p53对间充质干细胞抗肝纤维化作用的调控及其机制研究

Fibrosis is the endpoint of many chronic inflammatory diseases and is defined by an abnormal accumulation of extracellularmatrix components. Despite its slow progression, it leads to organ malfunction. Current fibrosis management therapies either are insufficiently effective or induce severe adverse effects. Fortunately，mesenchymal stem cells (MSCs) therapy seems to be a promising treatment. The value of their immunosuppressive effects and their antifibrotic potential are widely described. However，the therapeutic effect of MSCs on fibrosis is not satisfactory at present. How to improve the antifibrotic effects of MSCs, is still an urgent need..Recently, it was demonstrated that inflammatory microenvironment-induced autophagy downregulated the immunosuppressive function of MSCs. Inhibition of autophagy by knockdown of Beclin-1 significantly improved the therapeutic effects of MSCs on inflammation. However, directly impairing autophagy may link to the risk of tumor formation. Therefore，we turned to another method that could inhibit autophagy. Inhibition of p53 degradation by knockdown of mouse double minute 2(MDM2)，as reported， could inhibit autophagy，and should also contribute to prevention of the tumorigenesis risk resulted from inhibition of autophagy itself. This method gives rise to a novel strategy for improving antifibrotic effect of MSCs. And TGF-β1, an essential factor that contributed to liver fibrosis，were reported to have the capacity to inhibit p53 expression..The results of our previous study showed that knockdown of p53 significantly facilitated autophagy in the presence of TNF-α and IFN-γ. Furthermore, knockdown of p53 could inhibit the therapeutic effect of MSCs on CCl4-induced rat liver fibrosis. .Based on both the above literature and our previous work，we found that it was of great significance to investigate p53 function on modulation of antifibrotic effect of MSCs in the liver fibrosis microenvironment. Thus, we plan to establish mouse bone marrow mesenchymal stem cell lines with different levels of p53 via infection of lentiviral particles containing MDM2shRNA, p53 shRNA, control shRNA or p53. Finally, following the research clue，autophagy-immunity, and utilizing the Cre-loxP genetic lineage tracer technique，we look forward to demonstrate that inhibition of p53 degradation could improve the therapeutic effects of MSCs on experimental liver fibrosis via knockdown of MDM2.

纤维化是许多慢性炎性疾病发展的终点，常导致器官功能障碍,所产生的医疗负担日益沉重。间充质干细胞免疫抑制功能在抗纤维化研究中显示出较大潜力，但其疗效亟待提高。文献表明，抑制鼠双微体2表达，能够抑制p53降解,进而抑制自噬；而抑制自噬能够增强间充质干细胞在炎症微环境中的免疫抑制功能；我们的前期研究提示，p53稳定敲减，促进间充质干细胞自噬，抑制其抗大鼠肝纤维化作用。那么，抑制p53降解能否提高间充质干细胞抗肝纤维化疗效？由此，本项目拟构建p53降解抑制、表达抑制及p53高表达间充质干细胞株，以自噬-免疫功能为线索，结合Gli1遗传谱系示踪技术，探寻上述细胞在小鼠肝纤维化微环境中的功能与命运变化，揭示p53对间充质干细胞自噬、免疫功能与抗肝纤维化作用的调控机制；期望通过抑制p53降解的策略，提高间充质干细胞抗肝纤维化疗效，为开发临床细胞治疗技术提供支撑。

纤维化是许多慢性炎性疾病发展的终点，常导致器官功能障碍,所产生的医疗负担日益沉重。间充质干细胞（mesenchymal stem cells, MSCs）在抗纤维化研究中显示出较大应用潜力，但其疗效亟待提高。本项研究发现，抑制鼠双微体2（mouse double minute 2，MDM2）表达以抑制MSCs p53降解，或者抑制自噬相关基因Becn1表达，均能抑制肝纤维化微环境诱导的MSCs自噬，并提高MSCs前列腺素内过氧化物合酶2（prostaglandin-endoperoxide synthase 2，PTGS2）、前列腺素E2（Prostaglandin E2，PGE2）水平；肝纤维化病变小鼠的肝脾组织，对于通过尾静脉移植的MSCs，具有较强趋化募集作用；移植MSCs、CD4+T、CD8+T以及Gli1+细胞，在肝内均主要分布在纤维增生区域；在肝纤维化微环境下，抑制 MDM2或Becn1表达，可使MSCs抑制与其共培养的肝星状细胞(hepatic stellate cells，HSCs)的增殖活化；MDM2或Becn1稳定敲减的MSCs，能够同时抑制肝纤维化小鼠肝脾组织内CD4+T与CD8+T细胞的浸润，使肝内Gli1+细胞减少、肝组织病变减轻、肝功能改善，抗肝纤维化作用优于对照MSCs。上述结果提示，MSCs在肝纤维化微环境中，可通过旁分泌作用，既抑制免疫细胞，也抑制肌成纤维细胞，从而抑制炎症损伤与纤维性修复之间的恶性循环；同时提示，MSCs免疫抑制效应可能来自于其对肝脾T细胞免疫的协同抑制作用。综上，本项目所采用的抑制MSCs p53降解的策略，能够抑制MSCs自噬，促进PGE2的生成与分泌，抑制CD4+T细胞、CD8+T细胞、Gli1+细胞及HSCs，最终提升MSCs抗肝纤维化疗效，为进一步开发MSCs临床治疗技术提供了支撑基础。