Gab2促进髓源性抑制细胞（MDSCs）功能介导肝癌发展的机制探究

Our previous research revealed that deletion of Gab2 ameliorates symptoms of fatty liver, and inhibits the development of primary liver cancer by interfering IL-6 signaling networks. However, the underlying mechanism of Gab2-mediated the progression of Hepatic carcinoma remains unclear. Our previous studies found that mouse liver-specific Gab2 overexpression promoted the development of Hepatic carcinoma, tumor growth, and IL-6 secretion, enhanced the expression of MDSCs-related proliferative and activated genes (S100A8, S100A9, and Arg-1; Gr-1 positive staining cells number) and tumor angiogenesis factor (VEGF), accompanied by inhibiting the secretion of immunosuppressive factors (IL-4 and IL-13) that acted as the activator of T cells. It is confirmed that IL-6 can induce the function of MDSCs-mediated immunosuppression and angiogenesis, which are the important developing link of liver cancer. Hereby, we suspect that Gab2 possibly regulates the progression of Hepatic carcinoma through affecting IL-6-mediated the functional change of MDSCs. In the present project, we will perform in vivo and in vitro experiments applying liver-specific Gab2 overexpression transgenic mice, lentivirus interference and mouse MDSCs and T cells isolation bio-technology, to explore the effects of Gab2 on accumulation and function of MDSCs in the formation of Hepatic carcinoma, clear the role of IL-6 in the cause of Gab2-mediated the properties changes of MDSCs, illustrate the regulation mechanism of Gab2 on the functional changes of MDSCs in the progression of liver cancer in vitro, and reveal the clinic correlation between Gab2, IL-6, and MDSCs in liver cancer patients. This study will shed a new perspective to explore the underlying mechanism of Gab2 on the development of Hepatic carcinoma, and provide a novel insight for the prevention and treatment of Hepatic carcinoma in clinically.

我们的研究成果揭示敲除Gab2改善脂肪肝症状、干扰IL-6信号通路抑制肝癌发生，但其介导肝癌进程的机制不明确。我们前期研究发现，小鼠肝脏过表达Gab2促进肝癌发展及IL-6分泌，增加髓源性抑制细胞（MDSCs）标志物及血管生成因子合成，抑制免疫激活因子分泌。文献调查显示IL-6可诱导MDSCs促血管生成和免疫抑制功能，这两个功能是肝癌发生的重要环节。因此我们猜测Gab2可能通过IL-6介导MDSCs功能调控肝癌发展。本课题将从动物、细胞及临床样本层次，运用过表达Gab2小鼠、慢病毒干扰、MDSCs及T细胞分离等方法，探究Gab2对肝癌形成中MDSCs扩增和功能的影响；明确IL-6在Gab2调控MDSCs特性中的作用；体外阐明Gab2促进MDSCs功能调控肝癌进程的分子机制；揭示临床上Gab2、IL-6和MDSCs的相关性。本研究从新角度探究Gab2调控肝癌的作用机理，为肝癌防治提供新思路。

我们之前的研究成果已经揭示敲除Grb2相关结合蛋白2（Gab2）改善脂肪肝症状、干扰IL-6信号通路抑制Gab2介导的肝癌发生，但其介导肝癌进程的机制不明确。本研究旨在阐明Gab2介导肝癌发生过程中的免疫调节作用。本项目从动物、细胞、分子及临床样本层次，利用肝脏特异性过表达Gab2转基因小鼠，结合DEN诱导原发性肝癌模型、慢病毒干扰小鼠基因表达技术、体外MDSCs及T细胞分离培养、免疫染色和流式细胞分选等技术展开研究。研究发现与野生型组相比，肝脏特异性过表达Gab2小鼠(L-Gab2)在二乙基亚硝胺(diethylnitrosamine, DEN)诱导5个月后出现早期肝癌发生，在DEN诱导9个月后肿瘤生长加速。与DEN处理的野生型小鼠相比，在DEN刺激的L-Gab2小鼠中观察到更多的骨髓源性抑制细胞(MDSCs)。此外，在DEN暴露的L-Gab2小鼠中，MDSCs激活诱导的肿瘤血管生成能力和免疫抑制功能被过度激活，体现在血小板内皮细胞粘附分子(PECAM)和血管内皮生长因子(VEGF)增加，细胞毒性T细胞下降。机制上，DEN诱导的L-Gab2小鼠产生了更多的IL-6，而IL-6的缺失显著剥夺了Gab2过表达介导的肿瘤促进作用，并可损害MDSC介导的免疫抑制功能。从IL-6缺失的L-Gab2小鼠中分离的MDSCs或灭活MDSCs可部分恢复细胞毒性T细胞的免疫功能。值得注意的是，MDSCs基因集与临床肝癌标本中Gab2或IL6的增加有显著的相关性。综上所述，L-Gab2小鼠加速肝癌进展可能是通过激活IL-6激活MDSCs的途径。本研究为探索Gab2在肝癌发生过程中自身免疫耐受中的作用提供新的见解。