shRNA沉默C5aR对LPS诱导的大鼠肾小管上皮细胞损伤的影响  

Effect of silencing C5a receptor expression with shRNA on rat kidney epithelial cells with acute injury induced by lipopolysaccharide

作　　者：张泽英[1,2] 张辉[1] 赵昕[1] 黄爱妮[1] 蒋思婧[2] 

机构地区：[1]武昌工学院食品与环境工程学院,湖北武汉430065 [2]湖北大学生命科学学院,湖北武汉430062

出　　处：《江苏农业学报》2017年第6期1327-1332,共6页Jiangsu Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31070776)

摘　　要：为观察短发夹状RNA(Short hairpin RNA,shRNA)阻断大鼠C5a受体(C5a receptor,C5aR)基因表达对抑制脂多糖(Lipopolysaccharide,LPS)诱导的大鼠肾小管上皮细胞(NRK-52E细胞)损伤的影响,将构建好的针对大鼠C5aR基因的shRNA表达质粒转染NRK-52E细胞系,经G418筛选后,形成稳定的表达C5aR shRNA的细胞系。设置空白对照组(NC组)、正常对照组(LPS组)、阴性对照组(LPS+shRNAc组)和处理组(LPS+C5aR-shRNA组)。采用荧光定量反转录PCR(RT-PCR)检测细胞中白介素-6(Interleukin-6,IL-6)、肿瘤坏死因子α(Tumor necrosis factor-alpha,TNF-α)mRNA的表达,酶联免疫吸附方法(Enzyme-linked immunosorbent assay,ELISA)检测细胞上清液中TNF-α、IL-6和髓过氧化物酶(MPO)活性,流式细胞仪检测各组细胞凋亡率。结果显示,与正常对照组和阴性对照组相比,试验组中C5aR shRNA能有效抑制炎症因子TNF-α、IL-6的释放,降低细胞凋亡率和细胞中MPO活性(P<0.05)。可见,C5aR shRNA能显著抑制由LPS诱导的TNF-α、IL-6等炎症因子的释放,改善大鼠肾脏功能,对LPS诱导的肾脓毒症损伤有明显保护作用。To investigate the effect of silencing C5 a receptor(C5 aR) expression with shRNA on rat kidney epithelial cells with acute injury induced by lipopolysaccharide(LPS),eukaryotic expression vector of small hairpin RNA targeting rat C5 aR gene was constructed and transfected into rat renal tubular epithelial cell line NRK-52 E,after G418 selection,stable cell line expressing C5 aR shRNA was got. The experiment was designed as follows: blank control group(NC group),normal control group(LPS group),negative control group(LPS + shRNAc group),experimental group(LPS +C5 aR-shRNA group). After incubation with C5 aR shRNA,the expression of interleukin-6(IL-6) and tumor necrosis factoralpha(TNF-α) mRNA were detected by fluorescence quantitative reverse transcription PCR, the content of TNF-α,IL-6 and myeloperoxidase(MPO) in the supernatant of cells were tested by enzyme-linked immunosorbent assay(ELISA),and the apoptosis ratio of each group was detected by flow cytometry. The results showed that C5 aR shRNA in the experimental group could effectively inhibit the release of inflammatory factors TNF-α and IL-6,reduce the apoptosis rate and MPO content in cells(P< 0. 05) compared with that in the normal control group and negative control group. In conclusion,specific short hairpin RNA targeting C5 aR can significantly inhibit the release of inflammatory factors such as TNF-α and IL-6 induced by LPS,improve the renal function in rats and has obvious protective effect on renal sepsis injury induced by LPS.

关 键 词：C5A受体 脂多糖 基因沉默 脓毒症 

分 类 号：S852.2[农业科学—基础兽医学]