摘要
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(P<0.01)。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(P<0.01),且促使Col-4、FN以及TGF-β的表达水平下降(P<0.05)。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。
Objective To construct the lneRNA-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 eDNA 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 fibroneetin, collagen IV and TGFq31. 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 （ ＋ ）- 1700020II4Rik. The expressions of fibronectin, collagen Ⅳ and TGF-β1 were down-regulated by 1700020I14Rik. Conclusions The plasmid pcDNA3.1 （ ＋ ）-1700020I14Rik is able to effectively express the lncRNA-1700020I14Rik. Over-expression of 1700020I14Rik may protect mesangial cells from fibrosis conduced in high glucose medium.
出处
《基础医学与临床》
CSCD
2017年第6期781-785,共5页
Basic and Clinical Medicine
基金
国家自然科学基金(81570747)
关键词
长链非编码RNA
表达质粒
糖尿病
糖尿病肾病
long non-coding RNA
expression plasmid
diabetes mellitus
diabetic nephropathy
