人脑胶质瘤干细胞标志物CD133表观遗传调控的分子机制及其与临床预后相关性的研究

It's unclear about the regulatory mechanism of CD133 expression in gliomas nowdays, the correlation between immunohistochemical expression of CD133 with clinical prognosis is also controversy. To research the epigenetic regulatory mechanism of CD133 expression, evaluate the correlation between the CD133 promoter methylation status, CD133 expression, tumorigenesis and outcome of glioma patients, search for a novel type of prognostic and predictive markers in gliomas. We plan to: 1. Obtain The glioma specimens from patients for isolation and culture of primary cells, CD133+ and CD133- cells were sorted by flow cytometry. 2. Determine the correlation between the promoter methylation of CD133 and expression. For demethylation studies, cultured cells were treated with DAC, then to determine the varieties of CD133 expression. Analyze The oncogenic ability of gliomas with promoter hypomethylation and hypermethylation of CD133. Analyze The chemotherapy drug(TMZ) resistance of gliomas with promoter hypomethylation and hypermethylation of CD133. 3. For histone acetylation studies, cultured cells were treated with TSA or combined with DAC, then determine the varieties of CD133 experssion. 4. Evaluate the correlation between patients’ outcome and CD133 methylation status or CD133 expression levels. We expected that CD133 promoter methylation status and expression in glioma are correlated with patient's survival and chemotherapy resistance, the level of CD133 protein expression assessed by immunohistochemical staining and/or mRNA are determined by the CD133 promoter methylation status and histone deacetylation, CD133 promoter methylaiton status might be promising tools for diagnostic purposes.

人脑胶质瘤中CD133表达的调节机制尚不清楚，其与患者预后相关性也存在争议。为研究胶质瘤CD133的表观调控机制，阐明CD133表达、调控与胶质瘤发生及预后的关系，探寻脑胶质瘤诊断/预后判断的标志物，我们通过：1.收集脑胶质瘤新鲜样本，分离培养原代细胞球，流式分选CD133+与CD133-细胞；2.分析CD133表达与CD133启动子甲基化的关系，去甲基化药物处理后检测表达差异，将CD133启动子低甲基化和高甲基化细胞作致瘤性分析，化疗药替莫唑胺对两类细胞的增殖抑制及生存力分析；3.组蛋白去乙酰化酶抑制剂单独及联合去甲基化药物处理后检测CD133表达差异；4.CD133启动子甲基化、表达与胶质瘤预后相关性分析。我们预计胶质瘤中CD133表达受CD133启动子甲基化和组蛋白去乙酰化调控，CD133启动子甲基化状态、蛋白表达与患者预后及耐药性相关，CD133启动子甲基化可成为胶质瘤预后标志物。

人脑胶质瘤CD133蛋白表达的调控机制尚未阐明，其与患者预后存在一定相关性。为研究胶质瘤CD133的表观调控机制，阐明CD133表达、调控与胶质瘤发生及预后的关系，筛查脑胶质瘤预后分析的标志物，本课题通过：1.收集脑胶质瘤新鲜样本，分离培养原代细胞球，流式分选CD133+与CD133-细胞；2.分析CD133表达与CD133启动子甲基化的关系，去甲基化药物处理后检测表达差异；3.组蛋白去乙酰化酶抑制剂单独及联合去甲基化药物处理后检测CD133表达差异；4.CD133启动子甲基化、表达与胶质瘤预后相关性分析。我们成功的建立了胶质瘤肿瘤干细胞分离、培养、扩增体系，肿瘤干细胞具有很好的均一性，并成功培养出了高比例的CD133阳性细胞，比例在50%左右。去甲基化药物5-氮杂胞苷处理肿瘤干细胞之后，CD133的表达在mRNA水平以及蛋白水平都得到了一定的增高。而在曲古抑菌素A处理肿瘤干细胞之后，CD133的表达在mRNA水平以及蛋白水平都得到了一定的增高。多因素分析发现CD133启动子甲基化状态是独立于肿瘤分级、手术切除范围和患者年龄之外的影响患者预后的重要因素，CD133启动子甲基化与患者无进展生存期和总体生存期延长相关。CD133免疫组化结果显示，CD133的表达水平在胶质瘤样本中存在很大变异。分析结果显示，经免疫组化染色法测得的CD133蛋白表达水平和患者预后之间没有相关性（P>0.05），且与CD133启动子甲基化状态之间也不存在相关性（Kw =-0.165）。CD133启动子甲基化与患者无进展生存期和总体生存期延长相关，免疫组化法测得的CD133蛋白表达水平和患者预后及CD133启动子甲基化状态之间缺乏相关性，CD133启动子甲基化状态有望成为脑胶质瘤预后判断的分子标志物，针对CD133的表观遗传修饰，可以调控下游的CD133蛋白表达，且与胶质瘤患者预后可能存在相关性，因此下一步有望通过类似药物实验来控制胶质瘤的发生发展，为脑胶质瘤的治疗提供新的个体化的途径。