摘要
目的研究替莫唑胺(TMZ)对胶质瘤干细胞(GSC)错配修复基因hMLH1和hMSH2的影响及其与TMZ耐药的相关性。方法采用悬浮培养法分离培养GSC株。免疫荧光法检测CD133、Nestin和GFAP。细胞毒性实验计算TMZ半数抑制浓度(IC50)。甲基化特异性PCR(MSP)和Western blot分别检测hMLH1和hMSH2基因启动区甲基化和蛋白表达。结果(1)从U251和A172胶质瘤细胞系中成功分离胶质瘤干细胞株U251g和A172g。(2)U251g和A172g的IC50分别为1695.41μmol/L和724.63μmol/L。以IC50、150%IC50两种浓度对U251g和A172g诱导培养1周后对应U251g-1、U251g-2和A172g-1、A172g-2,IC50各为1913txmol/L、2555μmol/L和754μmol/L、1549μmol/L。(3)诱导后的U251g和A172g的hMLHI基因启动区发生异常甲基化和蛋白表达缺失,且A172g的甲基化水平随药物浓度的增加而增高;诱导后的U251g和A172g的hMSH2基因启动区未发生甲基化和蛋白表达缺失。结论GSC存在于U251和A172细胞系中,对TMZ不同程度耐药。TMZ诱导下,胶质瘤干细胞U251g和A172g错配修复系统功出现异常,主要表现为hMLH1基因启动区甲基化和蛋白表达缺失。
Objective To analyze the status of hMLH1 and hMSH2 of the glioma stem cells (GSC) during TMZ chemotherapy and the relationship between them and temozolomide -resistance of GSC. Methods GSC line U251g and A172g were isolated from the glioma cell line U251 and A172 respectively by suspension culture in the neural stem cell culture medium. CD133, Nestin and GFAP of GSC were examined by Immunofluorescence. The half inhibition concentration (IC50) of TMZ for U251g and A172g were calculated through Cytotoxic Test with CCK -8, then the corresponding two kinds of concentration of TMZ, IC50 and 150% IC50 as the condition culture mediums, were used to induce U251g and A172g. At the end, the IC50 of TMZ for U251g and A172g after induction were calculated. Methylation -specific PCR (MSP) and Western blot were used to detect gene promoter methylation and the corresponding protein expressions of hMLH1, hMSH2 of the GSC before and after induction. Results ( 1 ) The GSC line U251g and A172g were successfully isolated from the glioma cell line U251 and A172 respectively by suspension culture in the neural stem cell culture medium, which met the definition of cancer stem cells through the identification. (2) The IC50 of TMZ was 1 695.41 μmol / L for U251g, 724. 63 μmol for A172g, 1 913 tzmol / L for U251g - 1, 2 555 txmol / L for U251g -2 , 754 μmol / L for A172g - 1, 1 549 μmol / L for A172g - 2. ( 3 ) After TMZ induction, hMLH1 gene promoter abnormal methylation and the corresponding protein deletion were detected in U251g and A172g, and methylation level promoted by the increase of drug concentration in A172g. hMSH2 gene promoter was found no methylation and protein expression deletion in U251g and A172g. Conclusions There were GSC in the glioma cell line U251 and A172, which had different levels of temozolomide -resistance. During the process of treatment with TMZ, the function of mismatch repair system was abnormalized in U251g and A172g, mainly hMLH1 gene promoter abnormal methylation and the corresponding protein deletion.
出处
《中华神经外科杂志》
CSCD
北大核心
2013年第11期1101-1105,共5页
Chinese Journal of Neurosurgery
基金
国家自然科学基金资助项目(2060146)