶增殖物激活受体γ1(PPARγ1)全长表达基因质粒,观察PPARγ1过表达对系膜细胞细胞外基质(ECM)的作用。方法将野生型(WT)小鼠PPARγ1全长cDNA连接入真核细胞表达质粒pIRES2绿色荧光蛋白中,以脂质体介导的方法将该表达质粒pIRES2EGFPmPPARγ1/WT转染入系膜细胞,采用RTPCR、Western印迹、PPARγ与PPRE结合活性测定等方法鉴定PPARγ1在系膜细胞中的表达与活性,并且给予高糖(30mmol/L)刺激,观察PPARγ1过表达对TGFβ1、PAI1及纤连蛋白等ECM的作用。本研究中同时以一用相同方法构建的表达功能缺陷型(DN)PPARγ1的质粒pIRES2EGFPmPPARγ1/DN作为对照。结果限制性酶切及DNA测序结果均说明所构建的质粒为PPARγ1全长表达基因质粒。该质粒的表达在转染48h后达到高峰。PPARγ1过表达能显著减少高糖所致的系膜细胞的转化生长因子(TGF)β1、纤溶酶原激活剂抑制物(PAI)1的mRNA和培养细胞上清液中FN的高表达(P<0.05)。结论PPARγ1全长表达基因质粒的构建为进一步研究PPARγ1的效应和机制提供了有利的工具,此研究结果显示PPARγ1过表达可以抑制高糖引起的细胞外基质的积聚。Objective To construct a plasmid expressing peroxisome proliferator-activated receptor γ1 （ PPARγ1 ） and to study its antifibrotie effects on transfected mesangial cells under the condition of high glucose. Methods Wild type full length of mouse PPARγ1 （mPPARγ1/WT） eDNA was ligated into an expressing vector pIRES-EGFP. This constructed pIRES-EGFP-mPPARγ1/WT was then transfeeted into cultured glomerular mesangial cells facilitated by lipefeetin. The transfeetion efficiency was determined by RT-PCR, Western bloting for the expression level of PPARγ1, as well as by specific PPRE binding activity. The effects of over-expressed PPARγ1 on the expressions of TGF-β1, PAI-1 and FN in the mesangial cells stimulated by high glucose （30 mmol/L） was examined by RT-PCR and ELISA. A vector expressing dominant negative type of mPPARγ1, pIRES-EGFP- mPPARγ1/DN, lacking the biological activity of PPARγ1, was also constructed and transfeeted by using the same technology to serve as a control throughout the study. Results The accuracy of constructed and selected plasmids was confirmed by restriction enzymatic analyses and DNA sequencing. The expression level and activity of PPARγ1 in the mesangial cells reached the peaks 48 hours after transfeetion. Over-expressed PPARγ1 significantly inhibited the high glucose-induced increases in TGF-β1, PAI-1 and fibroneetin syntheses （ all P 〈 0. 05）. Conclusion The constructed expressing vector pIRES2-EGFP-mPPARγ1/WT provides a useful tool for further study on the effects and mechanisms of PPARγ1. Over-expressed PPARγ1 may protect the mesangial cells from fibrosis caused by high glucose.

关 键 词：转染 质粒 过氧化物酶增殖物激活受体γ1 系膜细胞 过氧化物酶增殖物激活受体Γ 全长CDNA 基因质粒 细胞外基质(ECM) 鉴定 真核细胞表达质粒 

分 类 号：R346[医药卫生—基础医学]