骨髓间充质干细胞移植调节肝脏巨噬细胞亚群对肝纤维化的影响

Liver fibrosis is an inevitable pathological stage forward progression to liver cirrhosis. And the research of new efficient therapy method for liver fibrosis is vital and meaningful in clinic. We found the cytokine of TNF-alpha,IL-6,INF-gamma which can promote M1 decreased, the the cytokine of IL-13, IL-4 which can promote M2 increased due to the transplantation of bone marrow mesenchymal stem cell (MSC) to xenogenic mice with liver fibrosis. Meanwhile the transplantation promoted the elevation of MMP13 mRNA expression and decreased the expression of TGF-beta1, and also decreased the mRNA expression of fibrosis related genes including collagen-1,collagen-4, alpha-SMA. We speculated transplantation of MSC would attenuate liver fibrosis through regulating macrophage subsets. After transplantation of MSC,we will measure the changed condition of macrophage subsets and demonstrate the increased MMP13 come from the activated M2 cell and MSC probably suppress the activation of M1 cell which can stimulate HSC secrete TGF-beta1, by the methods of qRT-PCR, ELISA, cell culture ,immunohistology and flow cytomteter. We wish this research can verify above suppose and provide essential data for profound research and reasonable evidence for MSC transplantation in clinic in future.

肝纤维化是慢性肝病进展至肝硬化的前期阶段,预后差,研究新的有效治疗手段并探索其机制具有重要临床价值。课题组前期研究发现小鼠肝纤维化时伴随巨噬细胞M1亚群相关细胞因子表达增加，M2亚群相关细胞因子无明显变化。而通过骨髓间充质干细胞（MSC）移植会引起M1亚群相关细胞因子表达下降，M2亚群相关细胞因子表达升高，同时肝纤维化明显减轻。我们推测MSC移植后通过旁分泌作用使肝脏巨噬细胞微环境发生改变，引起M1亚群巨噬细胞向M2亚群转变，从而进一步引起MMP13表达增高，以及抑制HSC活性，使肝纤维化减轻。本研究将对MSC移植后肝内相关细胞因子的变化与肝脏巨噬细胞亚群转变的关系，以及影响肝纤维化的MMP13、TGF-beta1与肝脏巨噬细胞亚群动态变化之间的联系进行检测，论证肝纤维化减轻的可能机制，为MSC移植治疗肝纤维化这一难题寻找细胞分子学证据。

背景：肝纤维化是肝病向肝硬化或肝癌进展的关键阶段，本项目对小鼠骨髓间充质干细胞移植是否能通过调节肝脏巨噬细胞亚群有效减轻小鼠肝纤维化进行研究。.方法：肝纤维化模型通过持续10周每周两次腹腔注射四氯化碳造模，骨髓间充质干细胞注射分别在第60天、70天进行两次尾静脉注射。对肝纤维化和巨噬细胞亚群相关标志进行免疫组化及基因表达的检测，并且小鼠 RAW264.7细胞及JS1细胞（肝星状细胞系）用于体外实验检测骨髓间充质干细胞对于肝纤维化的作用。.结果：骨髓间充质干细胞移植后, F4/80及CD206阳性的活化M2巨噬细胞以及MMP13表达明显增加， F4/80及iNOS阳性的活化M1巨噬细胞明显减少，并且肝组织IL-10表达提高，IFN-r,TNF-a, IL-6 表达下降。受到骨髓间充质干细胞条件培养液刺激的肝星状细胞分泌TGF-b1 和 collagen-1 减少，而 caspase-3 增加。骨髓间充质干细胞移植后有效的抑制了肝组织a-SMA, Sirius red 以及 collagen-1这些与四氯化碳注射密切相关的肝纤维化指标蛋白的表达。.结论：我们证明了骨髓间充质干细胞移植能够通过激活肝组织阳性表达MMP13的M2巨噬细胞，并抑制M1巨噬细胞活化从而进一步抑制肝星状细胞活化，这两者发挥协同作用减轻肝纤维化的进展。