外源Smad7转染肝星状细胞及对转化生长因子β1和Ⅰ、Ⅲ型胶原mRNA表达的影响  

Effect of exogenous Smad7 gene transfected hepatic stellate cells on mRNA expression of transforming growth factor beta 1,collagen Ⅰ and collagen Ⅲ

作　　者：杨小艳[1] 杨勇[2] 郑勇[3] 李睿[3] 周婷[4] 

机构地区：[1]石河子大学医学院第一附属医院老干一科,新疆维吾尔自治区石河子市832008 [2]石河子大学医学院第一附属医院心内二科,新疆维吾尔自治区石河子市832008 [3]石河子大学医学院第一附属医院消化内科,新疆维吾尔自治区石河子市832008 [4]石河子大学医学院第一附属医院中心实验室,新疆维吾尔自治区石河子市832008

出　　处：《中国组织工程研究与临床康复》2009年第50期9887-9891,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：教育部科学技术研究重点项目(207136);兵团博士基金资助课题(05JC08)~~

摘　　要：背景:Smad7是转化生长因子β信号转导途径的主要抑制性蛋白,具有抗纤维化的作用。目的:构建并鉴定大鼠Smad7真核表达质粒,观察外源Smad7可否有效转染肝星状细胞T6,并进一步研究其对转化生长因子β及Ⅰ、Ⅲ型胶原mRNA表达水平的影响。设计、地点:基因重组及细胞观察实验,于新疆石河子大学医学院第一附属医院完成。材料:pcDNA3.1(+)质粒为课题组保留;大肠杆菌DH5α系石河子大学医学院新疆地方与民族高发病教育部重点实验室所赠;肝星状细胞T6细胞由中国医学科学院肿瘤医院肿瘤研究所提供品。方法:采用基因重组技术将Smad7cDNA插入真核表达载体pcDNA3.1(+),构建大鼠Smad7真核表达质粒。脂质体介导转染肝星状细胞T6细胞,分为正常对照、空质粒及转染组,G418筛选,挑取阳性细胞。主要观察指标:反转录-聚合酶链反应法检测各组中Smad7、转化生长因子β及Ⅰ、Ⅲ型胶原mRNA的表达水平。结果:酶切和测序结果证实Smad7真核表达质粒构建成功。Smad7转染组与正常对照组、空质粒组比较:Smad7mRNA表达显著增加(P<0.01);转化生长因子β、Ⅰ型胶原mRNA表达减少(P<0.01);Ⅲ型胶原mRNA表达差异无显著性意义(P>0.05)。正常对照组、空质粒组Smad7、转化生长因子β及Ⅰ、Ⅲ型胶原mRNA表达差异无显著性意义(P值均>0.05)。结论:大鼠Smad7真核表达质粒构建成功,外源Smad7转染肝星状细胞T6细胞后可有效表达,并能降低转化生长因子β及Ⅰ型胶原mRNA表达水平。BACKGROUND： Smad7 is a major repressible protein in transforming growth factor β（TGF-β1） signal transduction pathway, which possess antifibrotic effects. OBJECTIVE： To construct rat Smad7 eukaryotic vector and to observe the mRNA expression level of TGF-β1, collagen I and collagen Ⅲ in rat hepaticstellate cells （HSC）-T6 cell. DESIGN, TIME AND SETTING： The gene recombination and cytology observation experiment was performed at the First Affiliated Hospital of Shihezi University School of Medicine. MATERIALS： pcDNA3.1（＋） plasmid was reserved in the laboratory. E coil DH5a was presented by Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Shihezi University School of Medicine. The HSC T6 cell was provided by Cancer Institute and Hospital, Chinese Academy of Medical Sciences. METHODS： Rat Smad7 cDNA was cloned into eukaryotic plasmid pcDNA3.1（＋） to construct Smad7/pcDNA3.1 （＋） plasmid and transfected it into HSC-T6 cells by Lipofectmine 2000. The experiment was divided into normal control, empty vector and Smad7 transfected groups, and the positive cells were selected by G418. MAIN OUTCOME MEASURES： The levels of Smad7, TGF-β1, collagen 1 and Ⅲ mRNA was detected by reverse transcriptase polymerase chain reaction, respectively. RESULTS： Smad7 eukaryotic vector was successfully constructed and confirmed by endonuilease digestion and sequencing. Compared to the control and empty vector groups, Smad7 mRNA expression was significantly higher in Smad7 transfected group （P 〈 0.01 ）; and TGF-β1 and collagen I mRNA expression was notably reduced （P 〈 0.01 ）. There was no statistically significant difference of the change of collagen Ⅲ mRNA expression among the three groups （P 〉 0.05）. The difference of Smad7, TGF-β1, collagen I and Ⅲ mRNA expression had no statistically significant between control and empty vector groups （Pall〉 0.05） CONCLUSION： Smad7 eukaryotic expression vector is successfully constructed. The Smad7 gene can effectively ex

关 键 词：SMAD7 真核表达质粒 转染 肝星状细胞 

分 类 号：R392.114[医药卫生—免疫学]