摘要
目的探讨骨髓间充质干细胞(bone marrow mesenchymal stem cells,MSCs)在体外对异体外周血B淋巴细胞的免疫调节作用。
方法密度梯度离心法从骨髓中分离单个核细胞,加入含10%胎牛血清的LG-DMEM培养液,将细胞的密度调整到2×10~5个/ml,接种到培养瓶中,48 h后更换新鲜培养液,以后每2～3 d换液1次。当细胞铺满培养瓶底90%以上时,按常规方法进行细胞传代培养。用流式细胞术检测培养细胞的CD3、CD4、CD8、CD13、CD22、CD33、CD34、CD44、HLA-DR表达情况。常规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)处理的绵羊红细胞(SRBC)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)刺激与或未与MSCs及其培养上清共培养3天的B淋巴细胞,应用MTT法测B淋巴细胞的增殖,ELISA法测培养上清中免疫球蛋白IgG、IgM的产生,应用流式细胞仪分别检测与MSCs共培养24h、48h后B淋巴细胞的凋亡。
结果实验中观察到,梯度离心所得单个核细胞接种2～3h细胞开始贴壁,24h后贴壁细胞数目不再增加,1～2w后开始迅速增殖,细胞多为梭形,培养至3w周左右贴壁细胞接近80%～90%融合。传代培养后,传代细胞在12～24 h内全贴壁,生长迅速,形态均一,大约7d左右达到完全融合。传代扩增细胞以流式细胞术检测,结果显示CD3、CD4、CD8、CD22、CD33、CD34和HLA-DR均表达阴性,高表达CD13,CD44。
在体外活化B淋巴细胞反应体系中加入异体MSCs,显示MSCs及其培养上清抑制由丝裂原Anti-IgM诱导的B淋巴细胞的增殖,且这种抑制程度和MSCs的细胞数量及其培养上清浓度有关。体外与不同比例MSCs共培养的B淋巴细胞,MSCsⅡ(B∶MSCs10∶1)、MSCsⅢ(B∶MSCs 1∶1)组A值明显低于未加MSCs的对照组A值(均P＜0.01),并且MSCsⅢ组A值显著低于MSCsⅠ(B∶MSCs 50∶1)组(P＜0.05)。用含有12.5%、25%、50%不同MSCs培养上清液浓度的培养基培养B淋巴细胞3d后,其中50%组A值同对照组相比有统计学意义(P＜0.05),并且显著低于12.5%组的A值(P＜0.05)。ELISA检测显示MSCs及其培养上清液抑制B淋巴细胞分泌免疫球蛋白,并且随着MSCs数量的及其上清浓度的增加,这种抑制也越明显。与B淋巴细胞加刺激物组相比,B∶MSCs 1∶1组及50%super组分泌的IgM明显减少(P＜0.01),而只有B∶MSCs 1∶1组分泌的IgG同对照组相比差异有统计学意义(P＜0.05)。
MSCs不诱导B淋巴细胞的凋亡;B淋巴细胞与MSCs共培养24h、48h,加或不加Anti-IgM,各组间细胞凋亡率组比较差异无统计学意义(P＞0.05)。MSCs对B淋巴细胞的抑制具有可逆性。收集和MSCs共培养3d的B淋巴细胞,重新用Anti-IgM刺激增殖,与未加Anti-IgM的共孵B淋巴细胞组A值相比,二者间差异有统计学意义(P＜0.0)。
结论MSCs对异体外周血B淋巴细胞存在免疫调节作用,并且这种调控机制是复杂的,不仅与MSCs细胞数量有关,还与细胞间的相互作用和MSCs分泌的细胞因子有关。
Object To study the immunoregulatory effects of bone marrow mesenchymal stem cells(MSCs)on allogeneic peripheral B lymphocytes in vitro.
Methods Mononuclear cells were acquired from bone marrow by density gradient centrifugation.Then,they were seeded at 2×10~5个/ml in DMEM-LG supplemented with 10 %fetal bovine serum(FBS)in culture flask.The medium was changed first time after 48 hours,and once every 2 or 3 days thereafter.When the cultures reached nearly 90%of confluence,cells were passaged with routine methods.Flow cytometry was used to detect the cells surface antigens,include CD3、CD4、CD8、CD13、CD22、CD33、CD34、CD44、HLA-DR.Mononuclear cells were isolated routinely from peripheral blood,then monocytes were eliminated by L-leucine methy ester;remained T lymphocytes were eliminated by AET-SRBC rosette method.The action of MSCs and its supematant on B lymphocytes proliferation in the presence of antihuman immunoglobubin M goat antibodies(Anti-IgM) was investigated by MTT;the IgG,IgM in the supernatant were detected by ELISA.The percent of apoptosis B lymphocytes,co-cultured with MSCs for 24 hours or 48 hours,was assayed by FACS.
Results In our study,it was observed that mononuclear cells from bone marrow have adhered to culture flask after 2 to 3 hours culture period.The quantity of adhered cells no further increased after 24 hours.After about a 3-week primary expansion period,MSCs nearly reached confluence,and these cells had a fibroblast-like morphology.After passaged, those adhered cells were homogenous population and proliferated rapidly.Analyzed by flow cytometry,MSCs were demonstrated that they were uniformly positive for CD13,CD44,but negative for CD3、CD4、CD8、CD22、CD33、CD34 and HLA-DR.The result demonstrated that there were no hematopoietic cells in these cells isolated and cultured in vitro.
The study shows when Allo-MSCs were added to B lymphocytes stimulated by Anti-IgM in vitro,MSCs and its supernatant inhibited B lymphocytes proliferation and Ig secretion.The inhibitory effect depended on the amount of MSCs and condition of its supernatant.The A value orB lymphocytes co-cultured with MSCs at different ratios,group MSCsⅡ(B:MSCs 10:1)and group MSCsⅢ(B:MSCs 1:1)were both significantly lower than that in control group(both P＜0.01),and group MSCsⅢsignificantly lower than that of group MSCsⅠ(B:MSCs 50:1)(P＜0.05).B lymphocytes were co-cultured with different condition of MSCs supernatant after 3 days,group 50%supematant was significantly lower than that in control group(P＜0.05),and significantly lower than group 12.5%(P＜0.05).Compared with the level of Ig of culture supematant of control group,the level of IgM of group B:MSCs 1:1 and group 50%super were decreased(P＜0.01),only the IgG of group B:MSCs 1:1 was significantly lower than that in control group(P＜0.05).
MSCs didn't induce apoptosis of B lymphocytes.The apoptosis rates ofB lymphocytes co-cultured with MSCs for 24h or 48h,in presence of Anti-IgM or not,were not a significant difference among the four groups.The effect of MSCs suppressed on B lymphocytes in vitro was reversible.B lymphocytes and MSCs co-cultured for 3 days,then B lymphocytes were isolated from MSCs and re-stimulated by Anti-IgM.Compared with group pure B lymphocytes,the value of A was significant different:(P＜0.01).
Conclusions MSCs have immunoregulatory effects on B lymphocytes,and its mechanisms were complex,not only correlating with the concentration of MSCs but also the action between cells and the secretory cytokine of MSCs.
引文
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