淋巴细胞介导的免疫排斥反应对MSCs肝内移植后定植生存能力的研究

Transplantation of bone marrow-derived mesenchymal stem cells(MSCs) has been suggested as a potential therapeutic approach for liver injury. However, the transplanted MSCs have low survival rate and short survival time in the host liver, which decreases the therapeutic efficiency to the injured liver. Some researchs hinted that NK cells and T cells mediated immune rejection may be related to the above phenomenan. In this project, allogenic MSCs will be labeled with CMV or Albumin (Alb) promoter-driven reporter genes and transplanted to the mice of liver injury model. Anti-asialo GM1 which can reduce the activity of NK cells and Tacrolimus which can supress T cell activation will be administrated to the mice. The survival, proliferation and hepatocyte differentiation of transplanted MSCs will be dynamically monitored through in vivo bioluminescent imaging.At the same time,the proportion and function of NK cells and T cells in host liver and spleen will be detected. Through in vivo and in vitro experiments, we intend to elucidate the action of immune rejection to the survival, proliferation and differentiation of transplanted MSCs and the related mechanism. The expected results of this project will enhance the treatment scheme optimination of MSCs transplantation and improve progress of its clinical application.

骨髓间充质干细胞移植（MSCs）是治疗肝损伤最具潜力的方法之一。但是，移植MSCs在受体肝脏生存率低、存活时间短等关键问题影响了其对损伤肝脏的治疗效果。有研究提示受体NK细胞和T细胞介导的免疫排斥反应与以上现象有密切相关性。本课题拟采用分别负载CMV和白蛋白（Alb）启动子报告基因的同种异体MSCs，对肝损伤模型小鼠进行肝内移植，分别采用anti-asialo GM1清除宿主体内NK细胞和Tacrolimus抑制T细胞介导的免疫排斥反应，通过生物发光成像活体动态示踪MSCs存活、增殖及向肝细胞的分化情况；同时检测肝脏和脾脏淋巴细胞的数量、功能，探讨淋巴细胞介导的免疫排斥反应对移植MSCs的存活、增殖和分化的作用及其机制。本课题的预期结果将有助于优化MSCs移植治疗方案，推动MSCs移植的临床应用。

间充质干细胞（MSCs）移植具有治疗肝损伤的潜能，而活体细胞示踪是研究干细胞在体内分布、存活及增殖的重要手段。免疫排斥可能是移植MSCs在损伤肝脏保持活性及生存的主要障碍。本项目采用报告基因 （CMV-luciferase2-mKate2）稳定标记MSCs，通过活体生物发光成像示踪移植后MSCs在小鼠体内存活和增殖情况，确立了MSCs移植的最佳剂量和途径。研究结果表明，相对于下腔静脉及肝内注射移植途径，经由肠系膜上静脉（SMV）移植的MSCs细胞可以均匀并相对较长时间地停留在肝脏内，因此是最佳的移植途径。进一步的活体成像结果显示，在对照组及NK细胞激活组小鼠，移植的MSCs数目随着时间有递减的趋势，但MSCs数目在NK细胞激活组小鼠减少得更快，提示NK细胞加速了移植MSCs的清除。同时，NK细胞的数量和标志物在MSCs移植后有明显提高，说明移植的MSCs激活了体内NK细胞，并在一定程度上介导了MSCs移植的排斥反应；IL-15/IL15-Rα激活的NK细胞可以进一步加快清除移植的MSCs，并且明显抑制MSCs的增殖、诱导MSCs的死亡。本研究成果为探索MSCs移植的最佳方法以及为克服免疫排斥提供了新的思路，进一步为优化及推广MSCs治疗肝损伤提供了重要的理论基础。