RIP3、HMGB1在HK-2细胞程序性坏死中的表达及作用研究  

Expression and effect of RIP3,HMGB1 in necroptosis of HK-2 cells

作　　者：曹彦卫 张世球 王川玲 俞容 朱永俊 Cao Yanwei;Zhang Shiqiu;Wang Chuanling;Yu Rong;Zhu Yongjun(Department of Nephrology,Hainan Medical University First Clinical Medical College,Haikou 570102,China)

机构地区：[1]海南医科大学第一临床医学院肾内科,海口570102

出　　处：《疑难病杂志》2025年第2期220-226,共7页Chinese Journal of Difficult and Complicated Cases

基　　金：国家自然科学基金(82060143)。

摘　　要：目的构建人肾小管上皮(HK-2)细胞程序性坏死的模型,观察受体相互作用蛋白3(RIP3)、高迁移率族蛋白1(HMGB1)在HK-2细胞程序性坏死中的表达和作用机制。方法于2022年5月—2024年1月在海南医科大学实验室进行实验。取HK-2细胞,分为control组、TNF-α组、TNF-α+Nec-1s组、TNF-α+GSK’872组、TNF-α+NSA组,分别给予相应干预。采用流式细胞术检测各组细胞凋亡、坏死率;TUNEL+RIP3荧光双染色结合激光共聚焦显微成像检测TUNEL+RIP3阳性细胞百分率;ELISA法检测HMGB1蛋白表达水平;qRT-PCR检测RIP3、HMGB1 mRNA表达水平;Western blot检测RIP3、HMGB1蛋白表达水平。结果与control组比较,TNF-α组HK-2细胞凋亡和坏死率、TUNEL+RIP3双阳性细胞百分率、HMGB1蛋白表达量及RIP3、HMGB1 mRNA和蛋白表达水平显著升高(q/P=56.786/<0.001、47.963/<0.001、24.186/<0.001、5.020/0.034、4.708/0.047、46.495/<0.001、26.837/<0.001)。与TNF-α组比较,TNF-α+Nec-1s组、TNF-α+GSK’872组、TNF-α+NSA组HK-2细胞凋亡和坏死率显著降低(q/P=44.243/<0.001、37.666/<0.001、30.324/<0.001),TUNEL+RIP3双阳性细胞百分率显著降低(q/P=35.176/<0.001、28.461/<0.001、21.104/<0.001),HMGB1蛋白表达量显著降低(q/P=39.043/<0.001、39.412/<0.001、41.510/<0.001),RIP3 mRNA表达显著降低(q/P=13.982/<0.001、5.386/0.022、8.811/<0.001),HMGB1 mRNA表达显著降低(q/P=7.219/0.003、6.318/0.008、4.658/0.049),RIP3蛋白表达显著降低(q/P=62.436/<0.001、46.495/<0.001、39.853/<0.001),HMGB1蛋白表达显著降低(q/P=20.982/<0.001、20.006/<0.001、28.301/<0.001)。TNF-α+Nec-1s组、TNF-α+GSK’872组、TNF-α+NSA组两两比较,HK-2细胞凋亡和坏死率、TUNEL+RIP3双阳性细胞百分率、RIP3蛋白表达水平差异均有统计学意义(P<0.05)。结论TNF-α能诱导HK-2细胞发生RIP3介导的程序性坏死并释放HMGB1分子。Objective To construct the model of HK-2 cell programmed necrosis of human renal tubular epithelium(HK-2),and observe the expression and role of receptor interaction protein 3(RIP3)and high mobility group protein 1(HMGB1)in HK-2 cell programmed necrosis.Methods The experiment will be conducted in the Laboratory of Hainan Medical University from May 2022 to January 2024.HK-2 cells were divided into control group,TNF-αgroup,TNF-α+Nec-1s group,TNF-α+GSK’872 group and TNF-α+NSA group.Flow cytometry was used to detect apoptosis and necrosis rates in each group of cells.TUNEL+RIP3 fluorescence double staining combined with laser confocal microscopy was used to detect the percentage of TUNEL+RIP3 positive cells.The expression of HMGB1 protein was detected by ELISA.The mRNA expression levels of RIP3 and HMGB1 were detected by qRT-PCR.The expression levels of RIP3 and HMGB1 were detected by Western blot.Results Compared with control group,the apoptosis and necrosis rate of HK-2 cells,the percentage of TUNEL+RIP3 double positive cells,the expression level of HMGB1 protein and the mRNA and protein expression levels of RIP3 and HMGB1 in TNF-αgroup were significantly increased(q/P=56.786<0.001,47.963/<0.001,24.186/<0.001,5.020/0.034,4.708/0.047,46.495/<0.001,26.837/<0.001).Compared to the TNF-αgroup,apoptosis and necrosis rate of HK-2 cells,percentage of TUNEL+/RIP3+double positive cells,HMGB1 protein expression and mRNA and protein expression levels of RIP3,HMGB1 in TNF-α+Nec-1s group,TNF-α+GSK’872 group and TNF-α+NSA group were significantly decreased(The apoptosis and necrosis rates of HK-2 cells:q/P=44.243/<0.001,37.666/<0.001,30.324/<0.001;percentage of TUNEL+/RIP3+double positive cells:q/P=35.176/<0.001,28.461/<0.001,21.104/<0.001;HMGB1 protein expression:q/P=39.043/<0.001,39.412/<0.001,41.510/<0.001;RIP3 mRNA expression:q/P=13.982/<0.001,5.386/0.022,8.811/0.001,HMGB1 mRNA expression:q/P=7.219/0.003,6.318/0.008,4.658/0.049,RIP3 protein expression:q/P=62.436/<0.001,46.495/<0.001,39.853/<0.001,HMGB1 protein e

关 键 词：程序性坏死 肿瘤坏死因-α 受体相互作用蛋白3 高迁移率族蛋白1 

分 类 号：R692.5[医药卫生—泌尿科学]