促凋亡蛋白Smac和Caspase9在内毒素诱导体外肝细胞凋亡中的作用  

The role of pro-apoptotic proteins smac and caspase9 in lipopolysaccharide-induced apoptosis of hepatocyte in vitro

作　　者：陈淼[1] 田德英[1] 陈安群[1] 张振刚[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院感染科,武汉430030

出　　处：《临床内科杂志》2008年第10期709-711,共3页Journal of Clinical Internal Medicine

摘　　要：目的探讨内毒素致肝细胞凋亡和内毒素血症在重症肝炎发病中的作用机制。方法培养LO2肝细胞并进行传代,采用内毒素10mg/ml分别在4小时、8小时、16小时和24小时进行刺激,并将其分为4小时、8小时、16小时和24小时组和对照组5组,采用流式细胞术和荧光染色检测肝细胞凋亡以及免疫组化法检测Smac和Caspase9的表达。结果内毒素直接作用的L02细胞在形态学上表现出胞浆浓缩核凝聚成团块、形成凋亡小体,应用Hoechst对内毒素刺激的L02细胞进行染色并在荧光显微镜下观察,Hoechst染色显示凋亡细胞呈蓝色荧光。流式细胞仪分析内毒素刺激4小时、8小时、16小时和24小时后晚期凋亡率分别为2.3%、4.0%、6.2%和18.2%,呈进行性增高(P<0.01),与对照组晚期凋亡率(0.3%)比较有显著性差异(P<0.05),Smac和Caspase9蛋白随着刺激时间的增长表达率逐渐增高(P<0.01),与对照组比较,各刺激组Smac和Caspase9蛋白表达显著增高(P<0.05)。各组Smac与Caspase9蛋白表达呈正相关关系(r=0.527,P<0.001)。且Smac和Caspase9蛋白在胞浆中呈棕黄色表达。结论内毒素在体外可以通过促凋亡蛋白Smac及增强下游通路Caspase9的活性直接诱导正常肝细胞凋亡。Objective To study the effect of endotoxin on the hepatic apoptosis and the role of endotoxin in severe hepatitis. Method To stimulate the L02 hepatocyte 4 h, 8 h, 16 h,24 h, respectively with endotoxin 10 mg/ml,to detect the hepatic apoptosis by flow cytometry and fluorescein stain, and to measure the expression of smae and caspase9 by immunohistochemistry as well. Result L02 cell morpho-logically displayed cytoplasm concentrated, nuclear-mass rally, the formation of apoptotie bodies after the stimulation of LPS;the LPS-stimulated L02 cells were stained with Hoechst and were observed under fluo-rescence microscope,which emit blue fluorescence. The late apoptotic rates were 2.3% ,4.0% ,6.2%, 18.2% , respectively with flow analysis after 4 h, 8 h, 16 h ,24 h with LPS stimulation. The apeptotic rates increased with time and there were significant differences （ P〈0.05 ） compared with the normal control group which apoptotic rate was 0.3% ,the expression of protein smac and caspase9 increased with the increasing of stimulation time（ P 〈 0.01 ）. Compared with the normal control group ,the expression of protein smac and easpase9 detected with immunohistochemistry significantly increased in all of the stimulation groups（ P 〈 0.05 ）. Caspase9 and smac protein diplayed brown in the cytoplasm, smae protein was positive correlated with the expression of Caspase9 （r=0.527,P〈0.001）. Condution LPS in vitro can induce apoptosis of normal liver cell by pro-apoptotie protein smae and can enhance the activity of Caspase9 of the downstream pathway.

关 键 词：细胞凋亡 内毒素 SMAC蛋白 重症肝炎 

分 类 号：R575.1[医药卫生—消化系统]