摘要
目的探讨转染p53上调凋亡调控因子(PUMA)的人脑胶质瘤细胞对替莫唑胺敏感性增强的作用机制。方法将人脑胶质瘤细胞U87MG分为正常对照组、空载体组和实验组进行培养,分别将感染复数(MOI)=50的空载体腺病毒(Ad-△BH3)和携带PUMA的重组腺病毒(Ad-PUMA)转染U87MG细胞,转染后用MTT比色法测定各组细胞的存活率并计算替莫唑胺(TMZ)IC50,流式细胞仪检测细胞周期的变化,应用TUNEL法检测细胞的凋亡情况。Western blot检测凋亡相关蛋白p53、Bax的表达。结果外源性PUMA基因在Ad-PUMA转染U87MG48h后,随着时间延长,PUMA表达使U87MG细胞的增殖能力降低并诱导凋亡,转染24h、48h、72h的生长抑制率分别为17.3%、35.6%、43.3%,凋亡率分别为20.3%、31.4%、45.4%。正常对照组、Ad-PUMA、Ad-△BH3组细胞的TMZ IC50分别为(15.0±1.9)μmol/L、(2.3±0.1)μmol/L和(14.4±1.6)μmol/L。PUMA表达的U87MG细胞DNA合成受到抑制,周期阻滞在G2期;Western blot检测显示p53表达在各组中差异无统计学意义(P>0.05)、PUMA和Bax在实验组中表达较对照组强,差异有统计学意义(P<0.01)。结论 Ad-PU-MA转染可有效增强人脑胶质瘤细胞U87MG对TMZ的敏感性,抑制人脑胶质瘤细胞U87MG的增殖,通过诱导细胞G2期阻滞促进其凋亡,促凋亡机制不依赖于p53。
Objective To investigate the effect of PUMA on the sensitivity of human glioblastoma U87MG cells to temozolomide and its related mechanisms.Methods U87MG cells under culture were divided into the normal control group,mock group and experiment group.After 24 hours of culture,the mock and experiment group were transfected with mock vector（Ad-△BH3）and the recombinant adenovirus carrying PUMA（Ad-PUMA）respectively at a multiplication of infection（MOI）of 50.The proliferation activity of cells and IC50 were detected by MTT assay,the apoptosis effect and the change of cell cycle determined by Hoechst stain and flow cytometry（FCM）technology respectively.The expression of related proteins was revealed by the method of Western blot.Results Exotic PUMA gene was expressed in U87MG cells transfected with Ad-PUMA,which could inhibit the proliferation of U87MG cells.The inhibition rate of proliferation 24,48,72 h after transfection was 17.3%,35.6%,43.3% and apoptosis rate was 20.3%,31.4%,45.4% respectively.Results：The TMZ IC50 values of PBS group,Ad-PUMA and Ad-△BH3 group cells were（15±1.9）,（2.3±0.14）and（14.4±1.6）μmol/L respectively,with the sensitivity to the TMZ of Ad-PUMA group cells increased by 7.0 folds.PUMA overexpressing U87MG cells showed the DNA synthesis was inhibited and arrested in G2 phrase.The results of Western blot showed that after 72 h of transfection the PUMA and Bax protein showed increased expression（P0.01）.There was no significant difference in the expression of p53 among these groups（P0.05）.Conclusions PUMA gene transfection greatly enhances the sensitivity of U87MG cells to TMZ-induced apoptosis and can effectively inhibit the proliferation and promote the apoptosis of U87MG cells.The mechanism of apoptosis mainly relates to induce cell cycle G2 arrest and apoptosis in vitro.
出处
《中华临床医师杂志（电子版）》
CAS
2011年第3期29-32,共4页
Chinese Journal of Clinicians(Electronic Edition)
基金
山西省卫生厅科技攻关项目(20100105
20100224)
