中华神经外科杂志
中華神經外科雜誌
중화신경외과잡지
Chinese Journal of Neurosurgery
2013年
11期
1101-1105
,共5页
李孟凯%孙彦辉%张亚楠%历俊华%李杰飞%黄铂渊%张红波
李孟凱%孫彥輝%張亞楠%歷俊華%李傑飛%黃鉑淵%張紅波
리맹개%손언휘%장아남%력준화%리걸비%황박연%장홍파
胶质瘤干细胞%错配修复基因%替莫唑胺
膠質瘤榦細胞%錯配脩複基因%替莫唑胺
효질류간세포%착배수복기인%체막서알
Glioma stem cells%Mismatch repair gene%Temozolomide
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1 695.41 μmol/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1 913 μmol/L、2 555 μmol/L和754 μmol/L、1 549 μmol/L.(3)诱导后的U251g和A172g的hMLH1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.
目的 研究替莫唑胺(TMZ)對膠質瘤榦細胞(GSC)錯配脩複基因hMLH1和hMSH2的影響及其與TMZ耐藥的相關性.方法 採用懸浮培養法分離培養GSC株.免疫熒光法檢測CD133、Nestin和GFAP.細胞毒性實驗計算TMZ半數抑製濃度(IC50).甲基化特異性PCR(MSP)和Western blot分彆檢測hMLH1和hMSH2基因啟動區甲基化和蛋白錶達.結果 (1)從U251和A172膠質瘤細胞繫中成功分離膠質瘤榦細胞株U251g和A172g.(2)U251g和A172g的IC50分彆為1 695.41 μmol/L和724.63μmol/L.以IC50、150％IC50兩種濃度對U251g和A172g誘導培養1週後對應U251g-1、U251g-2和A172g-1、A172g-2,IC50各為1 913 μmol/L、2 555 μmol/L和754 μmol/L、1 549 μmol/L.(3)誘導後的U251g和A172g的hMLH1基因啟動區髮生異常甲基化和蛋白錶達缺失,且A172g的甲基化水平隨藥物濃度的增加而增高;誘導後的U251g和A172g的hMSH2基因啟動區未髮生甲基化和蛋白錶達缺失.結論 GSC存在于U251和A172細胞繫中,對TMZ不同程度耐藥.TMZ誘導下,膠質瘤榦細胞U251g和A172g錯配脩複繫統功齣現異常,主要錶現為hMLH1基因啟動區甲基化和蛋白錶達缺失.
목적 연구체막서알(TMZ)대효질류간세포(GSC)착배수복기인hMLH1화hMSH2적영향급기여TMZ내약적상관성.방법 채용현부배양법분리배양GSC주.면역형광법검측CD133、Nestin화GFAP.세포독성실험계산TMZ반수억제농도(IC50).갑기화특이성PCR(MSP)화Western blot분별검측hMLH1화hMSH2기인계동구갑기화화단백표체.결과 (1)종U251화A172효질류세포계중성공분리효질류간세포주U251g화A172g.(2)U251g화A172g적IC50분별위1 695.41 μmol/L화724.63μmol/L.이IC50、150％IC50량충농도대U251g화A172g유도배양1주후대응U251g-1、U251g-2화A172g-1、A172g-2,IC50각위1 913 μmol/L、2 555 μmol/L화754 μmol/L、1 549 μmol/L.(3)유도후적U251g화A172g적hMLH1기인계동구발생이상갑기화화단백표체결실,차A172g적갑기화수평수약물농도적증가이증고;유도후적U251g화A172g적hMSH2기인계동구미발생갑기화화단백표체결실.결론 GSC존재우U251화A172세포계중,대TMZ불동정도내약.TMZ유도하,효질류간세포U251g화A172g착배수복계통공출현이상,주요표현위hMLH1기인계동구갑기화화단백표체결실.
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 U251 g 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 μmol / L for U251g-1,2 555 μmol / 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.
