CAJ | 학술논문

目的:探讨短发夹状RNA基因沉默补体受体C5aR及抑制LPS诱导的肾脏上皮细胞凋亡的作用效果.方法:构建针对大鼠补体受体C5aR基因编码区的短发夹状RNA(shRNA)真核表达载体质粒pRNAT-U6.1-C5aR shRNA,采用电穿孔的方法转染RK3E细胞,经G418筛选后,形成稳定的表达C5aR shRNA的细胞系.实验分为3组,①正常对照组:未转染的RK3E细胞;②阴性对照组:转染空载体pRNAT-U6.1的RK3E细胞系;③实验组:转染C5aR shRNA 的RK3E细胞系.经脂多糖(LPS)孵育12小时后,流式细胞仪检测各组细胞凋亡率,RT-PCR检测mRNA的表达,γ计数仪测定~(125)I标记的C5a与RK3E的结合情况.结果:与正常对照组和阴性对照组相比,实验组的细胞凋亡率显著降低(P＜0.01),C5aR mRNA水平显著降低(P＜0.01),~(125)I标记的C5a与RK3E结合活性显著下降.结论:针对C5aR的特异性短发夹RNA可以明显引起靶基因的沉默,进而抑制LPS诱导的肾脏上皮细胞凋亡的发生.
목적:탐토단발협상RNA기인침묵보체수체C5aR급억제LPS유도적신장상피세포조망적작용효과.방법:구건침대대서보체수체C5aR기인편마구적단발협상RNA(shRNA)진핵표체재체질립pRNAT-U6.1-C5aR shRNA,채용전천공적방법전염RK3E세포,경G418사선후,형성은정적표체C5aR shRNA적세포계.실험분위3조,①정상대조조:미전염적RK3E세포;②음성대조조:전염공재체pRNAT-U6.1적RK3E세포계;③실험조:전염C5aR shRNA 적RK3E세포계.경지다당(LPS)부육12소시후,류식세포의검측각조세포조망솔,RT-PCR검측mRNA적표체,γ계수의측정~(125)I표기적C5a여RK3E적결합정황.결과:여정상대조조화음성대조조상비,실험조적세포조망솔현저강저(P＜0.01),C5aR mRNA수평현저강저(P＜0.01),~(125)I표기적C5a여RK3E결합활성현저하강.결론:침대C5aR적특이성단발협RNA가이명현인기파기인적침묵,진이억제LPS유도적신장상피세포조망적발생.
Objective:To investigate RNA interference and apoptosis induced by LPS in kidney epithelial cells through silencing C5aR gene with small hairpin RNA (shRNA).Methods:We construct the eukaryotic expression vector of small hairpin RNA targeting rat C5aR gene,and transfected RK3E cell by electroporation,after G418 selection,so we got the stable cell line expressing C5aR shRNA.The experiment was designed into 3 groups:①normal control group,RK3E cells without transfection;②negative control group,RK3E cells transfected with blank vector;③ experimental group,RK3E cells transfected with C5aR shRNA.After incubation with LPS for 12 h,the ratio of apoptosis was tested by flow cytometry,the level of mRNA was tested by RT-PCR,and binding of ~(125)I-rrC5a to RK3E cells stimulated with LPS were performed to examine the expression of C5aR in RK3E cells.Results:Compared with the normal control group and negative control group,in the experimental group the ratio of apoptosis was significantly decreased(P＜0.01),and the expression of C5aR mRNA was significantly inhibited(P＜0.01),and binding of ~(125)I-rrC5a to RK3E cells was significantly decreased also.Conclusion:Hairpin shRNA targeting C5aR gene can lead to obvious gene silence in vitro and inhibit the cell apoptosis induced by LPS in kidney epithelial cell.