pEGFP-N1-过氧化物酶体增殖物激活受体γ载体构建及在兔骨髓间充质干细胞中的表达  

Construction of pEGFP-N1-peroxisome proliferators activated receptor gamma vector and its expression in rabbit bone marrow mesenchymal stem cells

作　　者：黄娇甜[1] 刘厂辉[1] 钟警[1] 曾艳辉[1] 

机构地区：[1]南华大学附属第一医院心血管内科,湖南省衡阳市421001

出　　处：《中国组织工程研究与临床康复》2009年第6期1081-1086,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：湖南省卫生厅科技计划项目“PPARγ在脂肪细胞早期分化及分化成熟中的作用”~~

摘　　要：背景:过氧化物酶体增殖物激活受体γ是细胞分化重要的调控因子,通过调节其他转录因子的表达,参与调节骨髓间充质干细胞的分化。目的:构建pEGFP-N1-过氧化物酶体增殖物激活受体γ真核表达载体,体外转染兔骨髓间充质干细胞,为过氧化物酶体增殖物激活受体γ受体功能和基于过氧化物酶体增殖物激活受体γ受体靶点的药物筛选提供分子研究平台。设计、时间及地点:单一样本观察,于2007-09/2008-07在南华大学附属一医院临床研究所完成。材料:选择雄性2～5月龄新西兰大白兔,用于分离培养骨髓间充质干细胞。方法:应用密度梯度离心法分离骨髓间充质干细胞,贴壁法不断纯化。从正常小鼠的肝组织中提取总RNA,反转录-聚合酶链反应法获得过氧化物酶体增殖物激活受体γcDNA,经XhoⅠ,ApaⅠ双酶切,定向克隆到真核表达载体pEGFP-N1,构建重组质粒pEGFP-N1-过氧化物酶体增殖物激活受体γ。主要观察指标:经酶切分析和测序鉴定重组质粒中过氧化物酶体增殖物激活受体γ基因的完整性和真实性,脂质体介导下体外转染骨髓间充质干细胞,荧光倒置显微镜下观察瞬时表达及转染效率,提取总RNA和总蛋白,进行反转录-聚合酶链反应和Westernblot检测。结果:获得的过氧化物酶体增殖物激活受体γ基因片段经酶切和测序鉴定正确,重组质粒经酶切和测序鉴定证实构建正确,成功转染骨髓间充质干细胞,在其中检测到目的基因及蛋白表达。结论:实验成功构建了pEGFP-N1-过氧化物酶体增殖物激活受体γ重组质粒,同时在转染的骨髓间充质干细胞中获得了过氧化物酶体增殖物激活受体γ的高效表达。BACKGROUND： Peroxisome proliferators activated receptor y （PPARy） is an important regulatory factor of cell differentiation and participates in regulating the differentiation of bone marrow mesenchymal stem cells （BMSCs） by adjusting the expression of other transcription factors. OBJECTIVE： To construct karyocyte expression plasmid pEGFP-N1-PPARy and transfect it into rabbit BMSCs in vitro to provide an ideal molecular platform for screening natural ligands of PPARy. DESIGN, TIME AND SETTING： The simple sample study was performed at the Clinical Institute of First Affiliated Hospital, Nanhua University from September 2007 to July 2008. MATERIALS： Male New Zealand rabbits aged 2-5 months were used to isolate and culture BMSCs. METHODS： BMSCs were isolated by the density gradient centrifugation method and purified by the adherence method. Total RNA was extracted form liver tissues of normal mice. The cDNA of PPARy was got by RT-PCR and was inserted into eukaryotic expression vector pEGFP-N1 after digested with Xho Ⅰ and Apa Ⅰ. Recombinant plasmid pEGFP-N1-PPARy was constructed. MAIN OUTCOME MEASURES： The integrity and validity of recombinant plasmid pEGFP-N 1-PPARy were identified by the enzyme digestion as well as sequence analysis. BMSCs were transfected in vitro via lipofectamine. The results of the transient expression and transfection efficiency were observed by fluorescence microscope. Total RNA and protein were extracted and analyzed through RT-PCR and Western Blot. RESULTS： PPARy gene sequence contained in pEGFP-N1-PPAR γ recombinant plasmid was verified correctly by enzyme digestion as well as sequence analysis. The construction was correct, which was confirmed by the enzyme digestion as well as sequence analysis. After being transfected into BMSCs, target gene and protein expression could be detected. CONCLUSION： pEGFP-N1-PPARy recombinant plasmid has been constructed successfully. High-performance expression of PPARy is obtained in the transfected BMSCs.

关 键 词：骨髓间充质干细胞 过氧化物酶体增殖物激活受体Γ 基因转染 

分 类 号：R394.2[医药卫生—医学遗传学]