摘要
目的检测视网膜母细胞瘤(RB)中,O6-甲基鸟嘌呤DNA甲基转移酶(MGMT)基因启动子区甲基化状态及mRNA、蛋白产物的表达情况,并探讨其与RB临床、组织病理学特征的可能关系。设计实验研究。研究对象石蜡包埋RB组织20例,RB细胞系4株(Y79、SO-Rb50、SO-Rb50/VCR、WERI-RB1)。方法采用甲基化特异性聚合酶链式反应(MSP)检测20例石蜡包埋RB组织及4株RB细胞系MGMT启动子区甲基化状态,并进一步以实时荧光定量PCR及蛋白印迹确认MGMT mRNA、蛋白的表达。收集患者临床资料(如性别、发病年龄等),并对RB组织标本HE、免疫组织化学染色切片进行组织病理学诊断,判断组织病理学特征(如肿瘤侵犯范围、虹膜新生血管等)。主要指标MGMT启动子区甲基化状态,MGMT mRNA及蛋白表达水平,患者临床及组织病理学特征。结果 20例石蜡包埋RB组织标本中,12例(60%)MGMT启动子区为部分/完全甲基化,余8例(40%)未甲基化,甲基化与未甲基化组的临床及组织病理学指标不具有统计学差异(P>0.05)。Y79、SO-Rb50、SO-Rb50/VCR 3株RB细胞系MGMT启动子区为部分甲基化;WERI-RB1细胞系MGMT启动子区为未甲基化。4株RB细胞系均有不同程度MGMT mRNA及蛋白的表达,WERI-RB1 MGMT mRNA表达水平(1.000±0.040)高于其他3株细胞系(Y79,0.617±0.026;SO-Rb50,0.356±0.020;SO-Rb50/VCR,0.389±0.017),差异具有统计学意义(P<0.05),WERI-RB1 MGMT蛋白表达水平(1.506±0.493)略高于其他3株细胞系(Y79,1.388±0.304;SO-Rb50,1.495±0.212;SO-Rb50/VCR,1.406±0.547),差异无统计学意义(P>0.05)。结论在RB组织及细胞系中,存在较高的MGMT启动子区甲基化率,MGMT甲基化状态可影响其产物表达水平。
Objective To detect the methylation status of the promoter of O6 methylguanine DNA meghyltransferase（MGMT） gene and the expression level of MGMT mRNA and protein.Design Experimental Study.Participants 20 paraffin embedded RB tissues,4 RB cell lines（Y79,SO-Rb50,SO-Rb50/VCR,WERI-RB1）.Methods A series of 20 paraffin embedded RB tissue samples and 4 RB cell line were subjected to mehtylation-specific PCR（MSP） analysis to evaluate the methylation status of the MGMT promoter.Further,the expression of MGMT mRNA and protein were studied by Realtime PCR and Western Blot.The clinical data（e.g.gender,age of onset） and pathological features（e.g.the realm of tumor encroachment,neovascularization in iris,NVI） were also collected.Main Outcome Measures Methylation status of the MGMT promoter,mRNA and protein expression level of MGMT,data of clinical and pathological features.Results Among the 20 paraffin embedded RB tissue samples,12 cases（60%） were partially/completely methylated,the remaining 8 cases（40%） showed MGMT promoter unmethylated.As to the 4 RB cell lines,Y79,SO-Rb50,SO-Rb50/VCR were partially methylated,while WERI-RB1 was unmethylated.MGMT mRNA and protein were expressed in all 4 RB cell lines.The mRNA expression level of WERI-RB1（1.000±0.040）was higher than the other 3 cell lines（Y79,0.617±0.026;SO-Rb50,0.356±0.020;SO-Rb50/VCR,0.389±0.017）,the differences among which were statistically significant（P0.05）.The protein expression of WERI-RB1（1.506±0.493） was slightly higher than the other 3 cells lines（Y79,1.388±0.304;SO-Rb50,1.495±0.212;SO-Rb50/VCR,1.406±0.547）,the differences among which were not statistically significant（P0.05）.Conclusions There was a comparatively high MGMT promoter methylation rate in RB pathologic samples and cell lines,which had a corresponding effect to the expression level of MGMT mRNA and pro-tein.
出处
《眼科》
CAS
2012年第2期121-126,共6页
Ophthalmology in China
基金
国家自然科学基金项目(81172393)
