文档介绍：靶向大鼠CTGF的shRNA表达载体的构建和作用分析
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作者:郎明健,曾秋棠,郭敏,杨汉东,闵新文
【摘要】目的: 构建并筛选靶向大鼠结缔组织生长因子(CTGF)的shRNA表达载体. 方法: 根据大鼠CTGF mRNA序列设计并构建4个靶向大鼠CTGF基因的shRNA干扰质粒;然后以脂质体转染试剂将目的质粒转染至大鼠心肌成纤维细胞,流式细胞技术分选出成功转染的阳性细胞. 通过RTPCR和Western Blot技术对成功构建的4个shRNA干扰质粒进行有效性筛选,分析其CTGF mRNA及蛋白表达水平. 结果: 酶切证实转录shRNA的目的基因片段已成功克隆入载体,经测序证明与设计的相同;在转染成纤维细胞48 h后,与空白对照组比较,有3组细胞CTGF mRNA及蛋白表达水平明显降低;而转染非特异阴性质粒对照组CTGF mRNA及蛋白表达水平无明显变化. 结论: 成功构建并筛选出靶向大鼠CTGF的shRNA真核表达质粒重组体,为进一步进行体内外心肌纤维化的RNA干扰研究奠定了基础.
【关键词】结缔组织生长因子;RNA干扰;质粒;转染;心内膜;心肌纤维化
【Abstract】 AIM: To construct and screen out the potent short
hairpin RNA (shRNA)expressing plasmids which target to rat connective tissue growth factor (CTGF). METHODS: Four shRNAexpressing plasmids targeting to rat CTGF mRNA (Accession No: NM_022266) were designed and constructed based on the sequence of rat CTGF mRNA. The binant plasmids were identified by restriction enzyme assay and nucleotide sequence analysis. Then the plasmids were transfected into rat cardiac fibroblasts using lipofectamine2000 reagent, and the positive cells were sorted by fluorescence active cell sorting (FACS) technique. The mRNA and protein expressions of CTGF were analyzed by RTPCR and Western Blot technique, pared with thatof nontransfection group and negative control plasmid. RESULTS: The 4 binant plasmid vectors expressing CTGFtargeting shRNA were identified by restriction enzyme assay and sequence analysis. Fortyeight hours posttransfection, the mRNA and protein expressions of CTGF decreased in 3 experimental pared with that in nontransfection group, but no changes occurred in negative control plasmid and lipofecta
mine2000 group. CONCLUSION: Four binant plasmids targeting to rat CTGF are essfully constructed, and 3 potent binant plasmids are screened out. This lays a foundation for further researches of RNA interference on cardiac fibrosis in vivo and in vitro.
【Keywords】 connective tissue growth factor; RNA interference; plasmid; transfect