B56γ调控TCRζ介导CML 病人T细胞免疫缺陷及其分子机制研究

Down-regulated expression of TCRζ is the most common cause of T cell immunodeficiency in chronic myelogenous leukemia (CML), Targeting the related regulatory molecules in TCRζ signaling pathway could restore the host T cells immune function, however, the regulation mechanism of TCRζ is still unclear. Recently, we found that the down expression of TCRζ might be associated with the abnormal high expression of B56γ which belongs to the regulatory subunit of protein phosphatase 2A. In this study, the expression of B56γ in normal primary T cells will be up- and down- regulated using transgenic technology and siRNA technology , respectively, to confirm the role of B56γ in regulation of TCRζ gene expression, and its molecular mechanism will be characterized through determining the expression and phosphorylation of B56γ and its downstream regulatory proteins, as well as the features of gene expression profiles by different molecular techniques such as Western blot and microarray and so on. Moreover, the expression of B56γ in T cells from CML patients will be down-regulated using siRNA technology, and the ability of T cell proliferation, apoptosis and activation will be detected to determine whether down-regulation expression of B56γ can improve T cell immunue function, and to explore whether B56γ is a potential candidate target molecule for CML immunotherapy. These results will provide more comprehensive information of molecular mechanism of T cell immunity and a novel evidence of TCRζ-based immune therapeutic strategies in CML.

TCRζ表达下调是引起慢性粒细胞白血病（CML）T细胞免疫缺陷的主要原因之一，靶向调控TCRζ及其相关分子可恢复T细胞免疫功能，但TCRζ的调控机制仍不甚明确。近期我们发现CML中TCRζ表达下调可能与PP2A调节亚基的B56γ异常增高有关。本研究拟利用转基因和siRNA技术调控正常原代T细胞中B56γ的表达，通过Western blot等方法检测B56γ及其下游调控蛋白的表达和磷酸化情况，通过基因芯片等分析T细胞基因表达谱变化特点，明确 B56γ是否参与T细胞中TCRζ表达调控及其相关分子机制；进一步利用siRNA技术下调CML患者T细胞的B56γ表达，通过检测T细胞的增殖、凋亡和活化情况，明确下调B56γ能否改善T细胞的免疫功能，探讨B56γ能否成为CML免疫治疗的靶分子，为CML病人T细胞免疫状况提供更全面的资料和基于TCRζ的免疫治疗策略提供新的依据。

本研究主要目的利用siRNA沉默和转基因上调T细胞中PP2A调节亚基的B56γ蛋白的表达，分析其相关信号通路分子表达变化，明确B56γ蛋白是否参与CML 病人T细胞TCRζ表达的调控及其相关分子机制。本研究首先利用基因芯片分析了3例初诊未治疗的CML患者T细胞及其上调TCRζ后T细胞的全基因组表达谱变化情况，发现在CML患者T细胞TCRζ基因的表达明显低于正常健康人，而B56γ基因明显高于正常健康人。随后收集30例初诊未治疗的CML患者T细胞进行RQ-PCR验证，结果与基因芯片相一致，并且B56γ基因与TCRζ基因表达呈负相关，提示B56γ可能参与CML患者T细胞TCRζ基因表达的调控；进一步利用siRNA下调B56γ基因表达，下调后Jurkart T细胞TCRζ和Elf-1的RNA和蛋白水平明显上调，而CREMα的RNA和蛋白水平则下调，IL-2的分泌水平也明显增加，结果证实B56γ参与TCRζ表达调控；最后利用转基因分别上调B56γ1和B56γ2两种转录本，上调后Jurkart T细胞只能在B56γ1组中观察到CREMα呈明显高表达，揭示B56γ1是参与TCRζ表达调控的主要转录本，它是通过PP2A/SP-1/CREMα/TCRζ途径来介导TCRζ基因转录抑制。本研究针对CML患者T细胞研究的同时，还利用我们所建立的方法在AML患者T细胞中实现TCRζ基因转导和上调T细胞功能的作用。总之，本研究率先在国内外发现在CML患者T细胞中B56γ基因异常高表达现象，并证实B56γ1参与到TCRζ基因表达的负调控，提示B56γ1可能成为CML免疫治疗的靶分子，为CML病人T细胞免疫状况提供更全面的资料和基于TCRζ的免疫治疗策略提供新的依据。本课题经过3年实施，已经完成了研究任务，部分前期的研究成果已经发表SCI论文2篇，学位论文2本，本项目同时协助培养博士和硕士研究生各一名。主要研究成果尚在总结和撰写论文。