表观遗传学修饰Mage-D4增强特异性CTL杀伤胶质瘤细胞的作用研究

Our previous study found that Mage-D4 expressed inglioma with heterogenesis.Therefore, killing ability of specific CTL for glioma cells would be affected. Based on the fact that Mage-D4 gen promoter enriched CpGsites we speculate that Mage-D4 expression may involve in epigenetic regulation. Therefore, following contents will be studied: (1) epigenetic regulation forMage-D4expression.The methylated status of Mage-D4 will be tested by BSP and Pyrosequence, and its clinical significance will be analyzed. Mage-D4 reporter gen plasmid was transfected into glioma cells after treatment with DAC and VPA. The activity of Mage-D4 promoter reportergene plasmid will be tested. That binding Mage-D4 promoter with transcription factor SP1, methylated and acetylated histone was analyzed by ChIP-PCR. (2) Treatment of DAC and VPA affects the reacting of killing tumor cells by Mage-D4 specific cytotoxic T lymphoycyte (CTL). With DC transfected with Mage-D4 expression vector or and incubated with recombinant protein specific, CTL was induced in vitro. Increasing the Mage-D4 and HLA-I expression with treatment of DAC and VPA was performed. The killing ability of Mage-D4 specific CTL was observed in vitro as well as in vivo. This project will interpret the regulating mechanism of Mage-D4 expression from epigenetic aspect and provide experimental data for treatment of glioma by epigenetic immunotherapy.

我们的研究发现，胶质瘤呈异质性表达Mage-D4,这将影响CTL的杀瘤作用，DNA去甲基化药物DAC能上调Mage-D4表达，因此推测表观遗传学修饰Mage-D4可增强CTL杀伤力。拟开展以下研究:⑴表观遗传学对Mage-D4表达调控作用:用BSP和焦磷酸测序分析胶质瘤Mage-D4甲基化与其表达和临床指标的关系;甲基化处理Mage-D4启动子报告基因载体并转染细胞,观察表观遗传学药物DAC和VPA对启动子的活化;ChIP-PCR检测启动子与转录因子Sp1、甲基化和乙酰化组蛋白的结合;⑵DAC和VPA对Mage-D4特异性CTL杀瘤效应的影响：用Mage-D4载体转染或重组蛋白孵育DC,诱导特异性CTL；从细胞、动物水平观察CTL对DAC和VPA诱导后对瘤细胞的杀伤作用。本项目从表观遗传学角度阐明Mage-D4表达调控机制,并为以Mage-D4为靶标的胶质瘤表观遗传学免疫治疗提供实验依据。

本项目研究发现：①表观遗传学调控方面： Mage-D4核心启动子区域为-358~+172 bp；在胶质瘤组织中，Mage-D4核心启动子的多个CpG位点的甲基化及总甲基化水平较低，甲基化与Mage-D4表达呈负相关；进一步将Mage-D4核心启动子报告基因载体进行甲基化处理，发现Mage-D4启动子活性下降，提示Mage-D4表达受甲基化调控。随之，用siRNA/基因转入和ChIP-PCR技术证实了转录因子Sp1能与Mage-D4启动子结合并促进Mage-D4的转录，且去甲基化和Sp1能协同提高Mage-D4的转录活性。② 特异性CTL杀瘤效应方面：表观遗传学药物（DAC、VPA和TSA）可单独或联合诱导SF763中Mage-D4 mRNA表达升高，但对SF126的诱导作用不明显，提示同一肿瘤的不同个体对观遗传学药物的反应存在差异；用细胞因子联合诱导HLA-A2+人外周血单核细胞为DC，Mage-D4重组蛋白致敏DC后，可诱导同体CD8+ T 淋巴细胞产生的特异性CTL，在体外可特异性杀伤Mage-D4阳性的胶质瘤细胞，且这种杀伤作用具有HLA-A2+限制性。③血清Mage-D4蛋白和抗体意义方面：胶质瘤患者血清Mage-D4蛋白和抗体含量均高于正常人；胶质瘤Mage-D4蛋白和抗体阳性率分别为27.71%（25/92）和42.4%（39/92），而所检测的正常人血清Mage-D4蛋白和抗体均为阴性。进一步ROC分析显示Mage-D4蛋白和抗体用于区分胶质瘤与正常人的ROC曲线下的面积(AUC)分别为0.744和0.843，将两者合并则AUC提高到0.910。上述结果提示：Mage-D4的表达与甲基化调控有关，将Mage-D4应用于胶质瘤免疫治疗和血清标记物诊断，具有潜在的开发前景。