长链非编码RNA-1700020I14Rik对高糖培养下小鼠肾系膜细胞纤维化的影响

目的探索长链非编码RNA表达质粒构建的方法,研究lncRNA-1700020I14Rik对肾系膜细胞纤维化的影响。方法从小鼠肾系膜细胞中提取RNA并反转录为c DNA作为模板,PCR法扩增目的片段,构建入载体pc DNA3.1(+)中。通过脂质体3000转染方法,将载体转染至高低糖培养的小鼠肾系膜细胞中。RT-q PCR法检测1700020I14Rik的表达水平;Western blot检测肾脏纤维化标记蛋白Col-4、FN及TGF-β1表达水平。结果1700020I14Rik在高糖培养的肾系膜细胞中显著性下调(P0.01)。与转染空质粒组相比,转染表达质粒的肾系膜细胞中1700020I14Rik表达水平升高(P0.01),且促使Col-4、FN以及TGF-β的表达水平下降(P0.05)。结论pc DNA3.1(+)-1700020I14Rik表达质粒能高表达1700020I14Rik,长链非编码RNA-1700020I14Rik可以缓解高糖培养下肾系膜细胞的纤维化发展。

Effect of lncRNA-1700020I14Rikon the fibrosis in mouse mesangial cells in high glucose concentration

Objective To construct the lncRNA-1700020I14Rik plasmid and detect its effect on the fibrosis of mice mesangial cell (MMC) cultured with high glucose medium.Methods RT-qPCR was used to measure the expression of 1700020I14Rik in MMC cultured with low glucose medium or high glucose.Total RNA was extracted from MMC and cDNA was got by RT-PCR.The whole fragment of lncRNA-1700020I14Rik amplified by PCR was constructed into plasmid pcDNA3.1(+) through PCR.Lipidosome 3000 was used to transfect the plasmid into the MMC cultured with high glucose medium and RT-qPCR was used to measure the expression level of 1700020I14Rik.Western blot was used to analyze the expression of fibronectin, collagen Ⅳ and TGF-β1.Results 1700020I14Rik was significantly down-regulated in MMC cultured with high glucose and it was significantly up-expressed in the MMC after transfecting with pcDNA3.1(+)-1700020I14Rik.The expressions of fibronectin, collagen Ⅳ and TGF-β1 were down-regulated by 1700020I14Rik.Conclusions The plasmidpcDNA3.1(+)-1700020I14Rik is able to effectively express the lncRNA-1700020I14Rik.Over-expression of 1700020I14Rik may protect mesangial cells from fibrosis conduced in high glucose medium.