胶质瘤干细胞样细胞中MGMT表达以及与替莫唑胺的耐药关系研究

MGMT Expression and Temozolomide Resistance in Glioma Stem-like Cells

背景与目的:O6甲基鸟嘌呤DNA甲基转移酶(O6-methylguanine DNA methyltranferase,MGMT)是一种能将鸟嘌呤DNA第六位氧氧原子上的甲基加合物移除和修复损伤DNA的酶,临床上能影响甲基化类化疗药物的疗效。胶质瘤干细胞样细胞被认为是胶质瘤复发的根源之一。本研究旨在探讨MGMT在胶质瘤干细胞样细胞中的表达以及与替莫唑胺耐药的关系。方法:采用悬浮克隆球形成法自胶质瘤细胞株U251、SKMG-4、SF295、SKMG-1、U373、U87、MGR1和MGR2中富集胶质瘤干细胞样细胞。应用免疫荧光技术检测胶质瘤干细胞样细胞相关分子标志;裸鼠移植瘤试验检测胶质瘤干细胞样细胞的成瘤能力。RT-PCR和Western blot检测胶质瘤干细胞样细胞中MGMT的表达;甲基化特异性PCR分析胶质瘤干细胞样细胞MGMT启动子甲基化状况;CCK-8法检测不同浓度替莫唑胺对胶质瘤干细胞样细胞和胶质瘤细胞增殖的作用。结果:分别自8个胶质瘤细胞株中成功富集胶质瘤干细胞样细胞:U251G、SKMG-4G、SF295G、SKMG-1G、U373G、U87G、MGR1G和MGR2G。胶质瘤干细胞样细胞高表达CD133、Nestin和Sox-2等干细胞标志,而且低表达GFAP和TUJ1。胶质瘤干细胞样细胞均能在裸鼠移植成瘤。MGMT在8株胶质瘤细胞及U87G、MGR1G和MGR2G中为阴性,而在U251G、SKMG-4G、SF295G、SKMG-1G和U373G中为阳性表达。替莫唑胺对胶质瘤干细胞样细胞和胶质瘤细胞的抑制作用差异具有显著性。胶质瘤干细胞样细胞与胶质瘤亲代细胞相比更加耐药(P<0.05)。另外,替莫唑胺对MGMT阳性及MGMT阴性胶质瘤干细胞样细胞IC50间的差异无统计学意义(P>0.05)。结论:MGMT阴性表达的胶质瘤细胞经干细胞样培养后,MGMT表达可转为阳性;胶质瘤干细胞样细胞较胶质瘤亲代细胞更耐受替莫唑胺;MGMT的表达与胶质瘤干细胞样细胞对替莫唑胺的耐受之间无明显关联,提示胶质瘤干细胞样细胞对替莫唑胺的耐药可能还有MGMT以外的机制参与。

BACKGROUND OBJECTIVE： O6-methylguanine DNA methyltranferase（MGMT） can remove the DNA alkylating adduct and repair the damaged DNA.MGMT is responsible for the chemoresistance to alkylating agents in gliomas.In addition,glioma stem-like cells（GSCs） has been demonstrated to be involved in the recurrence and treatment resistance in gliomas.In this study,we was aimed to investigate MGMT expression and temozolomide（TMZ） resistance in GSCs.METHODS： GSCs were enriched from glioma cell line U251,SKMG-4,SF295,SKMG-1,U373,U87,MGR1 and MGR2 through serum-free clone culture.Immunofluorescence staining and xenograft models were applied to characterize cancer stem cell features of glioma stem-like cells.RT-PCR and Western blot were used to detect the MGMT expression.Methylation-specific PCR（MSP） was applied to detect the status of MGMT promoter.CCK-8 assay was employed to test the growth inhibition effect of TMZ in both GSCs and parental glioma cell lines.RESULTS： GSCs were successfully enriched from 8 parental glioma cell lines： U251G,SKMG-4G,SF295G,SKMG-1G,U373G,U87G,MGR1G,and MGR2G.GSCs showed high immunoreactivity for neural stem cell markers CD133,Nestin and Sox-2,but low for markers of differentiated lineages such as GFAP and TuJ1.GSCs successfully generated xenograft tumors in nude mice.All the parental glioma cell lines and U87G,MGR1G and MGR2G were MGMT-negative while U251G,SKMG-4G,SF295G,SKMG-1G and U373G were positive for MGMT.GSCs were more resistance to TMZ compared to parental glioma cell lines（P 0.05）.However,there was no significant differences of TMZ IC50 between MGMT-positive and-negative GSCs（P 0.05）.CONCLUSONS： MGMT-negative glioma cell lines may convert to MGMT-positive GSCs through serum-free clone culture.GSCs are more resistant to TMZ,compared to parental glioma cell lines.However,there are no significant differences of TMZ resistance between MGMT-positive and-negative GSCs,suggesting that mechanisms other than MGMT are involved in TMZ.