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目的观察人脑胶质瘤U251细胞系中hMLH1、hMSH2基因启动子区的甲基化状态及其在肿瘤发生中的作用。方法采用甲基化特异性聚合酶链反应(MSP)法对人脑胶质瘤U251细胞系的hMLH1、hMSH2基因启动子CpG岛甲基化进行检测;培养人脑胶质瘤U251细胞系,MSP法检测加入5-aza-2’-deoxycytidine前后hMSH2基因在人脑胶质瘤U251细胞中的甲基化状态改变;逆转录-聚合酶链反应(RT-PCR)法检测加入5-aza-2’-deoxycytidine前后hMSH2在人脑胶质瘤U251细胞中的mRNA表达改变。结果人脑胶质瘤U251细胞中未发生hMLH1启动子甲基化,而发生了hMSH2启动子甲基化;5-aza-2’-deoxycytidine处理细胞株后,可逆转hMSH2启动子甲基化,细胞株的mRNA表达增加,加药前后的平均灰度比值分别为(0.40±0.18;0.85±0.32,P<0.01),差异有统计学意义。结论人脑胶质瘤U251细胞系中hMSH2基因启动子CpG岛高甲基化,5- aza-2’-deoxycytidine能完全逆转hMSH2基因高甲基化状态,可为临床诊断人脑胶质瘤提供新的检测指标和治疗靶点。
Objective To investigate the methylation status of hMLH1 and hMSH2 gene promoters in human glioma U251 cell line and its role in tumorigenesis. Methods Methylation-specific polymerase chain reaction (MSP) was used to detect the methylation of CpG island of hMLH1 and hMSH2 gene promoter in human glioma U251 cell line. The human glioma U251 cell line, MSP Methylation status of hMSH2 gene in human glioma U251 cells before and after 5-aza-2’-deoxycytidine was assayed. The expression of 5-aza-2 was detected by reverse transcription-polymerase chain reaction (RT-PCR) The mRNA expression of hMSH2 in human glioma U251 cells before and after ’-deoxycytidine change. Results hMLH1 promoter methylation did not occur in human glioma U251 cells, but hMSH2 promoter methylation occurred. After 5-aza-2’-deoxycytidine treatment, hMSH2 promoter methylation could be reversed, The mRNA expression of cell lines increased, the average gray value before and after dosing was (0.40 ± 0.18; 0.85 ± 0.32, P <0.01), the difference was statistically significant. Conclusions The hMSH2 promoter CpG islands in human glioma U251 cell line are hypermethylated. 5-aza-2’-deoxycytidine can completely reverse the hypermethylation status of hMSH2 gene, which may provide a new detection index for clinical diagnosis of human gliomas And treatment targets.