间充质干细胞对异体B淋巴细胞增殖和凋亡的影响

目的 探讨骨髓间充质干细胞(MSC)在体外对异体外周血B淋巴细胞增殖和凋亡的影响.方法 从骨髓中分离、培养MSC.从外周血中分离单个核细胞,L-亮氨酸甲酯去除单核细胞,以2-氨乙基硫脲溴化物(AET)处理绵羊红细胞(SRBC)的花环形成法,去除T淋巴细胞获得纯化的B淋巴细胞.用羊抗人IgM单克隆抗体(Anti-IgM,终浓度10μg/ml)刺激与不同比例的MSC(B:MSC 50:1、10:1、1:1)及不同浓度的MSC培养上清(12.5%、25%、50%)共培养的B淋巴细胞,72 h后四甲基偶氮唑蓝(MTT)比色分析测B淋巴细胞的增殖.B淋巴细胞与MSC等比例,加或不加Anti-IgM,共培养24、48 h后,流式细胞术检测B淋巴细胞的凋亡.结果 10:1、1:1组B淋巴细胞A值分别为0.418±0.103、0.365±0.114,显著低于对照组的0.679±0.049(P＜0.01),1:1组显著低于50:1组(P＜0.05);50%MSC上清组B淋巴细胞A值(0.504±0.099),同对照组相比差异有统计学意义(P＜0.05),并且显著低于12.5%组(0.669±1.023,P＜0.05).MSC不诱导B淋巴细胞的凋亡;B淋巴细胞与Msc共培养24、48 h,加或不加Anti-IgM,各组细胞凋亡率为(1.90±0.75)%、(2.33±1.01)%、(2.33±0.75)%、(1.39±0.63)%,组间比较差异无统计学意义(P＞0.05).结论 MSC及其上清抑制B淋巴细胞增殖,作用机制与MSC细胞数量和MSC分泌的细胞因子有关,但是在体外MSC不诱导B淋巴细胞的凋亡.

Effects of mesenchymal stem cells on proliferation and apoptosis of allogeneic B lymphocytes

Objective To study the influenee of bone marrow mesenehyrnal stem cells(MSC)on the proliferation and apoptosis of allogeneic peripheral B lymphocytes in vitro.Methods MSCs were jsolated from the peripheral blood of healthy volunteer and cultured.B lymphocytes were isolated from another healthy volunteer and co-cultured with the MSCs at the B:MSC ratios of 50:1,10:1,and 1:1 or with different concentrations of MSC supernatant(12.5%,25%,and 50%)for 72 h,in the presence of antihuman IgM immunoglobubin goat antibodies(Anti-IgM)at a final concenrtration of 10 μg/mlL.The proliferation of B lymphocytes was analyzed with MTT assay.B lymphocytes and MSC were co-cultured at the ratio of 1:1 for 24 h or 48 h,with or without addition of Anti-IgM.Flow cytometric was used to detect the apoptosis of B Jymphocytes.Results The A valne of B lymphocytos co-cultured with MSCs at different ratios were 0.521±0.093,0.418±0.103,and 0.365±0.114 respectively.The A values of Group10:1 and Ggroup1:1 were both significantly lower than that of the control group(0.679±0.049,both P＜0.01),and the A value of Ggroup1:1 was significantly lower than that of Group10:1(P＜0.05).The A value of B lymphocytes co-cultured with 50% MSC supernatant was 0.504±0.099,significantly lower than those of the control group and Group 12.5%(both P＜0.05).MSCs didn't induee apoptosis of B lymphocytes.The apoptosis rates of B lympbocytes co-cultured with MSCs for 24 h or 48 h,in presence or absence of Anti-IgM were 1.90%±0.75%,2.33%±1.01%,2.33%±0.75%,and 1.39%±0.63% respectively,without significant difference between any 2 of the four groups.Conclusion MSC and its sulpernatant inhibit B lymphocyte proliferation with the mechanism correlated with the MSC concentration and the MSC-secreted cytokine,but MSCs does not induce B lymphocytes apoptosis in vitro.