激活肝星状细胞对肝脏固有和浸润巨噬细胞分化的调控与肝癌复发

Most hepatocellular carcinomas (HCCs) develop from cirrhotic liver. Recently, it has been recognized that activated hepatic stellate cells (A-HSCs) are not only the key cell type responsible for liver fibrosis following a variety of chronic liver injuries, but also an important component participating in liver immune microenvironment regulation. In our previous study based on patient data, we found that A-HSCs in peritumoral cirrhotic liver tissues may induce the differentiation of macrophages to a M2 type, thus induce an immunosuppressive microenvironment associated with an increased tumor recurrence. Macrophages in the liver includes: resident macrophages (Kupffer cells, KC) and infiltrating macrophages (IM) from peripheral blood mononuclear cells. These two groups of macrophages are different in ontology and function. The impact of A-HSCs on the differentiation of these two groups of macrophages has not been reported yet and deserves further investigation. In this project, we plan to establish a liver fibrosis model with allogeneic bone marrow transplanted mice, which will help us to accomplish clear separation of resident and infiltrating macrophages (KC and IM). Base on this model, we plan: (1) to dissect the regulatory role of A-HSCs in resident and infiltrating macrophages differentiation with next generation sequencing, quantitative proteomics methods and in vitro co-culture system; (2) to clarify the origin of A-HSCs related type M2 macrophages; (3) to explore the mechanism of A-HSCs induced immunosuppression. This project will deepen our understanding of the mechanisms for the high incidence of liver cancer in patients with cirrhosis and the high postoperative recurrence of HCC in patients with cirrhotic background from a view of A-HSCs related immunosuppressive microenvironment, and will provide reliable evidence for the development of immunotherapy targeting activated hepatic stellate cells and macrophages to prevent HCC recurrence.

肝细胞肝癌 (HCC) 与肝硬化关系密切。激活肝星状细胞 (A-HSC) 不仅是介导肝纤维化、硬化的关键细胞成分，还参与调控肝脏免疫微环境。我们前期的临床队列研究提示癌旁硬化组织中的A-HSC可能通过诱导巨噬细胞 (MΦ) 向M2型分化、形成免疫抑制性微环境，从而增加HCC术后复发。但关于A-HSC对肝脏MΦ分化的影响尚无报道。最新研究表明损伤肝脏中的固有MΦ (Kupffer细胞，KC) 和浸润MΦ (IM) 是两个具有独立起源和不同功能的细胞群体。本课题拟建立骨髓移植小鼠肝纤维化模型，以区分固有和浸润MΦ，通过流式分选、转录组测序、定量蛋白组学分析并结合体外共培养模型，确定硬化肝组织中A-HSC相关M2型MΦ的来源，系统解析A-HSC对不同来源MΦ分化的调控作用，探讨A-HSC诱导肝脏免疫抑制、促进硬化背景HCC术后复发的机制，为靶向A-HSC和MΦ的HCC预防和治疗提供理论依据。

80-90%的肝细胞肝癌（HCC）发生在硬化肝脏。近年发现激活肝星状细胞（HSC）不仅是各种慢性肝损伤导致肝脏纤维化、硬化的关键细胞成分，还是参与肝脏免疫微环境调控的重要细胞成分。我们前期的病例资料研究发现癌旁硬化肝组织中激活HSC可能通过诱导巨噬细胞向调节型分化、形成免疫抑制微环境而增加HCC的复发。为阐明纤维化、硬化肝组织中调节型巨噬细胞的来源；探讨HSC对肝脏中固有和浸润巨噬细胞分化的影响，以及两者间相互作用对HCC发生发展的影响，本研究中课题组利用CD45.1/CD45.2异体骨髓移植小鼠，建立CCL4诱导的急性和慢性肝损伤模型，通过组织学和流式细胞术初步评估了急性和慢性CCL4肝损伤对肝脏固有和浸润巨噬细胞数量、分布和表型的影响；进一步通过流式分选，收集各组巨噬细胞样本用于转录组测序分析，在基因表达水平深入解析了正常肝脏巨噬细胞的来源和组成，急性和慢性CCL4肝损伤中肝脏固有和浸润巨噬细胞表型的动态变化；并通过体外原代HSC-肝脏巨噬细胞共培养体系、原代HSC-肝脏巨噬细胞-HCC细胞共培养体系，探讨了静止和激活HSC对肝脏巨噬细胞分化影响，以及静止和激活HSC调控肝脏巨噬细胞对HCC细胞恶性生物学行为的影响。本研究将加深对HSC在肝脏局部免疫调控中作用的认识，尤其是对肝脏不同来源巨噬细胞分化的影响，有助于从肝脏免疫微环境的角度阐明肝硬化患者进一步发展为HCC及硬化背景HCC患者术后晚期复发的机制，为靶向激活HSC和巨噬细胞的HCC预防和治疗提供理论依据。