骨髓间充质干细胞在体外肠上皮环境下免疫调节的作用机制

The immunoregulation mechanism of bone marrow mesenchymal stem cells on T lymphocyte in the existence of CaCo-2 in vitro

目的观察异基因骨髓间充质干细胞(mesenchymal stem cells,MSCs)在体外肠上皮细胞环境中产生免疫调节的机制。方法挑选培养状态良好的结肠癌细胞系CaCo-2,提取Wistar大鼠骨髓MSCs及新鲜脾淋巴细胞做共培养。实验分4组,实验组为脾淋巴细胞、骨髓MSCs、CaCo-2共培养+刀豆蛋白A(ConA),对照组为脾淋巴细胞、CaCo-2共培养+ConA,增殖组为脾淋巴细胞+ConA,空白组为单纯脾淋巴细胞。分别于培养0、24、48 h后,MTT检测T细胞活性,ELISA检测细胞因子肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)及转化生长因子-β(TGF-β)水平,流式检测Foxp3+调节性T细胞表达率。结果在单层肠上皮共培养环境下,与对照组比较,实验组T细胞活性显著降低(P<0.05),TNF-α也有显著降低(P<0.05),IL-10、TGF-β表达水平有显著升高(P<0.05);Foxp3+调节性T细胞表达率升高;T细胞活性随着时间延长有逐渐降低趋势(P<0.05)。结论在体外单层肠上皮细胞环境中,骨髓MSCs可以通过直接抑制T细胞活化产生TNF-α抑制免疫应答,并可间接通过自分泌及诱导调节性T细胞产生并分泌免疫抑制因子IL-10、TGF-β下调免疫反应,产生免疫耐受。

Objective To investigate the immunoregulation mechanism of allogeneic bone marrow mesenchymal stem cells （BMMSCs） on T lymphocyte cell in the existence of CaCo -2. Methods BMMSCs and T lymphocyte cells of Wistar rats were prepared from bone marrow and spleen, respectively. Human colon carcinoma cell line （ CaCo - 2 ） in good condition was selected as a model system for intestinal epithelium. Four groups were designed for experiment： experiment group （splenic lymphocytes/BMMSCs/CaCo-2 ＋ ConA）, control group （splenic lymphocytes/CaCo-2 ＋ ConA）, proliferation group （splenic lymphoeytes ＋ ConA）, and blank group （splenic lymphocytes）. The vitality and the expression of TNF - oL, IL - 10 and TGF - 13 were assessed at baseline, 24th and 48th hours. Results Compared with control group, the T lymphocyte cell proliferation was significantly reduced in experimental group, so was the TNF - ct, while the IL - 10 and TGF - 13 were significantly elevated （ P 〈 0. 05 ）. The regulatory T cells in experimental group were significantly increased than those in control group, though the activity of the regulatory T cell was significantly trimmed down with the extent of time （ P 〈 0. 05 ）. Conclusion In the existence of CaCo - 2, T lymphocyte cell proliferation is reduced, so is the TNF - a. But BMMSCs can secrete IL - 10 and TGF - 13 and boost the level of regulatory T cell to promote immune tolerance.