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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 mRNA of O6-methylguanine DNA methyltransferase (MGMT) gene and the expression of mRNA and protein in retinoblastoma (RB) Possible relationship of pathological features. Design experiment research. Participants were paraffin-embedded RB tissue 20 cases, RB cell line 4 (Y79, SO-Rb50, SO-Rb50 / VCR, WERI-RB1). Methods Methylation-specific polymerase chain reaction (MSP) was used to detect the methylation status of MGMT promoter region in 20 paraffin-embedded RB tissues and 4 RB cell lines, and further confirmed by real-time fluorescence quantitative PCR and Western blotting. mRNA, protein expression. The clinical data (such as gender, age at onset) of the patients were collected, and histopathological diagnosis of histological specimens of HE and immunohistochemical staining of RB specimens was performed to determine the histopathological features (such as the extent of tumor invasion and iris neovascularization). The main indicators MGMT promoter methylation status, MGMT mRNA and protein expression, clinical and histopathological features. Results In 20 paraffin-embedded RB specimens, 12 (60%) MGMT promoter regions were partially / completely methylated and the remaining 8 (40%) were unmethylated, methylated and unmethylated The clinical and histopathological parameters were not statistically different (P> 0.05). The MGMT promoter region of three RB cell lines of Y79, SO-Rb50 and SO-Rb50 / VCR were partially methylated. The promoter region of MGMT in WERI-RB1 cell line was unmethylated. The expression of MGMT mRNA and protein in all four RB cell lines was significantly different from that in the other three cell lines (Y79,0.617 ± 0.026; SO-Rb50,0.356 ± 0.020; (1.506 ± 0.493) was slightly higher than that of the other three cell lines (Y79, 1.388 ± 0.304, SO-Rb50 / VCR, 0.389 ± 0.017), and the difference was statistically significant Rb50, 1.495 ± 0.212; SO-Rb50 / VCR, 1.406 ± 0.547), the difference was not statistically significant (P> 0.05). Conclusion There is a high methylation rate of MGMT promoter in RB tissues and cell lines. The MGMT methylation status may affect the expression level of MGMT promoter.