调控FGL1-LAG-3通路协同LncRNA-Tim3增强CD8+T细胞抑制HCC生长的研究

Hepatocellular carcinoma (HCC) is one of the most common and malignant tumors. Chronic HBV infection is considered to be an important risk factor for the development of HCC. Immunotherapy of CD8+ T lymphocytes is considered to be one of the most effective strategies for HCC treatment.At present, there are many researches on the mechanism of HCC evading CD8+ T cell immune clearance, but they are only the tip of the iceberg. The mechanism of HCC immune escape is not comprehensive. The applicant's previous study found that T cell immunoglobulin mucin 3 (Tim-3), a member of the immunological checkpoint protein, acts as an inhibitor of T cells and its ligand Lnc-Tim3 induces CD8+ T lymphocyte death, which leads to malignant proliferation of HCC cells. At the same time, in December 2018, the internationally renowned immunologist, Professor Lie Ping Chen, first demonstrated in the Cell magazine that FGL1 (fibrinogen-like protein 1) is the major inhibitory partner of the immunological checkpoint LAG-3 (lymphocyte activation gene 3). The combination of the two can significantly inhibit the activity of CD8+ T cells in T lymphocytes and cause immune escape of tumor cells. The discovery of the FGL1-LAG-3 pathway has provided new ideas for our research. Therefore, combined with our research foundation and the latest research, we will construct the LAG-3-FGL1 immune escape pathway and Lnc-Tim3-Tim- 3 The double inhibition model of the immune escape pathway was divided into four experimental groups to observe the activity of CD8+ T cells and the growth of HCC cells, respectively, and to study the effect of FGL1-LAG-3 single pathway inhibition on enhancing CD8+ T cell activity. It can also explore the synergistic effect of dual-channel inhibition, provide a new theoretical basis for HCC immunotherapy research, and provide new ideas for clinical intervention and treatment.

肝细胞癌是常见的恶性肿瘤，CD8+T细胞的免疫疗法是HCC治疗有效的策略之一。目前在HCC逃避CD8+T细胞免疫清除的机制上的研究尚不全面。申请人前期研究发现T细胞免疫球蛋白粘蛋白3，是免疫检查点蛋白的成员，作为T细胞的抑制受体与其配体LncRNA-Tim3的相互作用诱导CD8+T淋巴细胞死亡，从而导致HCC细胞恶性增殖。近期陈列平教授也在Cell杂志上首次证明了纤维蛋白原样蛋白1是免疫检查点淋巴细胞活化基因3的主要抑制性配体，两者的结合可以显著抑制CD8+T细胞的活性。因此，本课题将构建LAG-3-FGL1通路和 Lnc-Tim3-Tim-3通路的双抑制模型，分成四个实验组，分别观察CD8+T细胞的活性和HCC细胞生长变化，既能研究FGL1-LAG-3单通路抑制对增强CD8+T细胞活性的作用又能探索双通路抑制的协同作用，为HCC的免疫治疗研究提供新的理论依据，为临床干预和治疗提供新思路。

CD8+T淋巴细胞的失活是肝细胞癌免疫逃逸实现恶性增殖的重要机制，人淋巴细胞活化基因-3（LAG-3）作为活化T细胞的负调节因子在肝癌的免疫逃逸中发挥作用，而FGL1是LAG-3的主要抑制性配体作用于CD8+T细胞，促进肿瘤免疫逃逸。此外，LncRNA-Tim3与其配体Tim-3结合诱导HCC中Bat3的核转位也能加剧CD8+T细胞死亡。当前，多个免疫逃逸通路的联合调控协同增强CD8+T细胞清除肿瘤细胞的活性是肿瘤免疫治疗的趋势，本课题我们结合已有研究基础和最新发现，着眼于探索LncRNA-Tim3和FGL1-LAG-3联合作用于增强CD8+T细胞遏制HCC肿瘤的生长活性的协同作用。本课题的主要研究内容包括以下几个方面：检测正常肝脏细胞和HCC细胞中FGL1表达水平的差异；单一敲除LAG-3和单一敲除FGL1状态下的HCC小鼠的免疫功能和肿瘤生长状态的变化比较；联合抑制LAG-3-FGL1和LncRNA-Tim3-Tim-3两条信号通路协同增强CD8+T细胞遏制HCC肿瘤的生长的效用表达。研究结果显示，联合靶点通路抑制的HepG2小鼠在包括肿瘤生长时间窗、生长速度和生长加速度的多维度肿瘤生长梯度曲线显著低于单一靶点通路抑制组小鼠。同时，无论是联合抑制组还是单抑制组，LAG-3有效抑制后，CD8+T、CD4+T细胞亚群表达活性显著高于对照组，而Treg细胞亚群表达、肿瘤细胞恶性增殖活性度及肿瘤生长情况均显著低于对照组。此外，无论是联合抑制组还是单抑制组，FGL1有效抑制后，CD8+T、CD4+T细胞亚群表达活性显著高于对照组，而Treg细胞表达、肿瘤细胞恶性增殖活性度及肿瘤生长情况均显著低于对照组。本次研究中，我们也发现，在FGL1、LAG3与Tim3的联合抑制组的CD8+T、CD4+T细胞亚群表达活性显著高于单抑制组，Treg细胞亚群的表达、肿瘤细胞恶性增殖活性度及肿瘤生长情况也均显著低于单抑制组。研究成果揭示了肿瘤免疫逃逸机制的复杂性、多重性，显示了不同免疫检查点因子免疫逃逸通路的平行性，提示单一免疫检查点的抑制对于遏制肿瘤逃避免疫杀伤的局限性。也凸显了多个免疫检查点因子信号通路联合抑制相比于单一通路的单控，将更显著调控CTL细胞的激活和对肿瘤细胞的敏感性毒性表达，并最终抑制HCC肿瘤细胞的恶性增殖的重要意义。

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