TIP-6通过诱导细胞自噬抑制培养的HepG2细胞和正常肝细胞增殖  

Cell proliferation inhibited by TIP-6 through autophagy in human hepatoma cell line HepG2 and human normal hepatocyte cell line L02

作　　者：高笑菲[1] 海军[1] 杜宇平[1] 王勤[1] 惠新平[2] 

机构地区：[1]兰州大学生命科学学院 [2]兰州大学化学化工学院应用有机国家重点实验室,甘肃兰州730000

出　　处：《细胞与分子免疫学杂志》2009年第10期883-886,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：甘肃省科技支撑计划项目资助(0708NKCA124)

摘　　要：目的:研究7-(4-甲氧基苯基)-5,8a-二苯基-1,2,3,7,8,8a-六氢咪唑[1,2-a]吡啶(TIP-6)对培养的人肝癌HepG2细胞和人正常肝细胞L02增殖的影响。方法:用MTT法和台盼蓝染色法检测TIP-6对细胞增殖的影响;相差显微镜观察细胞形态学的变化;流式细胞术(FCM)分析细胞周期改变;吖啶橙荧光染色观察自噬泡的变化;Annexin V/7-AAD和DAPI染色检测细胞凋亡;DNA电泳检测凋亡梯形带的产生。细胞免疫化学法测定NF-κB的表达。结果:TIP-6浓度为90～200μmol/L时,细胞增殖受到抑制,且与浓度、时间有关;当浓度为200μmol/L、处理72h时,两种细胞增殖数分别只有对照的12.10%和18.75%,空泡化也随剂量和时间的增加而加剧,空泡数目越来越多、体积越来越大;FCM结果显示,细胞被阻滞在G2/M期,且HepG2比L02细胞敏感。吖啶橙荧光染色证实自噬及自噬性细胞死亡会随着化合物浓度和时间增加而增多;AnnexinV/7-AAD、DAPI染色及DNA电泳均证实凋亡并非TIP-6诱导HepG2及L02细胞增殖抑制的主要形式;细胞免疫化学法的结果显示,随着核转录因子NF-κB表达量的上调,自噬泡会随之增加,细胞增殖却相应减少。结论:TIP-6可能诱导自噬抑制培养的肝癌细胞和肝细胞增殖。自噬性细胞死亡可能与NF-κB蛋白的活化有关。AIM： To investigate the effect of 7-（4-methoxyphenyl）-5, 8a-diphenyl-1,2, 3, 7, 8, 8a-hexahydroim- idazo[ 1, 2-a] pyridine （TIP-6） on cell proliferation in human hepatoma cell line HepG2 and human normal hepatocyte cell line L02. METHODS： Typan blue assay was used to check the effect of TIP-6 on cell proliferation. The changes of cell morphology were observed by the phase contrast microscope. Flow cytometry （FCM） was used to check cell cycle. Autophagy and autophagic cell death were detected after acridine orange （AO） staining under fluorescent microscopy. Apoptosis was analyzed by Annexin V/7-AAD, DAPI staining and DNA ladder. NF-κB expression was detected with cellular immunochemistry. RESULTS： Cell proliferation inhibiting effect was appeared when treated with TIP- 6 from 60 μmol/L to 200 μmol/L, which was correlated with treated concentrations and time. The proliferation rates were just 12.10% and 18.75% （ vs control） under 200 μmol/L 72 h in HepG2 and L02 respectively. Vacuolization were found more and more frequently with the increasing of TIP-6 concentrations and treated time prolonged. FCM results indicated that cells were blocked in G2/M phase, and more sensitive were found in HepG2 than L02. AO staining results indicated that the phenomenon of autophagy and autophagic cell death were occurred and appeared more potent with more TIP-6 and longer time treated. No apoptosis markers were found with Annexin V/7-AAD and DAPI staining, and no DNA ladders were found either, these indicated that TIP- 6 didnt induce apoptosis in these cells. NF-κB was found increased after treated with TIP-6, and the autophagic vacu- ole became more and more with the increasing of NF-κB protein, but the proliferation rates decreased at the same time. CONCLUSION： TIP-6 inhibited cell proliferation and induced autophagy and autophagic cell death in HepG2 and L02 cells. NF-KB activation may be involved in these effects.

关 键 词：TIP-6 HEPG2 L02 自噬 NF—κB 

分 类 号：Q255[生物学—细胞生物学]