脐带间充质干细胞上调系统性红斑狼疮患者调节性T细胞的机制研究  

Mechanism of umbilical cord mesenchymal stem cells in the up-regulation of regulatory T cells by transforming growth factor β1 in systemic lupus erythematosus

作　　者：鲁琳[1] 王丹丹[2] 李霞[2] 曾小峰[3] 孙凌云[1] 

机构地区：[1]南京医科大学附属鼓楼临床医学院风湿免疫科,210008 [2]南京大学医学院附属鼓楼医院风湿免疫科 [3]中国医学科学院北京协和医学院北京协和医院风湿免疫科

出　　处：《中华医学杂志》2013年第13期980-983,共4页National Medical Journal of China

基　　金：国家自然科学基金重大国际合作项目（81120108021）;国家自然科学基金（30972736）;国家“十一五”科技支撑计划（2008BA159802）;江苏省自然科学基金（BK2009034）;江苏省重大成果转化项目（BA2009124）;江苏省科教兴卫工程项目

摘　　要：目的探讨脐带间充质干细胞（UC-MSC）上调系统性红斑狼疮（SLE）患者外周血调节性T细胞（Treg）的作用机制。方法20例SLE患者及正常人外周血单个核细胞（PBMC）分别与UC-MSC以不同比例共培养72h，流式细胞术检测PBMC中CIM’CD25’Foxp3’Treg细胞比例。分别以SLE患者及正常人PBMC及血清刺激UC-MSC，实时荧光定量聚合酶链反应检测UC-MSCTGF-B1mRNA表达水平，酶联免疫吸附试验（ELISA）检测共培养上清TGF-p1表达水平。并构建TGF-B1小干扰RNA（siRNA）干扰UC-MSCTGF-p1表达及在共培养体系中加入TGF-B1抑制剂，探讨UC-MSC对SLE患者Treg细胞的调节作用。结果UC-MSC呈剂量依赖性上调SLE患者外周血Treg细胞，且不依赖细胞直接接触，而对正常人外周血Treg细胞无调节作用。与无刺激组及健康志愿者PBMC刺激组相比，SLE患者PBMC显著促进UC-MSCTGF-β1mRNA表达（1．00±0．09，1．95±0．62，4．20±2．34，P〈0．05），细胞培养上清TGF-β1水平显著升高。SLE患者血清（5％）促进UC-MSC表达TGF-β1mRNA（12．19±4．49），显著高于胎牛血清及正常人血清培养组（1．33±0．06，2．53±0．72），P〈0．01。TGF-β1siRNA干扰的UC-MSC对SLE患者外周血Treg细胞无上调作用（SLEPBMC＋TGF-β1siRNAUC-MSC组2．33％±0．99％，SLEPBMC组1．80％±0．65％，P〉0．05）。此外，TGFN特异性抑制剂SB431542显著抑制UC-MSC对SLE患者外周血Treg细胞的调节作用（UC-MSC＋SIM31542组4．58％±2．10％，UC-MSC组7．85％±3．54％，P〈0．05）。结论SLE患者免疫微环境可显著刺激UC-MSC表达TGF-β1，后者在UC-MSC上调SLE患者Treg细胞中发挥重要作用。Objective To explore the mechanism of umbilical cord mesenchymal stem cells （UC- MSC） in the up-regulation of peripheral regulatory T cells in patients with systemic lupus erythematosus （SLE）. Methods Peripheral blood mononuclear cells （PBMC） from 20 SLE patients and normal controls were co-cuhured with UC-MSC at different ratios respectively for 72 hours. And the proportions of CIM ＋ CD25 ＋ Foxp3 ＋ regulatory T cells were analyzed by flow cytometry. PBMC and sera from active SLE patients and normal controls were used to stimulate UC-MSC. The expressions of transforming growth factor β1 （TGF-β1） on UC-MSC were detected by real-time fluorescence quantitative polymerase chain reaction （real-time PCR）. The supernatant level of TGF-β1 was determined by enzyme-linked immunosorbent assay （ELISA）. And the TGF-β1 small interfering RNA （siRNA） was used to interfere with the TGF-β1 expression on UC-MSC and determine its effect on the regulation of SLE Treg ceils. TGF-β1 inhibitor was added into the culture system of UC-MSC and PBMC from active SLE patients to observe its role on the up- regulation of Treg ceils by UC-MSC. Results UC-MSC could dose-dependently up-regulate the peripheral CD4 ＋ CD25 ＋ Foxp3 ＋ Treg proportion in SLE patients. And such an effect was not dependent on cell-cell contact. UC-MSC had no regulatory effect on Treg cells in normal controls. Compared with the non- stimulated and normal PBMC stimulated groups, PBMC from SLE patients significantly promoted TGF-β1 mRNA expression on UC-MSC （ relative gene expression was 1.00 ± 0. 09, 1.95 ± 0. 62, 4. 20 ±2. 34 respectively, both P 〈 0. 05 ）. The supernatant level of TGF-β1 was significantly elevated in the presence of SLE PBMC. Sera of SLE patients （5%） enhanced TGF-β1 mRNA expression on UC-MSC and it was significantly higher than fetal bovine serum cultured group （ 12. 19 ±4. 49 vs 1.33 ±0. 06, P 〈 0. 01 ） and normal individual sera cultured group （2. 53 ± 0. 72, P 〈 0. 01 ）. Add

关 键 词：红斑狼疮 系统性 间充质干细胞 调节性T细胞 转化生长因子Β1 

分 类 号：R593.241[医药卫生—内科学]