YAP在特发性膜性肾病中的表达及其对C5b-9诱导足细胞损伤的作用  

Expression of YAP in idiopathic membranous nephropathy and its role in C5b-9-induced podocyte injury

作　　者：史添立[1] 罗贞[1] 焦石[2] SHI Tianli;LUO Zhen;JIAO Shi(Department of Nephrology,Haikou People’s Hospital,Haikou 570208,China;Department of Nephrology and Rheumatology,Bo’ai Hospital,Zhongshan 510603)

机构地区：[1]海口市人民医院肾内科,海口570208 [2]中山市博爱医院肾病风湿科,广东中山510603

出　　处：《中国比较医学杂志》2022年第3期1-8,共8页Chinese Journal of Comparative Medicine

基　　金：国家自然科学基金项目(81473618);海南省卫生计生行业科研项目(19A200057)。

摘　　要：目的 在特发性膜性肾病(IMN)中探究转录共激活因子Yes相关蛋白(YAP)的表达及其在膜攻击复合物C5b-9诱导足细胞损伤的作用。方法 免疫组织化学染色检测YAP蛋白在IMN患者肾组织及正常对照肾组织中的表达;选用亚溶解剂量的膜攻击复合物C5b-9(以下简称C5b-9)在体外建立人足细胞系MPC5的损伤模型,Western blot检测随C5b-9刺激时间的延长,足细胞中YAP蛋白的表达变化;使用siRNA瞬时转染技术,靶向沉默足细胞中YAP基因表达,qRT-PCR及Western blot检测转染效率;按足细胞处理的不同将其分为4组,正常培养的Control组,经C5b-9处理的C5b-9组,C5b-9处理转染si-NC或si-YAP的的C5b-9+si-NC组与C5b-9+si-YAP组;应用CCK-8实验检测上述各组细胞的增殖活力,Anenxin V-FITC双染色联合流式细胞数检测各组细胞的凋亡发生率,DCFH-DA法检测细胞中活性氧(ROS)含量,Western blot检测各组细胞中足细胞标志性蛋白Nephrin、WT1与紧密连接蛋白ZO-1、表型转换蛋白Snail及纤连蛋白Fibronectin的蛋白表达水平。结果 与正常对照肾组织相比,YAP在IMN中的表达显著增加(P<0.05);随C5b-9刺激时间的延长,足细胞中YAP蛋白的表达水平逐渐升高;转染si-YAP后,足细胞中YAP mRNA及蛋白水平均显著降低(P<0.05);与Control组相比,C5b-9组、C5b-9+si-NC组及C5b-9+si-YAP组细胞的增殖活力均显著降低(P<0.05),而凋亡率及ROS含量均显著增加(P<0.05);但与C5b-9组或C5b-9+si-NC组相比,C5b-9+si-YAP组细胞增殖活力显著增加(P<0.05),凋亡率及ROS含量显著减少(P<0.05);Western blot检测各组细胞中相关蛋白的表达结果显示,与Control组相比,C5b-9组、C5b-9+si-NC组及C5b-9+si-YAP组细胞中Nephrin、WT1及ZO-1蛋白表达均显著降低(P<0.05),Snail与Fibronectin蛋白表达均显著增加(P<0.05);而与C5b-9组或C5b-9+si-NC组相比,C5b-9+si-YAP组细胞中Nephrin、WT1及ZO-1蛋白表达显著增加(P<0.05),而Snail与Fibronectin蛋白表达显著减少(P<0.05)。结�Objective To investigate expression of YAP in idiopathic membranous nephropathy(IMN). Methods Expression of YAP protein in IMN patients and normal renal tissues was detected by immunohistochemistry. An injury model of the human podocyte line MPC5 was established in vitro with C5 b-9. Expression of YAP protein in podocytes was detected by Western blot. siRNA transient transfection was used to silence YAP gene expression in podocytes. The transfection efficiency was assessed by qRT-PCR and Western blot. Podocytes were divided into four groups: control, C5 b-9, C5 b-9+si-NC and C5 b-9+si-YAP groups. A CCK-8 assay was used to assess the proliferation activity, annexin V-FITC double staining combined with flow cytometry was used to assess apoptosis, DCFH-DA staining was used to detect the content of reactive oxygen species(ROS). Western blot was used to measure the apoptosis rate. The expression of podocyte marker protein nephrin, WT1, tight junction protein ZO-1, phenotype transition protein snail and fibronectin was detected by Western blot. Results Compared with normal renal tissue, the expression of YAP in IMN was increased significantly(P<0.05). With prolongation of the C5 b-9 stimulation time, the expression level of YAP protein was increased in podocytes(P<0.05). After si-YAP transfection, the mRNA and protein levels of YAP in podocytes were decreased significantly(P<0.05). Compared with the control group, cell proliferation in C5 b-9, C5 b-9+si-NC and C5 b-9+si-YAP groups was decreased significantly, while the apoptosis rate and ROS content were increased significantly(P<0.05). However, compared with C5 b-9 and C5 b-9+si-NC groups, cell proliferation in the C5 b-9+si-YAP group was increased significantly and the ROS content in cells was decreased significantly(P<0.05). Expression of nephrin, WT1 and ZO-1 in C5 b-9, C5 b-9+si-NC and C5 b-9+si-YAP groups was significantly lower than that in the control group(P<0.05). Compared with C5 b-9 and C5 b-9+si-NC groups, the protein expression of nephrin, WT1 and ZO-1 in

关 键 词：特发性膜性肾病 转录共激活因子Yes相关蛋白 膜攻击复合物C5b-9 足细胞 

分 类 号：R-33[医药卫生]