间充质干细胞对系统性红斑狼疮CD4^＋Foxp3^＋T淋巴细胞的调节

Regulation of mesenchymal stem cells on CD4^＋ Foxp3^＋ T cells in systemic lupus erythematosus

目的探讨同种异体骨髓间充质干细胞（MSC）体内外对系统性红斑狼疮（SLE）患者外周血CD4＋Foxp3^＋T淋巴细胞及人脐带MSC移植对MRLOpr鼠睥脏和淋巴结CD4＋Foxp3^＋T淋巴细胞水平的影响。方法血缘相关供者骨髓中分离培养MSC移植治疗5例SLE患者，采用流式细胞术检测移植前后外周血CD4＋FoxD3^＋T淋巴细胞百分率。7例SLE患者外周血单个核细胞（PBMC）分别与SLE患者和正常人骨髓MSC按不同比例体外共培养72h，检测共培养后PBMC中CD4＋Foxp3^＋T淋巴细胞百分率。MRL／1pr鼠输注脐带MSC后检测脾脏和淋巴结CD4＋Foxp3^＋T淋巴细胞百分率。结果SLE患者异基因骨髓MSC移植后1周外周血CD4＋Foxp3^＋T淋巴细胞百分率（4．8±1．6）％和移植后3个月（6．0±2．6）％均较移植前（2．1±1．2）％明显升高（5例，P〈0．05）。正常骨髓MSC与SLE患者PBMC共培养后CD4＋Foxp3^＋T淋巴细胞百分率明显升高（P〈0．05），且存在剂量依赖性，狼疮MSC也可上调SLE患者CD4＋Foxp3^＋T淋巴细胞水平，但作用较正常MSC弱（P〈0．05）；正常MSC培养上清也可上调SLE患者PBMC中CD4＋Foxp3^＋T淋巴细胞水平，但作用弱于MSC：PBMC=1：1组（P〈0．05）。MRL／Ipr鼠经1次或3次脐带MSC移植后脾脏CD4^＋Foxp3^＋T淋巴细胞百分率均较对照组高（P〈0．05），但淋巴结CD4＋Foxp3^＋T淋巴细胞百分率均较对照组低（P〈0．01），1次和3次移植组间差异无统计学意义：结论异基因甚至异种MSC移植可上调SLE患者或MRL／Ipr鼠CD4^＋Foxp3^＋T淋巴细胞水平，同时体外试验也得出相同结论，且体外上调作用呈一定剂量依赖性，CD4^＋Foxp3^＋T淋巴细胞水平上调可能是MSC移植治疗SLE有效的机制之一。

Objective To investigate the in rivo or in vitro immune regulatory effects of allogeneic bone-marrow mesenehymal stem cells （MSC） and human umbilical cord MSC on CD4^＋ Foxp3^＋ T cells in the peripheral blood of patients with systemic lupus erythematosus （SLE） and in the spleen of MRL/lpr mice. Methods Human MSC were isolated and expanded from bone marrow cells of healthy donors and infused into five SLE patients. The percentages of CD4^＋ Foxp3^＋ T cells in peripheral blood were detected by flow cytometry. Human peripheral blood mononuclear cells （PBMC） were prepared by centrifugation on a Ficoll Hypaque density gradient. The MSC and PBMC from unrelated donors （MSC： PBMC =1：1, l：10, 1：50） were added into 24-well plates. After 7211 of co-cuhure, the percentages of CD4^＋ Foxp3^＋ T cells were detected by flow cytometry. Twenty four 18-week-old MRUIpr female mice were divided into 3 groups and were injected with umbilical cord MSC （1 ×10^6 cells for one time, 1 ×10^6 cells for three times and 0.5 ml sodium chloride as control respectively）. The percentages of CD4^＋ Foxp3^＋ T cells in spleen and lymphoid nodes were detected by flow cytometry. Results The percentages of blood CD4^＋ Foxp3^＋ T cells at one week （4.8±1.6）% and at three months （6.0±2.6）% post MSC transplantation for patients with SLE were both higher than that before transplantation （2.1 ±1.2）% （n=5, P〈O.053. The co-cuhure of normal bone marrow MSC with PBMC from SLE patients resulted in a statistically significant increase of CD4^＋ Foxp3^＋ T cells percentage in PBMC on a dose dependent manner （P〈0.05）. The percentages of CD4^＋ Foxp3^＋ T cells of PBMC from SLE patients coultured with lupus MSC were lower than that of normal MSC （P〈0.05）. The cultured supernatant of normal MSC also upregulated the percentages of CD4^＋ Foxp3^＋ T cells of PBMC from SLE patients （P〈O.05）. The MRL/lpr mice that had been injected umbilical cord MSC for one time and three times had higher percentages of CD4^＋ Foxp3^＋ T cells in the spleen but lower in the lymphoid nodes as compared with controls （P〈0.01）, but without statistical significant difference. Conclusion Allogeneic or heterogeneic MSC transplantation upregulates the percentages of CD4^＋ Foxp3^＋ T cells in SLE patients or in MRL/Ipr mice. Upregulation of Treg population may be one of the mechanisms of MSC transplantation that is effective for SLE treatment.