脂肪源性干细胞对多发性硬化患者Th17的免疫调控作用  

Adipose-derived stem cells mediate immunosuppression of Th17 in multiple sclerosis

作　　者：林晓滨[1] 陈颖[2] 杨德壕 谢甬淋[3] 毕涌[2] 柯建明[2] 陈志博[2] 苏中钱 厉向[2] 张旭[2] 

机构地区：[1]温州医科大学附属第一医院超声影像科,浙江温州325000 [2]温州医科大学附属第一医院神经内科,浙江温州325000 [3]武警浙江省总队杭州医院康复科,浙江杭州310000

出　　处：《中国病理生理杂志》2016年第1期51-57,共7页Chinese Journal of Pathophysiology

基　　金：浙江省自然科学基金资助项目(No.LY14H130002;No.LY13H090010);温州市科技计划(No.Y20140278)

摘　　要：目的:探讨人脂肪源性干细胞(h ASCs)对多发性硬化(MS)患者外周血辅助性T细胞17(Th17)的免疫调控作用机制。方法:分离、纯化脂肪组织中的h ASCs。采用密度梯度离心法分离MS患者外周血单个核细胞(PBMCs),磁珠分选CD4+T细胞,体外刺激细胞向Th17极化,并加入不同比例的h ASCs(h ASCs∶CD4+T为1∶4和1∶10)共培养4 d,设立添加anti-LIF抗体组;流式细胞术检测共培养后Th17细胞占CD4+T细胞的比例,real-time PCR检测白细胞介素6受体(IL-6R)、白细胞介素23受体(IL-23R)、白血病抑制因子受体(LIFR)、维甲酸相关孤儿受体γt(RORγt)及白血病抑制因子(LIF)的mRNA水平变化;ELISA法检测共培养体系上清液中LIF的水平。结果:分离的h ASCs经流式细胞术鉴定可基本判定为h ASCs;PBMCs经磁珠法分选后获得90%以上纯度的CD4+T细胞。共培养后,1∶4组和1∶10组中Th17细胞所占比例下降,且存在高浓度抑制效应;共培养后RORγt、IL-6R和IL-23R的mRNA表达水平下降,LIFR和LIF的mRNA表达水平均升高;加入anti-LIF抗体后,Th17细胞比例回升至对照组水平;RORγt和IL-6R的mRNA表达水平回升;ELISA检测各组LIF的水平,共培养组LIF分泌均较对照组明显增多,加入anti-LIF抗体后明显减少。结论:h ASCs可抑制MS患者Th17细胞的分化,其作用可能与其分泌LIF、通过IL-6/LIF轴竞争性抑制有关。AIM： To investigate how human adipose-derived stem cells（ h ASCs） regulates the differentiation of Th17 cells in multiple sclerosis. METHODS： h ASCs were isolated from the adipose tissues. Magnetic-activated cell sorting（ MACS） kit was used to isolate CD4＋T cells from peripheral blood mononuclear cells（ PBMCs） which were isolated by density gradient centrifugation. The percentage of CD4＋T cells was detected by flow cytometry. The activated CD4＋T cells were co-cultured with h ASCs for about 4 d at different ratios of h ASCs to CD4＋T cells（ 1∶ 4 and 1∶ 10） in a Th17 polarised condition. Another group adding anti-leukemia inhibitory factor（ LIF） antibody was set up. Th17 cell proportion of the CD4＋T cells was determined by flow cytometry. The level of LIF in the supernatant of co-cultured system was measured by ELISA. The mRNA expression of retinoid-related orphan receptor γt（ RORγt）,interleukin-6 receptor（ IL-6R）,interleukin-23 receptor（ IL-23R）,LIF and leukemia inhibitory factor receptor（ LIFR） was detected by real-time PCR.RESULTS： The result of flow cytometry suggested there were mainly h ASCs,and the percentage of CD4＋T cells in the PBMCs were above 90% after MACS. The Th17 cell proportion decreased in 1∶ 4 and 1∶ 10 co-cultured groups in a dosedependent manner. The mRNA expression of IL-6R,IL-23 R and RORγt was downregulated and the expression of LIFR and LIF was up-regulated. When the anti-LIF was added into the co-cultured system,the ratio of Th17 cells increased and reached to the control level. The protein level of LIF obviously increased after co-cultured. After anti-LIF added,the mRNA expression of RORγt and IL-6R was up-regulated. CONCLUSION： h ASCs inhibits the differentiation of Th17 cells from multiple sclerosis patients through the competitive inhibition of LIF / IL-6 by secreting LIF.

关 键 词：多发性硬化 脂肪源性干细胞 TH17细胞 白血病抑制因子 白细胞介素6 

分 类 号：R741.05[医药卫生—神经病学与精神病学]