CAJ | 학술논문

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90～200 μLmol/L时,细胞增殖受到抑制,且与浓度、时间有关;当浓度为200 μmol/L、处理72 h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;Annexin V/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.
목적:연구7-(4-갑양기분기)-5,8a-이분기-1,2,3,7,8,8a-륙경미서[1,2-a]필정(TIP-6)대배양적인간암HepG2세포화인정상간세포L02증식적영향.방법:용MTT법화태반람염색법검측TIP-6대세포증식적영향;상차현미경관찰세포형태학적변화;류식세포술(FCM)분석세포주기개변;아정등형광염색관찰자서포적변화;Annexin V/7-AAD화DAPI염색검측세포조망;DNA전영검측조망제형대적산생.세포면역화학법측정NF-κB적표체.결과:TIP-6농도위90～200 μLmol/L시,세포증식수도억제,차여농도、시간유관;당농도위200 μmol/L、처리72 h시,량충세포증식수분별지유대조적12.10%화18.75%,공포화야수제량화시간적증가이가극,공포수목월래월다、체적월래월대;FCM결과현시,세포피조체재G2/M기,차HepG2비L02세포민감.아정등형광염색증실자서급자서성세포사망회수착화합물농도화시간증가이증다;Annexin V/7-AAD、DAPI염색급DNA전영균증실조망병비TIP-6유도HepG2급L02세포증식억제적주요형식;세포면역화학법적결과현시,수착핵전록인자NF-κB표체량적상조,자서포회수지증가,세포증식각상응감소.결론:TIP-6가능유도자서억제배양적간암세포화간세포증식.자서성세포사망가능여NF-κB단백적활화유관.
AIM: To investigate the effect of 7-(4-methoxyphenyl)-5, 8a-diphenyl-1,2, 3, 7, 8, 8a-hexahydroimidazo[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 TIP6 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 TIP6 didnt induce apoptosis in these cells. NF-κB was found increased after treated with TIP-6, and the autophagic vacuole 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-κB activation may be involved in these effects.