组蛋白H3K27甲基化异常修饰致局灶节段性肾小球硬化小鼠足细胞损伤的作用机制  

Mechanism of abnormal methylation of histone H3K27 inducing podocyte damage in focal segmental glomerulosclerosis mice

作　　者：张燕子 顾凤娟 隋晓露 许云鹏 张艾莎 谢婷妃 陈家辉 陈继红 Zhang Yanzi;Gu Fengjuan;Sui Xiaolu;Xu Yunpeng;Zhang Aisha;Xie Tingfei;Chen Jiahui;Chen Jihong(Department of Nephrology,the Second Affiliated Hospital of Shenzhen University,the People's Hospital of Baoan District,Shenzhen 518000,China;Department of Nephrology,Shenzhen Shiyan People's Hospital,Shenzhen 518000,China)

机构地区：[1]深圳大学第二附属医院深圳市宝安区人民医院肾内科,深圳518000 [2]深圳市宝安区石岩人民医院肾内科,深圳518000

出　　处：《中华肾脏病杂志》2025年第1期38-48,共11页Chinese Journal of Nephrology

基　　金：国家自然科学基金(82170712)。

摘　　要：目的分析组蛋白H3K27甲基化致足细胞损伤的靶目标信号通路,验证组蛋白H3K27甲基化通过靶目标信号通路致局灶节段性肾小球硬化(focal segmental glomerulosclerosis,FSGS)小鼠足细胞损伤的调控作用,探究组蛋白H3K27甲基化异常修饰致FSGS小鼠足细胞损伤的作用机制。方法(1)细胞实验:体外培养原代永生化小鼠肾足细胞MPC5,分为对照组、阿霉素组、阿霉素+GSK-J4(组蛋白去甲基化酶KDM6B抑制剂)组、阿霉素+香豆霉素A1(coumermycin A1,C-A1,JAK2激动剂)组、阿霉素+GSK-J4+C-A1组。采用透射电镜观察足细胞超微结构;免疫荧光检测足细胞组蛋白H3K27三甲基化(H3K27me3)、肾病蛋白Nephrin蛋白表达;获取足细胞的全基因组序列,筛选差异表达基因并行基因本体论(Gene Ontology,GO)及京都基因和基因组数据库(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析;实时定量PCR和Western印迹分别检测足细胞JAK2-STAT3信号通路基因和蛋白表达;酶联免疫吸附测定法检测足细胞中白细胞介素6(interleukin 6,IL-6)、单核细胞趋化蛋白1(monocyte chemotactic protein 1,MCP-1)、α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)及转化生长因子β1(transforming growth factor-β1,TGF-β1)水平。(2)动物实验:建立组蛋白甲基化酶同源序列增强子2(enhancer of zeste homolog 2,EZH2)基因敲除(EZH2^(podKo))-FSGS(尾静脉注射阿霉素)小鼠模型,分为EZH2^(ctrl)+对照组(n=20)、EZH2^(ctrl)+FSGS组(n=20)、EZH2^(podKo)+对照组(n=30)、EZH2^(podKo)+FSGS组(n=30)。采用HE染色观察肾脏病理学变化;免疫组化检测足细胞组蛋白H3K27me3蛋白表达;实时定量PCR和Western印迹分别检测肾组织中JAK2-STAT3信号通路基因和蛋白表达;酶联免疫吸附测定法检测肾组织IL-6、MCP-1、α-SMA及TGF-β1水平。结果(1)细胞实验:阿霉素组细胞核缩小、破裂,细胞质呈空泡状,线粒体和内质网肿胀,提示阿霉素肾病足细胞损伤模型建立成功。与对Objective To analyze the target signaling pathway of histone H3K27 methylation-induced podocyte injury,verify the regulatory effect of histone H3K27 methylation on podocyte injury in focal segmental glomerulosclerosis(FSGS)mice through target signaling pathway,and explore the mechanism of abnormal methylation of histone H3K27-induced podocyte injury in FSGS mice.Methods(1)Cell experiments:primary cultured immortalized mouse podocytes MPC5 were cultured in vitro,and divided into control group,adriamycin(ADR)group,ADR+GSK-J4(histone demethylase,KDM6B inhibitor)group,ADR+coumarin A1(C-A1,JAK2 agonist)group and ADR+GSK-J4+C-A1 group.The transmission electron microscope was used to observe ultrastructure of podocytes.Immunofluorescence was used to detect the protein expression of H3K27me3 and nephrin in podocytes.The whole genome sequence of podocytes was obtained,the differentially expressed genes were screened,and Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were used for enrichment analysis.Real time-quantitative PCR and Western blotting were used to detect the gene and protein expression of JAK2-STAT3 signaling pathway in podocytes respectively.Enzyme-linked immunosorbent assay was used to detect interleukin 6(IL-6),monocyte chemotactic protein 1(MCP-1),α-smooth muscle actin(α-SMA)and transforming growth factorβ1(TGF-β1).(2)Animal experiments:EZH2 gene knock out(EZH2^(podKo))-FSGS(tail vein injection of ADR)mouse models were established,and divided into EZH2^(ctrl)+control group(n=20),EZH2^(ctrl)+FSGS(n=20),EZH2^(podKo)+control group(n=30)and EZH2^(podKo)+FSGS group(n=30).HE staining was used to observe the morphology of kidney tissues.Immunohistochemistry was used to detect the H3K27me3 protein expression in podocytes.Real time-quantitative PCR and Western blotting were used to verify the gene and protein expression of JAK2-STAT3 signaling pathway respectively.Enzyme-linked immunosorbent assay was used to detect IL-6,MCP-1,α-SMA and TGF-β1 of kidney tissues.Results(1)Cell experiments:Co

关 键 词：组蛋白类 足细胞 肾小球硬化症 局灶节段性 JAK2-STAT3信号通路 

分 类 号：R73[医药卫生—肿瘤]