CDE诱导慢性肝损伤过程中肝细胞特异性过表达细胞周期抑制因子P21促进胆管上皮细胞转分化为成熟肝细胞研究  

Hepatocyte-specific cell cycle inhibitor protein P21 overexpression promotes transdifferentiation of biliary epithelial cells into mature hepatocytes in mice with CDE-induced chronic liver injury

作　　者：刘清桂 王紫君 王敏君 杜涵 陈费 Liu Qinggui;Wang Zijun;Wang Minjun(Department of Cell Biology,Naval Medical University(Second Military Medical University),Shanghai 200433,China)

机构地区：[1]海军军医大学基础医学院细胞生物学教研室,上海市200433

出　　处：《实用肝脏病杂志》2023年第4期466-471,共6页Journal of Practical Hepatology

基　　金：上海市自然科学基金资助项目(编号:21ZR1477400);上海市人才发展计划项目(编号:2021080)。

摘　　要：目的探讨慢性损伤模型细胞周期抑制因子P21(P21)表达对肝损伤程度和胆管上皮细胞转分化为肝细胞的影响。方法利用谱系示踪技术建立胆管上皮细胞示踪模型小鼠;在胆管上皮细胞示踪小鼠,利用腺相关病毒构建肝细胞特异性P21过表达模型;利用胆碱缺乏/乙硫氨酸补充饮食(CDE)构建小鼠严重慢性肝损伤模型;采用蛋白印迹和免疫组织荧光法检测胆管上皮细胞标记和P21过表达水平,以过表达P21为实验组,过表达空载体为对照组,取损伤2 w和损伤后恢复2 w的小鼠肝组织,采用蛋白印迹、免疫组织化学和免疫组织荧光等技术检测肝损伤程度、细胞增殖水平、胆管上皮细胞转分化效果和转分化细胞特性。结果在CDE诱导慢性肝损伤小鼠,肝细胞特异性过表达P21组肝脏外观表面失去原有光泽,肝质量比为(4.5±0.1)%,显著低于对照组【(5.2±0.2)%,P<0.05】;血清AST、ALT和TBIL水平分别为(385.4±12.7)U/L、(423.8±32.4)U/L和(21.3±1.4)μmol/L,显著高于对照组【分别为(175.9±11.4)U/L、(214.8±23.5)U/L和(10.5±0.9)μmol/L,P<0.05】;过表达P21组肝细胞Ki67阳性比例为(0.1±0.1)%,显著低于对照组【(8.2±1.5)%,P<0.05】,S期标志蛋白CyclinA2阳性比例为(0.1±0.2)%,显著低于对照组【(3.2±0.7)%,P<0.05】,而两组间G1期特异性蛋白CyclinD1阳性比例无显著性差异【(43.2±3.5)%对(42.0±2.8)%,P>0.05】,提示肝细胞过表达细胞周期抑制蛋白P21使肝细胞阻滞于G1/S期;肝细胞过表达细胞周期抑制蛋白P21组在损伤恢复2周时出现了带有绿色荧光标记的肝细胞样克隆,这群细胞表达HNF4α肝细胞标志物。进一步分析发现绿色肝细胞样克隆具有成熟肝细胞的特征,比如表达Alb、CYP3A4、CYP2E1和CYP2D6等。结论在肝损伤过程中,P21过表达抑制肝细胞增殖能力,导致肝损伤程度加剧,从而促进胆管上皮细胞转分化为具有功能的成熟肝细胞。Objective The aim of this experiment was to investigate the effects of hepatocyte-specific cell cycle inhibitor protein P21(P21)overexpression on transdifferentiation of biliary epithelial cells(BEC)into mature hepatocytes in mice with choline-deficient and ethionine-supplemented(CDE)-induced chronic liver injury.Methods We established a mice with rosa-stop^(flox/flox)-EGFP by biliary epithelial cells lineage tracing via Krt19-Cre^(ERT),and the mice were injected with the adeno-associated virus serotype 8 encoding Cdkn1a under the control of the CAG promoter(AAV-CAG-P21),a virus which infected only hepatocytes.The overexpression of P21 and the bile duct epithelial cell tracing model were determined by Western blot and immunohistochemistry.Then,the male Krt19-Cre^(ERT) and Rosa-stop^(flox/flox)-EGFP mice injected with AAV-CAG-P21(experiment group)and AAV-CAG-MCS(control group)were fed with CDE diet for 2 weeks.The liver tissues were collected for liver damage and cell proliferation analysis.The mice received 2 weeks normal diets after CDE diet-induced liver injury to observe biliary epithelial cells transdifferentiation into hepatocytes,and the liver tissues were collected from all mice and analyzed by immunohistochemistry and immunofluorescence.Results The mice with P21 overexpression in hepatocytes from CDE diet-fed mice developed severe liver injury with pale liver surface,decreased significantly the ratio of liver weight/body weight from(4.5±0.1)% to(5.2±0.2)%(P<0.05)and increased serum AST levels from(175.9±11.4)U/L to(385.4±12.7)U/L(P<0.05),ALT levels from(214.8±23.5)U/L to(423.8±32.4)U/L(P<0.05)and bilibubin levels from(10.5±0.9)μmol/L to(21.3±1.4)μmol/L(P<0.05);the immunohistochemistry results showed an impaired hepatocyte proliferation in mice with P21 overexpression;the hepatocytes were arrested at G1/S transition-phase with decreased Ki67+hepatocytes from(8.2±1.5)% to(0.1±0.1)%,with lower S phase marker CyclinA2 expression from(3.2±0.7)%to(0.1±0.2)and similar CyclinD1(G1 phase marker)expre

关 键 词：肝细胞 胆管细胞 细胞周期抑制因子P21 转分化 小鼠 

分 类 号：R575[医药卫生—消化系统]