人端粒酶逆转录酶(hTERT)介导MDSC趋化发挥免疫抑制作用的分子机制研究

There is a strong correlation between the accelerated tumor growth and malignant progression and the increase of MDSC in the microenvironment of tumor. The literature and our own studies have indicated that the quantity of MDSC increased with the expression of hTERT in tumor. However, no clear mechanism is known. Our preliminary experiment revealed that hTERT seems to transcribe CCL2.Based on literature and software predictions,we thus speculate that hTERT might combine with β-catenin as an assistant transcribing factor, which help it to enter into nuclei. Via the Wnt signal pathway, the expression of CCL2 may be controlled by intranuclear transcribing factor, leading eventually to the immunological suppression effect by MDSC. We plan to use human colon cancer cell SW480 infected with hTERT overexpressed plasmid introduced by lentivirus,and confirm the molecular mechanism of the CCL2 expression and chemotaxis of MDSC controlled by hTERT, with double fluorescence enzyme assay,flowcyte test and other experiments both in vitro and in vivo. Furthermore, we will attempt to treat tumors in HHD transgenetic mouse with hTERT vaccine. These experiments might elucidate the immunological supressing mechanism and provide the foundational understanding and optimization of hTERT based immunotherapy.

肿瘤微环境中髓源性抑制细胞(MDSC)的聚集增多与肿瘤生长增快、恶性增强密切相关。文献及我们研究均提示：随着肿瘤中人端粒酶逆转录酶(hTERT)表达增高，MDSC数量也随之增加，但机制不清。预实验中我们发现hTERT可转录调控单核细胞趋化蛋白-1(CCL2),结合文献及软件预测,推测hTERT可作为一种辅助转录因子结合β-catenin并促其入核,通过Wnt信号通路的核转录因子来调控CCL2的表达,最终趋化MDSC至癌巢发挥免疫抑制作用。本课题拟采用慢病毒介导的hTERT过表达质粒转染人结肠癌细胞SW480，再通过收集肿瘤标本、建立荷瘤模型，经双荧光素酶、流式等实验证实hTERT调控CCL2表达趋化MDSC的分子机制，进一步行HHD转基因小鼠皮下成瘤，给予hTERT疫苗等治疗，阐明hTERT介导的免疫抑制机制,为优化基于hTERT的免疫治疗提供理论依据。

背景与目的：结直肠癌是主要的致死性疾病之一，其发病机制涉及多种分子机制。人端粒酶逆转录酶（hTERT）是促进结直肠癌发展的关键酶，但其调控机制尚不清楚。趋化因子（C-C基序）配体2（CCL2）在结直肠癌发病机制中具有重要作用，但其上游调控机仍然不明。本研究旨在发现hTERT与CCL2之间的相互作用。方法：采用免疫组化检测hTERT和CCL2在结直肠癌组织及癌旁组织中的表达关系。用慢病毒或质粒调节HCT116细胞中hTERT和CCL2的表达。用CCK8和Transwell研究hTERT和CCL2在细胞生长和迁移中的作用。用荧光素酶报告基因、免疫荧光和ChIP检测hTERT与CCL2的调节作用。用COIP检测hTERT促进β-catenin/TCF-4复合物形成的作用。结果：结直肠癌组织中hTERT和CCL2的表达均明显高于癌旁组织。此外，在hTERT和CCL2表达水平较高的肿瘤区域可观察到MDSCs。体外功能检测显示hTERT通过增加CCL2表达显著促进HCT116细胞的迁移和侵袭能力。过表达hTERT可以促进β-catenin的核转位，并与下游效应分子TCF-4形成复合物，最终激活CCL2的启动子。结论：本研究发现了hTERT对CCL2的调控作用，并阐明了其具体机制，为结直肠癌的发病机制提供了新的认识。