低剂量免疫抑制剂诱导人脐带间充质干细胞自噬并促进CXCR4的分泌  

Promotion of autophagy and the secretion of CXCR4 of human umbilical cord mesenchymal stem cells induced by low-dose immunosuppressive durgs

作　　者：雷艳[1] 赵红州[1] 李荣春[1] 付云烽[1] 施小华[1] 林娜[1] 陈津[1] 詹世淮 谭建明[1] 黄梁浒[1] 

机构地区：[1]福州总医院福建省移植生物学重点实验室,350025

出　　处：《中华器官移植杂志》2017年第1期39-44,共6页Chinese Journal of Organ Transplantation

基　　金：国家自然科学基金（81270431）;福州总医院院内课题国际合作研究专项（2016G01）

摘　　要：目的 分析低剂量免疫抑制剂诱导人脐带间充质干细胞（hUC-MSC）产生自噬,并对趋化因子受体CXCR4分泌的影响.方法 低剂量的免疫抑制剂他克莫司和西罗莫司分别处理MSC后,流式细胞术检测hUC-MSC细胞表面标志物变化,WST-1法检测hUC-MSC细胞增殖的影响;并通过qRT-实时聚合酶链反应（PCR）检测自噬基因LC3B、Atg5、Beclin1 mRNA水平的变化,及Western blot检测他克莫司和西罗莫司诱导的自噬相关蛋白LC3B、Atg5、Beclin1和p-ULK1的表达;同时流式细胞术也分析了在hUC-MSC产生细胞自噬的状态下趋化因子受体CXCR4的分泌.结果 低剂量的免疫抑制剂他克莫司和西罗莫司并不引起hUC-MSC细胞表面标志物分子的表达明显改变,且低剂量的他克莫司对hUC-MSC完全没有细胞毒性作用,而西罗莫司对hUC-MSC的细胞增殖有一定的抑制作用,24 h和48 h的存活率分别为73.66％或68.81％,P＜O.05具有统计学意义.他克莫司和西罗莫司都能通过抑制PI3K/AKt/mTOR信号通路激活hUC-MSC自噬,自噬相关蛋白LC3B、Atg5和Beclin1表达显著上调,并诱导CXCR4分泌上调.结论 低剂量免疫抑制剂他克莫司和西罗莫司能诱导人脐带间充质干细胞hUC-MSC产生自噬并促进CXCR4的分泌.Objective To investigate the effect of human umbilical cord mesenchymal stem cells （hUC-MSCs） on autophagy and the secretion of chemokine receptor CXCR4 induced by low-dose immunosuppressive durgs.Methods Flow cytometry was used to detect the changes of hUC-MSCs surface markers after treatment with low-dose tacrolimus and rapamycin.The effect of treatment with tacrolimus and rapamycin on proliferation of hUC-MSCs was analyzed with WST-1 assay.Regular RT-PCR was applied to analyze the mRNAs expression of ligands such as LC3B,Atg5 and Beclin1 in hUC-MSCs.Western blotting was carried out to detect the expression of LC3B,Atg5,Beclin1 and p-ULK1 in hUC-MSCs after treatment with tacrolimus and rapamycin.The secretion of chemokine receptor CXCR4 in hUC-MSCs was analyzed under the state of autophay by flow cytometry.Results Flow cytometry analysis confirmed low-dose immunosuppressive drugs tacrolimus and rapamycin did not cause changes in hUC-MSCs phenotypes significantly.Low-dose tacrolimus had no cytotoxic effect on hUC-MSCs,while,rapamycin could inhibit the proliferation of hUC-MSCs after 24 h or 48 h,with survival rate being 73.66％ and 68.81％ （P＜0.05） of controls,respectively.Moreover,both tacrolimus and rapamycin could inhibit PI3K/AKt/mTOR signaling pathway to activate hUC-MSCs autophagy,and the related proteins of LC3B,Atg5 and Beclin1 increased significantly and induced the up-regulation of CXCR4 secretion.Conclusion Our results here demonstrated that low-dose tacrolimus and rapamycin induce autophagy in hUC-MSCs and promote the secretion of CXCR4.

关 键 词：他克莫司 西罗莫司 间充质干细胞 细胞自噬 CXCR4 

分 类 号：R96[医药卫生—药理学]