MST1在同型半胱氨酸促肝细胞凋亡过程中的表达及意义  

Significance of expression of MST1 in homocysteineinduced hepatocyte apoptosis

作　　者：孔繁琪[1] 马胜超[2] 赵丽[3] 和杨杨 刘现梅 周龙霞[1] 张辉[3] 张鸣号[1] 金少举[4] 姜怡邓[1] 

机构地区：[1]宁夏医科大学基础医学院病理学与病理生理学系,宁夏回族自治区银川市750004 [2]四川大学基础医学与法医学院,四川省成都市610065 [3]宁夏医科大学检验学院临床检验诊断学教研室,宁夏回族自治区银川市750004 [4]宁夏医科大学药学院,宁夏回族自治区银川市750004

出　　处：《世界华人消化杂志》2015年第16期2523-2531,共9页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;Nos.81460121;81360073;81360053~~

摘　　要：目的:探讨MST1(mammalian sterile 20-like kinase 1)在同型半胱氨酸(homocysteine,Hcy)促肝细胞凋亡中的作用.方法:动物水平:实验分为4组,每组12只小鼠.正常对照组:5周龄♂鼠(SPF级C57BL/6J)饲以普通饮食;Apo E-/-对照组:5周龄♂纯合子Apo E-/-鼠饲以普通饮食;Apo E-/-高蛋氨酸组:5周龄♂纯合子Apo E-/-鼠饲以高蛋氨酸饮食(普通饮食中加入1.7%蛋氨酸);Apo E-/-叶酸和维生素B12干预组:5周龄♂纯合子A p o E-/-鼠饲以高蛋氨酸饮食加0.006%叶酸和0.0004%维生素B12.喂养14 wk后,全自动生化分析仪测定血清Hcy水平;透射电镜、DAPI染色观察肝组织凋亡情况;实时定量聚合酶链反应(q RT-PCR)及Western blot分别检测MST1 m RNA和蛋白表达水平.细胞水平:分为3组:正常对照组(0μmol/L Hcy);高同型半胱氨酸(hyperhomocysteinemia,HHcy)组(100μmol/L Hcy);干预组(100μmol/L Hcy+叶酸+维生素B12);干预肝细胞48 h.Annexin VFITC/PI双标记流式细胞术检测肝细胞凋亡水平的变化;实时定量聚合酶链反应(q RTPCR)及Western blot分别检测MST1 m RNA和蛋白表达水平.结果:动物水平:(1)各组小鼠喂养20 wk后,测定血清Hcy水平,Apo E-/-高蛋氨酸组血清H c y水平分别是正常对照组和A p o E-/-对照组的2.3倍和1.9倍(P<0.01).干预组血清Hcy水平较Apo E-/-高蛋氨酸组降低28%(P<0.01),提示造HHcy模型成功;(2)电镜结果显示,A p o E-/-高蛋氨酸组小鼠肝细胞出现凋亡的倾向,染色质凝聚浓缩或边缘化,线粒体肿胀或浓缩、嵴断裂或变短,粗面内质网和滑面内质网明显肿胀、断裂、连贯性破坏、核糖体脱落,糖原溶解;(3)DAPI染色后观察,正常对照组和Apo E-/-对照组中细胞核大小均一、核圆,核膜光滑;Apo E-/-高蛋氨酸饮食组一些细胞出现核膜皱缩、细胞核呈现出各种不规则形状,提示可能存在凋亡;Apo E-/-干预组较Apo E-/-高蛋氨酸组细胞核损伤有所减轻;(4)与正常对照组和Apo E-/-对照组相比,Apo E-/-高蛋氨AIM：To study the role of the mammalian sterile20-like kinase 1（MST1） gene in homocysteineinduced hepatocyte apoptosis.METHODS：Five-week-old C57BL/6J mice of SPF grade were divided into four groups：a normal control group,an ApoE7＂ group,a high methionine diet group,and an intervention group（n- 12 each）.In the normal control group,normal mice were fed a normal diet.In the ApoE7＂ group,male ApoE7＂ mice were fed a normal diet.In the high methionine diet group,male ApoE7＂ mice were fed a high methionine diet.In the intervention group,male ApoE7＂ mice were fed a high methionine diet plus folic acid and vitamin B12.Transmission electron microscopy and DAPI staining were used to determine the level of apoptosis in hepatic tissue.qRT-PCR and Western blot were used to determine the expression of MST1.Hepatocytes were then cultured in the presence or absence of homocysteine（100 μmol/L） alone or 100μmol/L homocysteine plus folic acid and vitamin B12;flow cytometry was used to determine the level of hepatocytes apoptosis,and the expression of MST1 was detected by qRT-PCR and Western blot.RESULTS：After the mice were fed for 14 wk,serum homocysteine level in the high methionine diet group was 2.3 and 1.9times as high as that in the normal control group and the ApoE＂7＂ group（P 〈 0.01）,respectively.Serum homocysteine level in the intervention group was 28%lower than that in the high methionine diet group（P 〈 0.01）.These findings suggest that the model was successfully established.Electron microscopy showed that in the high methionine diet group,there were chromosome swelling or condensation,mitochondrial swelling,marked endoplasmic reticulum swelling and break,which suggested the trend of cell apoptosis in hepatic tissue.Compared with the normal control group and ApoE＂7＂ group,hepatic apoptosis level in the high methionine diet group was higher.However,hepatic apoptosis level in the intervention group was lower than that in the high methionine diet group.Compared with the normal

关 键 词：同型半胱氨酸 肝细胞凋亡 MST1 叶酸 维生素B12 

分 类 号：R575[医药卫生—消化系统]