大鼠骨髓间充质干细胞对免疫耐受的作用

Effect of mesenchymal stem cells from rat bone marrow on immunological tolerance

目的:体外分离培养大鼠骨髓间充质干细胞(Bone Marrow Mesenchymal Stem Cells,BMSCs),观察和检测其生长特性及表面标记,探讨它对免疫耐受的作用。方法:通过密度梯度离心、贴壁筛选法分离培养大鼠BMSCs,观察细胞形态学变化,应用流式细胞术测定其表面分子CD34、CD44的表达。将培养第3代的BMSCs经尾静脉输注大鼠,应用流式细胞技术检测大鼠外周血中CD4+CD25+Treg比率,通过3H-TdR掺入实验检测BMSCs对大鼠胸腺细胞自发增殖反应的影响。结果:体外培养的大鼠BMSCs呈贴壁生长,似成纤维细胞样,表面分子CD44表达阳性率为98,但CD34表达阴性。输注BMSCs的大鼠外周血中CD4+CD25+Treg占2.5±0.69,明显高于对照组(0.8±0.14)和PBS输注组(1.0±0.23),P<0.05;胸腺细胞自发增殖活性(cpm值3275.41±1736.05)明显低于正常对照组(7368.25±2532.13)和PBS输注组(6143.71±2380.47),P<0.05。结论:体外培养可获得形态单一、生长稳定、增殖活性较强的BMSCs,BMSCs能增强大鼠的免疫耐受作用。

Objective：To investiate biological properties of Mesenchymal stem cell（BMSC） from rat bone marrow in vitro,and their effects in inducing immune tolerance.Methods：BMSCs were obtained from rat bone marrow by density gradient centrifugation and selected by cell attachment.The morphology of BMSCs was observed by electron microscope.The expression of surface molecules of CD34 and CD44 was analyzed by flow cytometry.The cells were injected ex vivo intravenously after in vitro culture and then CD4^＋ CD25^＋ Treg ratio were determined.The proliferation of thymic cells was estimated by 3H-TdR incorporation method.Results：BMSCs were fibroblast-like cells.The expression ratio of CD44 in BMSCs was 98%,but expression of CD34 was negative.After administration of BMSCs,the ratio of CD4^＋CD25^＋Treg（2.5%±0.69%） from peripheral blood of rats was higher than in normal group（0.8%±0.14%） and PBS control group（1.0%±0.23%）,P〈0.05.Proliferation of thymic cells（cpm is 3 275.41±1 736.05）was lower than in both normal group（7 368.25±2 532.13） and PBS administration group（6 143.71±2 380.47）,P〈0.05.Conclusion：BMSCs can be cultured well in vitro.They have strong inhibitory effects on proliferation of thymic cells and reinforce immune tolerance.