替莫唑胺对胶质瘤U251干细胞livin及肿瘤坏死因子α的影响  

Effect of Temozolomide on livin and TNF-α in cancer stem cells isolated from glioblastoma U251 cells

作　　者：李根华[1] 郭强[2] 韩光魁[2] 杨冬旭[2] 冯嵩[2] 于喜贞[2] 颜世清[2] 靳峰[2] 

机构地区：[1]新乡医学院,河南省453003 [2]济宁医学院附属医院神经外科暨神经肿瘤学实验室

出　　处：《中华脑科疾病与康复杂志（电子版）》2013年第2期42-46,共5页Chinese Journal of Brain Diseases and Rehabilitation（Electronic Edition）

基　　金：国家自然科学基金项目资助(81071779);山东省中青年科学家科研奖励基金资助(BS2010YY006)

摘　　要：目的探讨替莫唑胺(TMZ)对胶质瘤U251细胞及其干细胞livin及肿瘤坏死因子α(TNF-α)mRNA表达和细胞增殖的影响。方法使用免疫磁珠方法分离、培养胶质瘤U251干细胞,使用不同药物浓度(0、25、50、100、200、400μmol/L)的TMZ在不同时间点(24 h、48 h、72 h)干预U251细胞及其干细胞,RT-PCR技术测定TMZ干预前后livin和TNF-αmRNA表达的变化规律,CCK-8检测TMZ干预后细胞增殖变化趋势。结果 U251干细胞增殖速度明显低于U251细胞。U251干细胞livin及TNF-αmRNA表达量明显高于U251细胞(livin mRNA:t=11.206,P=0.000;TNF-αmRNA:t=16.462,P<0.05),相同时间不同浓度TMZ能抑制U251细胞及其干细胞livin基因的表达,并随着TMZ浓度增加,抑制效应增强(U251细胞:t=-8.899、-19.049、-28.280、-29.651、-32.459,P均<0.05;U251干细胞:t=-15.899、-24.180、-25.652、-43.395、-43,128,P均<0.05);相同干预时间内低浓度的TMZ诱导TNF-α表达增多(U251细胞:t=10.134、14.428、7.873,P均<0.05;U251干细胞:t=4.292,P<0.05),但随着药物浓度增加,诱导作用降低,TNF-α表达量降低(U251细胞:t=-8.958,-22.488,P均<0.05;U251干细胞:t=-11.006、-11.145、-11.596,P=0.000)。400μmol/L TM2干预下,随着干预时间延长,TMZ对U251及其干细胞的中livin基因表达抑制加强(U251细胞:t=-21.406、-25.825、-25.964,P=0.000;U251干细胞:t=-18.238、-21.441、-22.886,P=0.000),而TNF-αmRNA表达抑制作用减弱(U251细胞t=-26.924、-20.677、-14.253,P=0.000,U251干细胞:t=-47.291、-20.204、-18.394,P=0.000),表达量呈现增加趋势。结论 TMZ能降低livin和TNF-αmRNA的表达,启动抗凋亡信号通路,抑制胶质瘤U251细胞及其干细胞增殖;但TMZ降低胶质瘤U251细胞livin和TNF-αmRNA的表达量比胶质瘤干细胞低,表现为对U251细胞的增殖抑制程度远强于U251干细胞,这可能是肿瘤耐药的机制。Objective To analyse the change of expression of livin, TNF-α gene in stem cells isolated from glioblastoma U251 cells and cell proliferation with chemotherapy drug temozolomide （TM2） intervention. Methods The cancer stem cells from glioblastoma U251 cells were isolated by using immune magnetic beads. The cell proliferation rate was detected by CCK-8. The expression of livin, TNF-α and the trend of cell proliferation were detetced by using RT-PCR and CCK-8 after the intervention with different drug concentrations（0,25,50,100,200,400 μmol/L）at different time points（24 h,48 h,72 h）. Results The CD133 ＋ stem cells speed of cell proliferation was significantly lower than U251 cells in the same time and condition of culture. The mRNA expression of [ivin,TNF-α in U251 stem cell were significantly higher than U251 cell（t = 11. 206,P = 0. 000 ; t = 16. 462, P 〈 0. 05 ）. At the same intervention time, the expression of livin in U251 cell and it＇s stem cell was inhibited after the intervention with TMZ, the inhibition effect enhanxed with increasing of drug concentration （ U251 cells ： t = - 8. 899, - 19. 049, - 28.280, - 29. 651, -32. 459,all P 〈0. 05 ;U251 stem eells：t = - 15. 899, -24. 180, -25. 652, -43. 395, -43. 128,all P 〈 0. 05）. At the same intervention time, the mRNA expression of TNF-α was induced increased in low drug concentration （ U251 cells ： t = 10. 134,14. 428,7. 873, all P 〈 0. 05 ; U251 stem cells ： t = 4. 292, P 〈 0. 05 ）, but its expression was decreased with increaseing of drug concentration（U251 cells：t = -8. 958, -22. 488, all P 〈 0. 05 ; U251 stem cells ： t = - 11. 006, - 11. 145, - 11. 596, all P 〈 0. 05 ）. The inhibition of cell expression of livin with TMZ became stronger（ U251 cells ： t = - 21. 406, - 25. 825, - 25.964, P = 0. 000 ; U251 stem cells ： t = - 18. 238, - 21. 441, - 22. 886, P = 0. 000 ） and the inhibition of cell expression of TNF-α turned to weaker（ U251 ceils ： t = - 26. 924, - 20. 677, - 14,253, P = 0.

关 键 词：神经胶质瘤 干细胞 肿瘤坏死因子Α 替莫唑胺 LIVIN 

分 类 号：R739.4[医药卫生—肿瘤]