一贯煎调节SDF-1/CXCR4信号通路促进骨髓干细胞迁移分化治疗肝硬化的机制研究

China is burdened with a large population suffering liver disease, and chronic hepatitis B accounts for the largest percentage. Other liverish cases rise by alcohol, drug and autoimmune. Worse still, varieties of liver trauma eventually may develop terminal liver diseases such as liver cirrhosis, failure and cancer, all of which become one of the primary causes that endanger human lives..Although liver transplantation is adopted as a more effective treatment for end-stage liver disease, such three significant bottlenecks as donor lack, immune rejection and expensive cost hamper the extensive development and application..Researches conducted recently indicate that stem cell transplantation has a distinct advantage in end-stage liver disease treatment, or rather, bone marrow stem cells can contribute to the restoration of liver damage, symptom improvement and an increase of survival rate..Preliminary studies of the research group directed by Professor Liu Ping have found that, Yiguanjian, a Chinese medicinal classic prescription, not only inhibits liver inflammation and fibrous tissue hyperplasia of cirrhotic rat induced by carbon tetrachloride (CCl4), but also promotes liver oval cells (HOCs) to differentiate to hepatocytes and biliary epithelial cells of cirrhotic rat by dimethylnitrosamine (DMN), improve liver function, and hereby reconstitute liver tissue. Therefore, Yiguanjian-induced stem cells differentiation mechanism to liver is worthy of further investigation..Mechanism studies of directed differentiation of bone marrow stem cells have found that, the homing migration of those mediated by combination of Stromal cell-derived factor -1(SDF-1) with its receptor CXCR4, which plays an important role in the regeneration of a variety of tissue and organ damage. Therefore, it is of great significance to further explore Yiguanjian in terms of activation and regulation mechanism of stem cells migration and differentiation to liver by SDF-1/CXCR4- mediated signal transmission pathways of (P13-K)/AKT..Therefore, the study applies green fluorescent protein (GFP) transgenic mice to lable bone marrow stem cells, set up mice cirrhosis model by DMN, and then treat with Yiguanjian, and applies Double Immuno Confocal Laser Scan Microscopy (DICLSM), Immunoblot, RT-PCR, and other methods, in vivo and vitro experiments to confirm the bone marrow stem cells migration and differentiation to liver by SDF-1/CXCR4-mediated (P13-K)/AKT signaling pathway, thus improving the liver function and pathology, and promoting cirrhosis to repair and reconstruction. .The study will clarify the mechanism of action of Yiguanjian treating liver cirrhosis, and molecular pharmacological mechanisms in Yiguanjian regulating bone marrow stem cells directed differentiation into liver, and meanwhile clarify part of the scientific connotation that "kidney breeds marrow, marrow breeds liver" and " liver and kidney share homologous source" in traditional Chinese medicine.

肝硬化时骨髓干细胞可被动员向肝脏迁移并定向分化以修复重构肝组织，SDF-1/CXCR4介导的信号通路在其中起重要作用。前期研究发现中药方剂一贯煎可促进骨髓干细胞向损伤的肝脏迁移并向肝细胞和胆管细胞分化，改善肝功能和肝脏病理。本课题进一步研究一贯煎影响骨髓干细胞肝内迁移分化、治疗肝硬化的作用机制。通过将GFP转基因小鼠骨髓干细胞移植入实验小鼠，制备骨髓干细胞标记的DMN肝硬化小鼠模型，应用经典方剂一贯煎治疗。观察肝硬化小鼠肝功能及肝组织病理改变，围绕SDF-1/CXCR4介导的信号通路，体内外实验研究一贯煎通过对SDF-1/CXCR4信号通路活化和调节作用，诱导骨髓干细胞向肝脏迁移与定向分化，促进肝硬化组织结构修复。阐明中药方剂一贯煎在肝硬化治疗中改善肝功能，重构肝组织的作用机制，以及一贯煎影响骨髓干细胞向肝脏迁移与肝内定向分化的分子药理机制，同时阐明中医"肾生髓、髓生肝"的部分科学内涵。

1.项目背景：肝硬化等终末期肝病是危及人类生命的主要原因之一，临床疗效差，死亡率高。研究表明，干细胞可以促进损伤肝脏修复、改善症状及提高存活率；SDF-1/CXCR4介导的信号通路在干细胞迁移归巢中起重要作用。前期研究已发现中医经典方剂一贯煎可通过促进肝硬化大鼠的骨髓干细胞向肝脏迁移并向肝细胞和胆管细胞分化，从而逆转肝硬化。因此本课题将进一步研究一贯煎影响骨髓干细胞定向分化的作用机制。.2.主要研究内容：通过应用CD90标记骨髓干细胞技术，建立DMN小鼠肝硬化模型，一贯煎进行治疗。观察肝功能及肝脏病理学改变，围绕SDF-1/CXCR4介导的信号通路，体内外实验研究一贯煎通过对该信号通路活化和调节作用，诱导骨髓干细胞定向分化。.3.重要结果：体内研究阐明，一贯煎通过上调SDF-1/CXCR4轴介导的MAPK/ERK1/2信号通路，促使骨髓间充质干细胞肝向分化，从而逆转肝硬化。体外研究进一步证实，一贯煎能够通过SDF-1诱导骨髓间充质干细胞的肝向分化，并且能够协同HGF、SDF-1促进BM-MSCs肝向分化作用。.4.关键数据：体内研究：二甲基亚硝胺注射后，各组小鼠一般状况、肝功能及病理学情况明显变差，免疫荧光共定位水平增加，α-SMA、CXCR4、ERK1/2、NF-kB P65及β-catenin的表达水平均明显上调。当使用一贯煎药物治疗后，各组小鼠一般状况、肝功能及病理学情况均明显改善，免疫荧光共定位水平持续增加，CXCR4、ERK1/2的表达水平继续上调，而α-SMA、NF-kB P65及β-catenin的表达水平下调（P﹤0.05）。体外研究：HGF、SDF-1、一贯煎药物血清组均检测出ALB、CK-18的表达，在各组中的表达呈时间依赖性，且任意两者联用，ALB、CK-18的表达量均高于单独应用，HGF、SDF-1、一贯煎药物血清三者联用出现ALB、CK-18的时间最早且表达量最多，差异有统计学意义（P﹤0.05）。.5.科学意义：阐明一贯煎在肝硬化治疗中改善肝功能、重构肝组织的作用机制，以及影响骨髓干细胞向肝脏迁移分化的分子药理机制，同时阐明中医“肾生髓、髓生肝”的部分科学内涵。