敲除Usp13促进棕榈酸诱导的小鼠肝实质细胞凋亡  

Knockdown of Usp13 Promotes Palmitic Acid-induced Apoptosis in Mouse Hepatocytes

作　　者：王婷 刘凯 李柯颖 陈旭 任广明 杨晓明[1,2,3] WANG Ting;LIU Kai;LI Ke-ying;CHEN Xu;REN Guang-ming;YANG Xiao-ming(School of Basic Medical Sciences,Anhui Medical University,Hefei 230032,China;Beijing Institute of Lifeomics,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Qingdao University,Qingdao 266071,China)

机构地区：[1]安徽医科大学基础医学院,合肥230032 [2]军事科学院军事医学研究院生命组学研究所,北京100850 [3]青岛大学,青岛266071

出　　处：《中国生物工程杂志》2022年第4期9-16,共8页China Biotechnology

基　　金：国家自然科学基金青年科学基金(82001666)资助项目。

摘　　要：目的:研究去泛素化酶Usp13对棕榈酸诱导的肝实质细胞凋亡的影响,并对其机制进行初步探究。方法:两步胶原酶灌注法分离小鼠原代肝实质细胞并采用棕榈酸处理;甘油三酯酶法及油红O染色检测肝实质细胞中脂质的累积;MTS和LDH试剂盒检测细胞活力;流式细胞术检测细胞凋亡;qPCR检测凋亡相关基因的表达以及免疫印迹法检测Caspase-3的活化。结果:敲除Usp13基因并不影响棕榈酸诱导下肝实质细胞的甘油三酯累积,但导致细胞损伤加重,凋亡增加。机制研究显示,敲除Usp13促使肝实质细胞中抗凋亡基因Bcl-2、Bcl-xl表达下调,促凋亡基因Bid、p53表达上调,以及Caspase-3活化增强。结论:初步揭示了Usp13调控棕榈酸诱导下肝实质细胞凋亡的机制,为深入研究Usp13调控非酒精性脂肪性肝炎的发生、发展奠定了基础。Objective:To study the effect of ubiquitin-specific protease 13(Usp13)on the apoptosis of mouse hepatocytes stimulated by palmitic acid,and to explore the underlying molecular mechanism.Methods:Mouse primary hepatocytes were isolated by two-step collagenase perfusion technique,and then stimulated with palmitic acid after cultured in vitro.Lipid accumulation was detected by triglyceride quantitative method and oil red O staining.Cell viability was measured by MTS and LDH assay.The apoptosis of hepatocytes was tested by Annexin V-FITC/7-AAD assay.Finally the expression levels of anti-apoptotic genes and pro-apoptotic genes were detected by qPCR and the activation of Caspase-3 was detected by Western blotting.Results:Knockdown of Usp13 did not affect the accumulation of triglyceride in palmitic acid-induced hepatocytes,but the cell viability was decreased and apoptosis was increased.Studies show that knockdown of Usp13 resulted in the down-regulated expression of anti-apoptotic genes Bcl-2 and Bcl-xl,and the up-regulated expression of pro-apoptotic genes Bid and p53.The Caspase-3 activity in Usp13 knockdown hepatocytes was enhanced after being exposed to palmitic acid.Conclusion:This study revealed the mechanism of Usp13 regulating the apoptosis of hepatocytes induced by palmitic acid,which laid a foundation for further study of Usp13 regulating the occurrence and development of non-alcoholic steatohepatitis.

关 键 词：非酒精性脂肪性肝炎 Usp13 细胞凋亡 棕榈酸 小鼠原代肝实质细胞 

分 类 号：Q819[生物学—生物工程]