同种异基因大鼠间充质干细胞移植对体内CD4^+CD25^+T细胞的影响

Effects of allogeneic rat mesenchymal stem cell transplantation on CD4^+CD25^+T regulator lymphocytes in vivo

背景:间充质干细胞的免疫抑制作用近年来逐渐得到证实并开始应用于临床,但相关研究成果主要来源于体外细胞实验。目的:观察同种异基因大鼠骨髓间充质干细胞从静脉输入后对体内CD4+CD25+调节性T细胞的影响。设计、时间及地点:以动物为对象的对照观察实验,于2008-03/09在南方医科大学珠江医院移植免疫研究所完成。材料:Wistar大鼠6只用于制备骨髓间充质干细胞,SD大鼠20只作为输注骨髓间充质干细胞的受体鼠。方法:从Wistar大鼠骨髓分离培养间充质干细胞,取第3~5代细胞进行实验。①体外淋巴细胞增殖实验:首先将间充质干细胞悬液从尾静脉注入SD大鼠体内10d后脱臼处死,取大鼠脾脏制备脾淋巴细胞。实验分5组:分别为脾淋巴细胞/骨髓间充质干细胞为1∶10,1∶50,1∶100+ConA5μg组,脾淋巴细胞+ConA5μg组(增殖组),单纯淋巴细胞组(空白组),检测共培养体系中CD4+CD25+/CD4+T淋巴细胞比率。②体内注射骨髓间充质干细胞实验:将5×109L-1,5×108L-1,5×107L-1间充质干细胞及PBS静脉输入SD大鼠体内,10d后取受体鼠胸腺、脾脏、外周血检测CD4+CD25+/CD4+T淋巴细胞比率。主要观察指标:①淋巴细胞增殖体系中CD4+CD25+/CD4+比率的变化。②SD大鼠胸腺、脾脏、外周血CD4+CD25+/CD4+比率的变化。结果:①体外淋巴细胞共培养体系中,1∶10组T细胞亚群CD4+CD25+/CD4+细胞百分率较增殖组显著升高(P<0.01)。②体内输注间充质干细胞达5×109L-1的受体鼠外周血和脾脏CD4+CD25+/CD4+T细胞比率上升,与输注PBS组相比有显著差异(P<0.05),而在胸腺中各组比率无明显差异。结论:高浓度同种异基因大鼠间充质干细胞不仅可以在体外实验中增加CD4+CD25+/CD4+T细胞比率,静脉输入后仍具有相同作用。

BACKGROUND： More recently, the immunosuppressive treatment of mesenchymal stem cells （MSCs） has been also been confirmed and proposed in patients. However, relevant study outcomes are from in vitro cell study. OBJECTIVE： To investigate effects of allogeneic mesenchymal stem cells on CD4^＋CD25^＋T regulator lymphocytes injecting via the vein. DESIGN, TIME AND SETTING： A controlled animal trial was performed at the Institute of Transplantation Immunology, Zhujiang Hospital, .Southern Medical University between March and September 2008. MATERIALS： Six Wistar rats were used to prepare MSCs, and twenty Sprague Dawley rats were recipients. METHODS： Mesenchymal stem cells were harvested from Wistar rat bone marrow. The third to fifth cells were used for this study. ① In vitro lymphocyte proliferation study： MSC suspension was infused into Sprague Dawley rats via the tail vein. Ten days later, these rats were sacrificed by luxation. The spleen was obtained to prepare splenic lymphocytes. There were 5 groups, comprising splenic lymphocyte/bone marrow MSCs 1： 10, 1： 50, 1：100 ＋ ConA 5 u g groups, splenic lymphocyte ＋ ConA 5 u g group （proliferation group）, and lymphocyte group （blank group）. CD4^＋CD25^＋ / CD4^＋ T lymphocyte ratio was detected in coculture medium. ②In vivo bone marrow MSC infusion study： 5×10^9/L, 5×10^8/L, 5×10^7/L MSCs and phosphate buffered saline were infused into allogeneic Sprague Dawley rats via intravenous infusions. Proportion of CD4^＋CD25^＋ / CD4^＋ T cells in peripheral blood, spleen and thymus were analyzed 10 days later. MAIN OUTCOME MEASURES： ①Variation of CD4^＋CD25^＋/CD4^＋ cultured MSCs with T lymphocytes in vitro; ②The changes in CD4^＋CD25^＋/CD4^＋ in thymus, spleen and peripheral blood. RESULTS： In vitro cocultured system, blood and spleen, percentage of subgroup CD4^＋ CD25^＋ T/CD4^＋ cells was significantly greater in the 1 ： 10 groups compared with the proliferation group （P 〈 0.01）. Ratio of CD4^＋CD25^＋/CD4^＋ T cells in the peripheral blood and spleen was increased in receptor rats infused with 5×10^9/L MSCs, which had significant difference compared with the phosphate buffered saline group （P 〈 0.05）. No significant difference was detected in the thymus in each group. CONCLUSION： High concentration of MSCs increases the proportion of CD4^＋CD25^＋ / CD4^＋ not only in vitro, but also in vivo via the vein infusion.