骨髓间充质干细胞对异体外周血B淋巴细胞的免疫调节作用

发布时间：2018-01-25 13:02

  本文关键词： 骨髓间充质干细胞 细胞培养 流式细胞术 B淋巴细胞 增殖 免疫球蛋白 凋亡 免疫调节　出处：《兰州大学》2008年硕士论文　论文类型：学位论文

【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。
[Abstract]:Objective to investigate the bone marrow mesenchymal stem cells of bone marrow mesenchymal stem cells. The immunomodulatory effect of MSCs on peripheral blood B lymphocytes in vitro. Methods Mononuclear cells were isolated from bone marrow by density gradient centrifugation, and LG-DMEM medium containing 10% fetal bovine serum was added to adjust the cell density to 2 脳 10 ~ 5 / ml. The fresh culture medium was changed after 48 h of inoculation in the culture bottle, and then changed every 2 ~ 3 days. When the cells were covered with the culture bottle bottom 90% or more. The cell culture was carried out by conventional method. The CD3T CD4, CD8, CD13, CD22, CD32, CD34 and CD44 of the cultured cells were detected by flow cytometry (FCM). HLA-DR expression. Mononuclear cells were routinely isolated from peripheral blood to remove monocytes from L- leucine methyl ester. The rosette formation of sheep red blood cell (SRBC) treated with 2-aminoethyl thiourea bromide was studied. Purified B lymphocytes were obtained by removing T lymphocytes. B lymphocytes were stimulated with or not co-cultured with MSCs and its supernatant for 3 days with sheep anti-human IgM monoclonal antibody Anti-IgM. MTT method was used to measure the proliferation of B lymphocytes and Elisa was used to detect the immunoglobulin IgM production in the supernatant. Flow cytometry was used to detect MSCs co-culture for 24 hours. Apoptosis of B lymphocytes was observed after 48 hours. Results it was observed that the mononuclear cells inoculated with gradient centrifugation for 24 hours after inoculation for 2 hours began to adhere to the wall, and the number of adherent cells did not increase after 2 weeks, and the cells began to proliferate rapidly, and most of the cells were fusiform. After 3 weeks of culture, the adherent cells were close to 80% and 90% fused. After subculture, the cells were adherent to the wall completely within 1224 h. The cells grew rapidly and the morphology was uniform. After about 7 days, the cells were fused completely. The results of flow cytometry showed that CD3 + CD4 + CD8 + CD22 + CD33 was detected by flow cytometry. Both CD34 and HLA-DR were negative and CD13 CD44 was highly expressed. MSCs and its culture supernatant inhibited the proliferation of B lymphocytes induced by mitogen Anti-IgM. The inhibition degree was related to the number of MSCs cells and the concentration of supernatant. The A value of MSCs 鈪，

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