乙型肝炎肝硬化患者骨髓间充质干细胞对自体CD4^+T细胞体外免疫效应研究  

The immunoregulation effect of bone marrow mesenchymal stem cells cultured with autologous CD4^+T cells from HBV-related cirrhosis

作　　者：许烂漫[1] 林秀丽[1] 林镯[1] 李骥[1] 陈迎晓[1] 董培红[1] 陈永平[1] 

机构地区：[1]温州医科大学附属第一医院,温州医科大学肝病研究所,温州市肝脏病学重点实验室,浙江省温州325000

出　　处：《现代实用医学》2017年第6期707-711,841,共6页Modern Practical Medicine

基　　金：国家自然科学基金资助项目(81570514;81500477);浙江省自然科学基金(LY15H030017;LQ15H030006);温州市科技局项目(Y20150014);北京医学奖励基金(YJHYXKYJJ-162);温州市终末期肝病的预警与干预治疗科技创新团队(C20150005)

摘　　要：目的比较乙型肝炎(乙肝)肝硬化组和正常组之间骨髓间充质干细胞(BMMSCs)对自体CD4+T细胞的体外免疫效应,探索BMMSCs对乙肝肝硬化患者的免疫调节机制。方法所有临床标本均为温州医科大学附属第一医院乙肝肝硬化患者(10例)骨髓和外周血,另选取健康志愿者5例(正常组)。两组CD4+T细胞与自体BMMSCs分别以1∶0、1∶10、1∶20、1∶40比例共培养,72 h后收集细胞及培养上清,用ELISA法测上清中白介素-6(IL-6)、肿瘤坏死因子-(TNF-)、转化生长因子-(TGF-)及白介素-17(IL-17)水平;实时定量PCR测胞内Treg细胞、Th17细胞特异性转录因子叉头状转录因子3(FoxP3)m RNA、维甲酸相关孤独受体t(rRORt)mRNA的表达量。结果乙肝肝硬化组1∶0培养上清中IL-6、TNF-、IL-17含量显著高于正常组(均<0.05)。与不同比例BMMSCs共培养后,IL-17、TNF-水平显著性下降(均<0.05),并随培养比例的加大更为显著。IL-6水平在1∶0、1∶10、1∶40比例肝硬化组较正常组低,差异均有统计学意义(均<0.05)。与正常组相比,乙肝肝硬化组TGF-出现显著升高,并随培养比例的增加而增加(均<0.05)。乙肝肝硬化组Fox P3m RNA的表达在1∶20、1∶40比例与正常组相比升高均更为显著(均<0.05);而ROR t m RNA的表达受到抑制,1∶10、1∶20、1∶40均出现显著性下降(均<0.05)。结论乙肝肝硬化来源的BMMSCs对自体CD4+T细胞具有负向免疫调节和抑炎作用,并可能与肝硬化炎症微环境密切相关。Objective To study the different immunoregulation effects of bone marrow mesenchymal stem cells （BMMSCs） cultured with autologous CD4＋ T cells from HBV-related cirrhosis patients and normal people.Methods All samples （bone marrow and peripheral blood） were collected from the First Affiliated Hospital of Wenzhou Medical University. There were two groups： HBV-related cirrhosis group （/2= l〇 ） and the normal group （r2=5）.BMMSCs from both groups were co-cultured with autologous CD4＋ T cells as the ratios of 1：0,1：10,1：20,1：40. Then cultured supernatants and cells were collected for ELISA （ IL-6, TNF-a, TGF-p and IL-17） and real-time PCR （FoxP3、 ROR-yt mRNA） detection. Results The levels o f inflammatory cytokines such as IL-6, TNF-a and IL-17 in the ratio of 1：0 were more higher than those in normal group （allP 〈 0.05）. After co-culture with BMMSCs in dif-ferent ratios, the levels of TNF-a and IL-17 decreased significantly in cirrhosis group （allP 〈 0.05）. The level of IL-6 in cirrhosis group was lower than that in normal group with ratios of 1：0, 1： 10, 1：40 （all P 〈 0.05）. However, the level of TGF-p in cirrhosis group increased significantly compared to that in normal group （P 〈 0.05）. In cirrhosis group, there was a significant elevation of FoxP3 mRNA and a significant reduction of RORyt mRNA by the co-cul-ture of BMMSCs in a dose dependent manner compared with those in the normal group （all_P 〈 0.05）.Conclusions There are negative immunoregulation effect and anti-inflammatory potential of BMMSCs cultured with autolog-ous CD4＋ T cells from HBV-related cirrhosis, and the inflammatory environment of cirrhosis may contribute to this.

关 键 词：乙型肝炎 肝硬化 骨髓间充质干细胞 CD4+T细胞 

分 类 号：R575.2[医药卫生—消化系统]