脂多糖通过Cdk5/p25/p35调节人腹膜间皮细胞TNF-α和IL-1β的表达  

Lipopolysaccharide regulates the expression of TNF-αand IL-1βin human peritoneal mesothelial cells through Cdk5/p25/p35

作　　者：李祖楷 冯俊霞 徐嘉琪 李静纯 高学娟[3] 张云芳[1,2] Li Zukai;Feng Junxia;Xu Jiaqi;Li Jingchun;Gao Xuejuan;Zhang Yunfang(The Third School of Clinical Medicine,Southern Medical University,Guangzhou 510800,China;Department of Nephrology,Huadu District People's Hospital,Southern Medical University,Guangzhou 510800,China;Institues of Life and Health Engineering,Jinan University,Guangzhou 510632,China)

机构地区：[1]南方医科大学第三临床医学院,广州510800 [2]南方医科大学附属花都医院肾内科,广州510800 [3]暨南大学生命与健康工程研究院,广州510632

出　　处：《国际泌尿系统杂志》2022年第5期853-858,共6页International Journal of Urology and Nephrology

基　　金：国家自然科学基金(81800675);广州市花都区科技计划项目(20-HDWS-033);广东省自然基金项目(2016A030313420)。

摘　　要：目的观察脂多糖(LPS)作用下人腹膜间皮细胞(PMCs)表达细胞周期蛋白依赖性激酶5(Cdk5)及其协同激活蛋白p25/p35以及对炎症介质分泌的影响。方法体外培养人腹膜间皮细胞株(HMrSV5),以293T细胞作为阳性对照。在不同浓度LPS作用6 h及LPS(1μg/mL)作用不同时间点收集细胞。LPS(1μg/mL)或不同浓度1NMPP1(一种Cdk5信号通路抑制剂)预处理2 h后再加LPS,作用6 h后收集细胞培养上清。用实时定量PCR检测Cdk5 mRNA的表达,用蛋白质印迹和免疫荧光检测Cdk5以及p25/p35的表达,用ELISA检测肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)的分泌。结果Cdk5及其共激活物p25/p35蛋白在HPMCs中均表达。免疫荧光分析显示Cdk5主要分布在HPMCs的胞浆中。LPS(1μg/mL或2μg/mL)处理6 h后,HPMCs中Cdk5的表达显著上调。LPS处理后Cdk5的蛋白表达呈浓度和时间依赖性增加,峰值为1μg/mL和6 h。LPS处理可诱导HPMCs分泌大量细胞因子TNF-α和IL-1β,而随着浓度增加的1NMPP1,可显著降低TNF-α和IL-1β的分泌(P<0.05)。结论Cdk5信号通路可能参与LPS诱导的腹腔分泌TNF-α和IL-1β等炎症介质。Objective To observe the expression of cyclin-dependent kinase 5(Cdk5)and its synergistic activation protein p25/35 in human peritoneal mesothelial cells(PMCs)under the action of lipopolysaccharide(LPS)and its effect on the secretion of inflammatory mediators.Methods The human peritoneal mesothelial cell line(HMrSV5)was cultured in vitro.Cells were collected at different concentrations of LPS for 6 hours and LPS(1μg/mL)at different time points.LPS(1μg/mL)or 1NMPP1(an inhibitor of Cdk5)was pretreated with different concentrations for two hours,then LPS was added,and the cell culture supernatant was collected after 6 hours of LPS treatment.Real-time quantitative PCR was used to detect the expression of Cdk5 mRNA,Western blot and immunofluorescence were used to detect the expression of Cdk5 and p25/p35,and ELISA was used to detect the expressions of inflammatory factors TNF-αand IL-1β.Results Both Cdk5 and its co-activator p25/p35 protein were expressed in HPMCs.Immunofluorescence analysis showed that Cdk5 was mainly distributed in the cytosol of HPMCs.After LPS(1μg/mL or 2μg/mL)treatment for 6 h,the expression of Cdk5 in HPMCs was significantly up-regulated.The protein expression of Cdk5 increased in a concentration-and time-dependent manner after LPS treatment,with a peak value of 1μg/mL and 6 h.LPS treatment can induce HPMCs lots cytokines secreted TNF-αand IL-1β,and with the increase of concentration of 1NMPP1,can significantly reduce the TNF-αand IL-1βsecretion(P<0.05).Conclusions The Cdk5 signal pathway may be involved in the secretion of TNF-αand IL-1βfrom the abdominal cavity induced by LPS.

关 键 词：腹膜透析 脂多糖类 细胞周期蛋白依赖激酶5 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]