组织金属蛋白酶抑制剂-1低表达系膜细胞模型的建立

Establishment of TIMP1 low-expression model in mesangial cells

  • 摘要: 目的 利用3种不同组织金属蛋白酶抑制剂-1(tissue inhibitors of metalloproteinase-1,TIMP-1) siRNA转染大鼠系膜细胞,建立高效、稳定的TIMP-1低表达系膜细胞模型,为肾疾病研究奠定实验基础。 方法 分别设计合成3条TIMP-1特异性siRNA序列,并以标记了绿色荧光基团6-羧基荧光素(6-carboxy-fluorescein,FAM)的无义siRNA作为对照,利用脂质体转染方法转染原代大鼠系膜细胞,收集转染后48 h的细胞,利用荧光显微镜观察绿色荧光,明确其转染成功率;同时用Trizol法分离提取系膜细胞的总RNA,利用Taqman探针方法检测试验组及对照组TIMP-1的mRNA表达水平。 结果 荧光显微镜观察,利用脂质体转染方法,siRNA转染效率可以达到90%以上;Taqman探针荧光定量PCR结果显示,3种不同序列的siRNA均有抑制TIMP-1表达的作用(P< 0.01),且siTIMP1-1的效果最佳。 结论 本研究利用siRNA转染技术,成功建立了TIMP-1低表达系膜细胞模型。

     

    Abstract: Objective Tocompoundthree different small interference RNAs specific for TIMP-1 and establish low-expression model of TIMP-1 in mesangial cells of rats, in order toprovide experimental basis for renal diseases. Methods Three different small interference RNA sequences that were specific for TIMP-1 were designedand compounded. The nonsense siRNA markedwith green fluorescent perssad(6-carboxy-fluorescein, FAM) was usedas control groupand the three siRNA groups were transfectedintoculturedmesangial cells in rats by liposome transfection method, then the transfectedcells were collectedat 48 hours after transfection toidentify the transfection efficiency with fluorescent microscope. At the same time, the total RNA in mesangial cells was extractedwith Trizol, the TIMP-1 mRNA expression level of all four groups was detectedby Taqman probe technique. Results The fluorescent microscope revealedthat the transfection efficiency was over than 90% in nonsense siRNA group, while the PCR results demonstratedthat TIMP-1 mRNA expression level in all three groups was lower than control group(P< 0.01), which suggestedthat our three siRNAs hadinhibitedthe expression of TIMP-1, and siTIMP1-1 hadthe best effect for it showedthe lowest expression level. Conclusion Our study has successfully establishedthe low-expression cell model of TIMP-1 in primary MCs with siRNA transfection technique.

     

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