论文部分内容阅读
目的 :构建能在真核细胞表达的含大鼠金属蛋白酶组织抑制物 1(tissueinhibitorofmetalloproteinase 1,TIMP 1)反义基因片段的TA克隆载体 ,并在体外培养的肾小球系膜细胞表达 ,为进一步研究TIMP 1反义基因片段抑制肾组织纤维化的作用奠定基础。 方法 :用RT PCR法扩增大鼠TIMP 1cDNA片段 (313bp) ,并将其反向克隆到真核表达载体pCR 3.1中 ,用脂质体将大鼠TIMP 1反义基因片段导入培养的大鼠肾小球系膜细胞 ,半定量RT PCR法检测系膜细胞TIMP 1表达的变化。 结果 :①用限制性内切酶鉴定TIMP 1cDNA片段插入方向 ,测序结果证实扩增片段为目的片段 ;②导入TIMP 1反义基因后 ,肾小球系膜细胞内TIMP 1表达明显减少。 结论 :①成功地构建了含大鼠TIMP 1反义基因片段的TA克隆载体 ;②该载体能在体外培养的系膜细胞中表达 ;③TIMP 1反义基因片段能有效地阻抑培养的大鼠系膜细胞中TIMP 1的表达。
OBJECTIVE: To construct a TA cloning vector containing the antisense gene fragment of rat tissue inhibitor of metalloproteinase 1 (TIMP1) expressed in eukaryotic cells and to express it in cultured glomerular mesangial cells To study the role of TIMP-1 antisense gene fragment in inhibiting renal fibrosis lay the foundation. METHODS: The rat TIMP 1 cDNA fragment (313 bp) was amplified by RT-PCR and cloned into the eukaryotic expression vector pCR 3.1. The antisense gene fragment of rat TIMP 1 was introduced into cultured rat Glomerular mesangial cells, semi-quantitative RT PCR assay of mesangial cell TIMP 1 expression changes. Results: (1) The insertion direction of TIMP-1 cDNA was identified by restriction endonuclease, and the sequence was confirmed by sequencing. (2) TIMP-1 expression in mesangial cells was significantly decreased after the introduction of TIMP-1 antisense gene. CONCLUSION: ① TA cloning vector containing rat TIMP-1 antisense gene fragment was constructed successfully; ② The vector was able to express in vitro mesangial cells; ③TIMP 1 antisense gene fragment can effectively inhibit the growth of cultured rat The expression of TIMP-1 in mesangial cells.