Yang Jing, Wang Qing-Hai, Zeng Qiu-Tang, Mao Xiao-Bo
Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2007 Aug;15(4):785-9.
The study was purposed to investigate the immune regulatory effects of human bone marrow mesenchymal stem cells (hMSCs) on Foxp3 expressing CD4(+)CD25(+) regulatory T cells and to explore the mechanism of immune modulation by hMSCs. Human MSCs were isolated and expanded from bone marrow cells, and identified with cell morphology, and the phenotypes were assessed by immunohistochemistry. Human peripheral blood mononuclear cells (hPBMNCs) were prepared by centrifugation on a Ficoll Hypaque density gradient. The hMSCs (1 x 10(3), 1 x 10(4), 1 x 10(5)) were added into wells containing hPBMNCs (1 x 10(6)) from an unrelated donor in the presence of rhIL-2. After 5 days of co-culture, the percentage of CD4(+)CD25(+) T cells was detected by flow cytometry. T cell proliferation was assessed by [(3)H] thymidine incorporation using a liquid scintillation counter. The expression of Foxp3 in CD4(+)CD25(+) T cells was detected by reverse transcription polymerase chain reaction (RT-PCR). Cytokines (TGF-beta, IL-12, IFN-gamma, IL-10) concertrations of cultured supernatants were measured with ELISA. The results indicated that in all the experiments, the presence of hMSCs with hPBMNCs resulted in a statistically significant decrease in T cell proliferation, in dose-dependent manner. The increase of percentage of CD4(+)CD25(+) T cells in the peripheral CD4(+) T cell was observed after coculturing lymphocytes with hMSCs (p < 0.01). The expression of Foxp3-mRNA (Foxp3/beta-actin) in hMSCs groups was significantly higher than that in the control and was negatively associated with the value of CPM representing T proliferation. The levels of TGF-beta and IL-10 were higher in hMSCs groups than that in the control, and the levels of TGF-beta and IL-10 correlated positively with Foxp3-mRNA expression and the percentage of CD4(+)CD25(+) T cells. However, the secretion of IL-12 and IFN-gamma was significantly attenuated by hMSCs coculture, and there was no correlation with Foxp3-mRNA expression and the percentage of CD4(+)CD25(+) T cells. It is concluded that the Foxp3 expressing regulatory T cells may play an important role in the immune regulatory by hMSCs. Its mechanism is related to that the hMSCs-mediated TGF-beta and IL-10 convert CD4(+)CD25(-) T cells into CD4(+)CD25(+) T regulatory T cells, which specifically inhibits the proliferation of T cells.
本研究旨在探讨人骨髓间充质干细胞（hMSCs）对表达Foxp3的CD4(+)CD25(+)调节性T细胞的免疫调节作用，并探索hMSCs免疫调节的机制。从骨髓细胞中分离并扩增人骨髓间充质干细胞，通过细胞形态进行鉴定，并用免疫组织化学评估其表型。通过在Ficoll Hypaque密度梯度上离心制备人外周血单个核细胞（hPBMNCs）。将hMSCs（1×10³、1×10⁴、1×10⁵）加入含有来自无关供体的hPBMNCs（1×10⁶）的孔中，并加入重组人白细胞介素-2（rhIL-2）。共培养5天后，通过流式细胞术检测CD4(+)CD25(+) T细胞的百分比。使用液体闪烁计数器通过[³H]胸苷掺入法评估T细胞增殖。通过逆转录聚合酶链反应（RT-PCR）检测CD4(+)CD25(+) T细胞中Foxp3的表达。用酶联免疫吸附测定（ELISA）法检测培养上清液中细胞因子（转化生长因子-β（TGF-β）、白细胞介素-12（IL-12）、干扰素-γ（IFN-γ）、白细胞介素-10（IL-10））的浓度。结果表明，在所有实验中，hMSCs与hPBMNCs共同存在导致T细胞增殖在统计学上显著降低，呈剂量依赖性。淋巴细胞与hMSCs共培养后，外周CD4(+) T细胞中CD4(+)CD25(+) T细胞的百分比增加（p < 0.01）。hMSCs组中Foxp3信使核糖核酸（Foxp3/β-肌动蛋白）的表达明显高于对照组，且与代表T细胞增殖的每分钟计数（CPM）值呈负相关。hMSCs组中TGF-β和IL-10的水平高于对照组，TGF-β和IL-10的水平与Foxp3信使核糖核酸表达及CD4(+)CD25(+) T细胞的百分比呈正相关。然而，hMSCs共培养显著减弱了IL-12和IFN-γ的分泌，且与Foxp3信使核糖核酸表达及CD4(+)CD25(+) T细胞的百分比无关。结论是，表达Foxp3的调节性T细胞可能在hMSCs的免疫调节中起重要作用。其机制与hMSCs介导的TGF-β和IL-1将CD4(+)CD25(-) T细胞转化为CD4(+)CD25(+)调节性T细胞有关，后者特异性抑制T细胞增殖。
