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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72 h,流式细胞术检测PBMC中CI4+ 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-MSC TGF-β1mRNA表达水平,酶联免疫吸附试验(ELISA)检测共培养上清TGF-β1表达水平.并构建TGF-β1小干扰RNA(siRNA)干扰UC-MSC TGF-β1表达及在共培养体系中加入TGF-β1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-MSC TGF-β1 mRNA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-β1 mRNA(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-β1 siRNA干扰的UC-MSC对SLE患者外周血Treg细胞无上调作用(SLE PBMC+ TGF-β1 siRNA UC-MSC组2.33％±0.99％,SLE PBMC组1.80％±0.65％,P＞0.05).此外,TGFβ1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+ SB431542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-cultured with UC-MSC at different ratios respectively for 72 hours.And the proportions of CD4 +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 (realtime 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 cells TGF-β1 inhibitor was added into the culture system of UC-MSC and PBMC from active SLE patients to observe its role on the upregulation of Treg cells 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 nonstimulated 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).Additionally,TGF-β1 siRNA interfered UC-MSC failed to up-regulate Treg cells in SLE patients.Furthermore,TGF-β1 specific inhibitor SB431542 significantly inhibited the regulatory role of UC-MSC on Treg cells in SLE patients Conclusion Immune microenvironment in SLE patients can significantly stimulate the TGF-β1 expressiou on UC-MSC and plays an important role in the up-regulation of Treg cells in patients.