高糖环境培养、lncRNA 58992过表达SV40中lncRNA 58992和纤维化标志物表达观察  

Expression changes of lncRNA 58992 and fibrosis markers in glomerular mesangial cell line SV40 with overexpression of lncRNA 58992 under high glucose environment

作　　者：丁明璐 王小花[1] 刘海峰[1] DING Minglu;WANG Xiaohua;LIU Haifeng(Mudanjiang Medical College,Mudanjiang 157000,China)

机构地区：[1]牡丹江市牡丹江医学院,黑龙江牡丹江157000

出　　处：《山东医药》2022年第15期54-57,共4页Shandong Medical Journal

基　　金：国家自然科学基金(81700544);黑龙江省自然科学基金(YQ2019H033);牡丹江医学院研究生创新科研项目(YJSCX-MY46)。

摘　　要：目的 观察高糖环境培养的肾小球系膜细胞系SV40中长链非编码RNA 58992(lncRNA 58992)的表达变化,以及过表达lncRNA 58992的肾小球系膜细胞系SV40中纤维化细胞因子α-平滑肌肌动蛋白(α-SMA)、纤维连接蛋白(FN)和Ⅰ型胶原(ColⅠ)的表达变化。方法 将肾小球系膜细胞系SV40分为3组,分别为NG+58992组、HG组、NG组。NG+58992组经lipofectamine 8000转染质粒pcDNA 3.1-lncRNA 58992 24 h,再经低糖(5 mmol/L)环境培养24 h。HG组低糖环境培养24 h后再经高糖(25 mmol/L)环境培养24 h。NG组以低糖环境培养48 h。采用qRT-PCR检测肾小球系膜细胞系SV40中lncRNA 58992、纤维连接蛋白(FN)、Ⅰ型胶原(ColⅠ) mRNAs;采用Western blotting检测肾小球系膜细胞系SV40中FN和α-平滑肌肌动蛋白(α-SMA);细胞免疫荧光法检测ColⅠ、α-SMA和FN蛋白。结果 HG组、NG组中lncRNA 58992的相对表达量分别为1.45±0.22、1.00±0.03,两组比较,P<0.05。NG+58992组、HG组、NG组FN mRNA相对表达量分别为8.78±1.86、13.17±1.95、1.00±0.01,ColⅠmRNA相对表达量分别为2.40±0.41、4.16±0.83、1.00±0.06;NG+58992组、HG组、NG组FN、Col I mRNA相比,P均<0.05。NG+58992组、HG组、NG组FN蛋白相对表达量分别为0.75±0.24、0.91±0.17、0.27±0.13,α-SMA蛋白相对表达量分别为0.28±0.03、0.59±0.06、0.14±0.03,与NG组相比,NG+58992组、HG组中FN、α-SMA蛋白相对表达量高(P均<0.05)。细胞免疫荧光实验结果表明,NG组中ColⅠ、α-SMA和FN蛋白均有少量的红色荧光(该蛋白在细胞中存在本底表达),而HG组和NG+58992组中ColⅠ、α-SMA和FN的红色荧光均增强。结论 高糖环境培养的肾小球系膜细胞系SV40中lncRNA 58992表达上调;过表达lncRNA 58992可使肾小球系膜细胞系SV40中α-SMA、FN和ColⅠ的表达上调、促进SV40的纤维化发生。Objective To observe the changes in the expression of long non-coding RNA 58992(lncRNA 58992) in glomerular mesangial cell line SV40 under high glucose environment and the changes in the expression of fibrotic cytokines α-smooth muscle actin(α-SMA),fibronectin(FN),and collagen type Ⅰ(Col Ⅰ) in glomerular mesangial cell line SV40 overexpressing lncRNA 58992.Methods Glomerular mesangial cell line SV40 was divided into three groups,NG+58992 group,HG group,and NG group.Cells in the NG+58992 group were transfected with plasmid pcDNA 3.1-lncRNA 58992 by lipofectamine 8000 for 24 h and then were treated with normal glucose(5 mmol/L)for 24 h.cells in the HG group were treated with normal sugar for 24 h,followed by high glucose treatment(25 mmol/L) for 24 h.cells in the NG group were treated with normal glucose for 48 h,and then were treated with normal glucose for 48 h.The expression levels of lncRNA 58992,FN and Col Ⅰ mRNAs in SV40 cells were measured by qRT-PCR.The expression levels of FN and α-SMA in the SV40 cells were detected by Western blotting;and the expression levels of Col Ⅰ,α-SMA and FN proteins were detected by cellular immunofluorescence.Results The relative expression levels of lncRNA 58992 in the HG group and NG group were 1.45±0.22 and 1.00±0.03,respectively,with statistically significant difference between two groups(both P<0.05).The relative expression levels of FN mRNA in the NG+58992 group,HG group,and NG group were 8.78±1.86,13.17±1.95,and 1.00±0.01,respectively;and the relative expression of Col Ⅰ mRNA in the NG+58992 group,HG group,and NG group were 2.40±0.41,4.16±0.83,and 1.00±0.06,respectively;there were statistically significant difference in the FN and Col Ⅰ mRNA expression levels between two groups(both P<0.05).The relative expression of FN protein in the NG+58992 group,HG group and NG group were 0.75±0.24,0.91±0.17,and0.27±0.13,respectively;the relative expression levels of α-SMA protein were 0.28±0.03,0.59±0.06,and 0.14±0.03,respectively;and the relative expre

关 键 词：长链非编码RNA 58992 肾小球系膜细胞 Α-平滑肌肌动蛋白 纤维连接蛋白 Ⅰ型胶原 

分 类 号：R394.2[医药卫生—医学遗传学]