Perilipin 2在小鼠肝脏CGI-58特异性敲除所致肝脏微泡型脂肪变性中的作用  

Role of Perilipin 2 in microvesicular hepatic steatosis induced by CGI-58 specific knockout in mice

作　　者：张艺馨 李杰 万小勤 蒋孝清 陈江慧 邓芳 李旻典 张倩 包鑫余 张志辉 ZHANG Yixin;LI Jie;WAN Xiaoqin;JIANG Xiaoqing;CHEN Jianghui;DENG Fang;LI Mindian;ZHANG Qian;BAO Xinyu;ZHANG Zhihui(Department of Cardiovascular Diseases,Center for Circadian Metabolism and Cardiovascular Diseases,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038;Key Laboratory of Geriatric Cardiovascular and Cerebrovascular Diseases of Ministry of Education,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038;College of Basic Medical Sciences,Chongqing Medical University,Chongqing,400016;Department of Pathophysiology,Faculty of High Altitude Military Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China;Department of General Practice,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038)

机构地区：[1]陆军军医大学(第三军医大学)第一附属医院心血管内科代谢生物钟与心血管疾病中心,重庆400038 [2]陆军军医大学(第三军医大学)第一附属医院老年心脑血管疾病教育部重点实验室,重庆400038 [3]重庆医科大学基础医学院,重庆400016 [4]陆军军医大学(第三军医大学)高原军事医学系病理生理学教研室,重庆400038 [5]陆军军医大学(第三军医大学)第一附属医院全科医学科,重庆400038

出　　处：《陆军军医大学学报》2024年第15期1701-1712,共12页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(81873663);重庆市研究生科研创新项目(CYS21515)。

摘　　要：目的探究Perilipin 2(Plin2)在肝脏CGI-58特异性敲除所致脂肪肝的作用,并比较Plin2和Plin3在脂滴生成和脂质蓄积的作用效能。方法将20只7周龄CGI-58^(Flox/Flox)小鼠(雄鼠10只,雌鼠10只)根据性别采用完全随机分组法分为对照组(NC组,n=5,注射靶向肝脏实现过表达Cre蛋白及对照Micro-RNA的腺相关病毒)和实验组(Mi-KD组,n=5,注射靶向肝脏实现过表达Cre蛋白及靶向Plin2的Micro-RNA的腺相关病毒),记录小鼠体质量变化,检测小鼠代谢相关表型和肝脏病理学的差异,RT-qPCR检测小鼠肝脏脂质代谢和胆固醇代谢相关基因的改变;以AML-12小鼠肝细胞作为细胞模型,构建SiRNA实现AML-12细胞Plin2/Plin3敲低,Bodipy染色和酶比色法检测对比正常培养和OA诱导下Plin2/Plin3敲低后AML-12细胞脂滴生成和脂质蓄积。结果Mi-KD小鼠肝脏中的PLIN2蛋白水平降低了99%以上;Mi-KD缓解了CGI-58特异性敲除雌鼠的肝肿大(P=0.0195)和肝细胞损伤(P=0.0004),降低了组织学NAS评分(P=0.0002)和肝脏甘油三酯含量(P=0.0166),显著改善了肝脏CGI-58特异性敲除所致的肝脏微泡型脂肪变性;敲低Plin2使AML-12细胞内甘油三酯含量呈下降趋势,敲低Plin3显著降低AML-12细胞内甘油三酯含量(P=0.0014);在OA诱导下,Plin2/Plin3敲低均显著降低OA诱导的AML-12细胞内甘油三酯的蓄积(P<0.05)。结论肝脏Plin2在CGI-58敲除所致的微泡型脂肪变性的发展过程中至关重要,Plin2和Plin3均参与肝细胞脂滴生成与脂质蓄积,且Plin3的作用强于Plin2。Objective To explore whether hepatocyte Perilipin-2(Plin2)is involved in the development of fatty liver related to comparative gene identification-58(CGI-58)deficiency mice and compare the effects of Plin2 and Plin3 on lipid droplet formation and lipid accumulation.Methods Based on CGI-58^(Flox/Flox) mice as animal model,the adeno-associated viruses targeting mouse liver,CGI-58 knockout and Plin2 knockdown were achieved by co-expression Cre protein and micro-RNA targeting Plin2(Mi-KD).Then CGI-58 deficiency mice were used as control(NC)to detect the differences in metabolic phenotype and liver pathology.AML-12 mouse hepatocytes were used as cellular model and interfered with siRNA to achieve Plin2/Plin3 knockdown in AML-12 cells.Lipid droplet formation and lipid accumulation were compared with Bodipy staining and enzyme colorimetry in basal condition or lipid-overloaded condition(OA inducement)after Plin2/Plin3 knockdown.Results Plin2 knockdown(Mi-KD)reduced PLIN2 protein level by>99%in mouse livers.Mi-KD decreased hepatomegaly(P=0.0195)and liver injury(P=0.0004),while reduced the histological NAS score(P=0.0002)and hepatic triglyceride content(P=0.0166)in the CGI-58 deficiency female mice.Mi-KD prevented microvesicular hepatic steatosis in the CGI-58 deficient female mice.Plin3 knockdown significantly reduced the triglyceride content in basal condition of hepatocytes(P=0.0014),and Plin2 knockdown just showed a decreased trend.Plin2 or Plin3 knockdown significantly reduced the triglyceride content separately in lipid-overloaded hepatocytes(P<0.05).Conclusion Hepatocyte Plin2 is essential in the development of microvesicular hepatic steatosis caused by CGI-58 deficiency.Both Plin2 and Plin3 are involved in lipid droplet formation and lipid accumulation in hepatocytes,and Plin3 shows a stronger effect.

关 键 词：肝脏微泡型脂肪变性 围脂滴包被蛋白2 比较鉴定基因-58 钱林-多尔夫曼综合征 脂滴 

分 类 号：R394.3[医药卫生—医学遗传学] R575.5[医药卫生—基础医学] R589.2