间充质干细胞调节MERTK蛋白促进慢加急性肝衰竭小鼠巨噬细胞M2型极化的机制研究

Acute-on-chronic liver failure (ACLF) is a severe disease with high mortality. Our previous study demonstrated that infusion of mesenchymal stem cells (MSC) for ACLF patients improved the survival rate. But its specific mechanism is unclear. We also found that M1 macrophages were polarized in ACLF patients, which enhanced its pro-inflammatory ability. Study suggested that the expression of MERTK protein of ACLF patients increased during resolution phage, which would promote the polarization of M2 macrophages via JAK1/STAT1 pathway and the resolution of inflammation. Our preliminary results showed that MSC can up-regulate the expression of MERTK. Thus, we propose our hypothesis as follows, MSC promote polarization of M2 macrophages by up-regulating the expression of MERTK and thereby activating JAK1/STAT1 pathway. In this project, flow cytometry, immunohistochemical staining, Western Blot, RT-PCR, gene over-expression, RNA interference and other methods were used to discuss the mechanism of MSC regulating macrophages’ function through MERTK, via cell and animal experiments. The conclusion will provide theoretical basis for promoting MSC application and optimizing cell therapy for ACLF patients.

慢加急性肝衰竭（Acute-on-chronic liver failure, ACLF）病死率高。申请人前期研究发现，静注骨髓间充质干细胞（Mesenchymal stem cells，MSC）可提高ACLF患者存活率，但具体机制不明确。申请人还发现在ACLF患者中巨噬细胞M1型极化，促炎能力增强。研究提示肝衰竭患者在恢复期巨噬细胞中MERTK蛋白表达增加，该蛋白可通过激活JAK1/STAT1通路促使巨噬细胞M2型极化，促进炎症修复。预实验结果提示MSC可上调MERTK表达。由此，我们提出以下科学假说：MSC通过上调MERTK的表达，进而激活JAK1/STAT1通路促进巨噬细胞M2型极化。本项目拟采用流式细胞、免疫组化、Western Blot、RT-PCR、基因过表达、RNA干扰等方法，通过细胞与动物实验，探讨MSC通过MERTK调节巨噬细胞功能的机制，为推广MSC的应用提供理论依据。

研究背景：慢加急性肝衰竭（ACLF）是在慢性肝病基础上发生的急性肝功能失代偿，病死率高达63%-72%，严重威胁人民健康。申请人所在团队的前期临床研究表明，经外周血注射间充质干细胞（MSCs）安全性好，可提高ACLF患者24周存活率，但具体机制仍未明确。研究表明：①巨噬细胞M1型极化引起的炎症损伤在ACLF的发生发展中起到重要作用；②在肝衰竭患者恢复期，巨噬细胞中Mertk蛋白表达升高，提示Mertk蛋白可能促进肝衰竭的炎症修复；③MSCs具有免疫调节功能，可缓解炎症损伤。据此，本课题旨在通过体内外实验，结合生信分析，研究MSC修复ACLF炎症损伤的具体机制。.研究结果：我们首先建立了ACLF小鼠模型，成功分离、培养及鉴定小鼠骨髓MSCs。随后我们在动物实验中证实了MSCs尾静脉输注可改善ACLF小鼠肝功能及48小时存活率，改善肝组织炎症及坏死情况。进一步地，在动物实验中，我们发现MSCs输注可以促进小鼠M2型巨噬细胞极化，缓解肝脏炎症损伤，并可促进Mertk蛋白表达。我们通过生信分析，检测和分析MSCs治疗的ACLF小鼠肝脏转录组学，发现参与MSCs阻断ACLF进展的候选枢纽基因和通路，结合生信分析结果及体外细胞实验，我们发现Mertk可通过激活JAK1/STAT6信号通路，促进巨噬细胞M2型极化，缓解肝脏炎症损伤。进一步地，我们通过将LPS预处理的巨噬细胞与MSCs共培养，证实了MSCs可上调Mertk表达，激活JAK1/STAT6信号通路进而促进M2型巨噬细胞极化，缓解ACLF炎症损伤。同样地，在动物实验中，我们也发现输注MSCs可以上调Mertk，激活JAK1/STAT6信号通路进而促进M2型巨噬细胞极化。.研究结论：本课题研究结果提示MSCs可通过上调巨噬细胞Mertk蛋白表达，进而激活JAK1/STAT6信号通路，促进巨噬细胞M2型极化，缓解ACLF炎症损伤。阐明了MSCs输注救治ACLF的新机制，为MSCs的临床运用提供了理论依据，并为肝衰竭救治提供了新的靶点及思路。