一氧化氮在人骨髓间充质干细胞抑制淋巴细胞增殖中的机制研究

发布时间：2018-08-19 08:59

【摘要】：大量实验证明骨髓间充质干细胞(Mesenchymal Stem Cells,MSCs)能抑制T细胞的增殖、应答,而且在异基因造血干细胞联合MSC移植治疗恶性血液病的实践中,MSC促进移植物的植入,促进受体造血恢复,抑制移植物抗宿主病(GVHD)的发生,降低GVHD的发生率和严重程度。目前,有关MSCs调节免疫方面的报道很多,尤其是MSCs对淋巴细胞抑制效应的研究,但是MSCs免疫抑制的机制仍不清楚。有实验报道小鼠MSC通过产生一氧化氮(nitrooxide,NO)介导其对小鼠脾淋巴细胞的抑制作用,NO抑制T细胞增殖可能是通过影响了JaK3/STAT5信号转导通路中STAT5酪氨酸蛋白激酶的磷酸化而实现的。而在异基因淋巴细胞作为刺激因素的混合淋巴细胞培养反应(Mixedlymphocyte reaction,MLR)中,NO是否在人MSCs(hMSCs)抑制外周血淋巴细胞增殖的过程中发挥作用呢?基于此,我们分离培养hMSCs,建立以异基因淋巴细胞作为刺激因素的MLR,探讨反应体系中NO的产生,及其在hMSC对淋巴细胞增殖、凋亡和淋巴细胞FOXP3 mRNA表达的影响中的作用。无菌抽取健康志愿者骨髓,制备骨髓细胞悬液,采用全骨髓贴壁培养法分离骨髓源间充质干细胞,传代培养至P4/P5消化后应用。分别以外周血T淋巴细胞为反应细胞,丝裂霉素C灭活的异基因淋巴细胞为刺激细胞建立混合淋巴细胞培养体系(MLR)。MSC/MLR共培养体系中MSC与反应T淋巴细胞数量比为1:10,在有或无诱导性一氧化氮合酶(iNOS)抑制剂(L-NAME)存在下,以CCK-8法检测T淋巴细胞增殖,Giess法检测各MLR体系上清中NO含量,流式细胞术检测各MLR体系中淋巴细胞凋亡比例,Real-time PCR监测各MLR体系中FOXP3mRNA表达水平。实验结果显示,1 hMSC对淋巴细胞反应性增殖具有抑制作用,表示淋巴细胞增殖活性的OD值为0.49±0.03,加入L-NAME后,共培养体系中NO产生减少同时淋巴细胞的增殖有所恢复,OD值为0.92±0.03。2 hMSC干预下,共培养体系中淋巴细胞凋亡比例为(20.83±1.69)%。加入L-NAME后,反应体系中的淋巴细胞凋亡比例为(9.56±0.96)%,与hMSC干预组相比有统计学意义(P＜0.05)。3hMSC干预下,共培养体系中淋巴细胞Foxp3 mRNA的表达水平升高,为(1.56±0.34)%,与对照组(0.74±0.15)%相比,有显著性差异(P＜0.05),加入L-NAME后,Foxp3 mRNA的表达水平为(0.81±0.18)%。通过实验我们得出以下结论,NO在hMSC抑制异基因抗原诱发的淋巴细胞增殖中起重要作用,其机制可能是通过促进凋亡、影响FOXP3的表达进而影响调节性T细胞的功能。
[Abstract]:A large number of experiments have proved that bone marrow mesenchymal stem cells (Mesenchymal Stem) can inhibit the proliferation and response of T cells. Moreover, in the practice of allogeneic hematopoietic stem cells combined with MSC transplantation in the treatment of malignant hematological diseases, bone marrow mesenchymal stem cells can promote graft implantation and promote hematopoietic recovery. To inhibit the occurrence of graft-versus host disease (GVHD) and to reduce the incidence and severity of GVHD. At present, there are many reports about MSCs regulating immunity, especially the effect of MSCs on lymphocyte inhibition, but the mechanism of MSCs immunosuppression is still unclear. It has been reported that mouse MSC inhibits T cell proliferation by producing nitric oxide (no) mediated by nitric oxide (no). The inhibition of T cell proliferation by no may be achieved by affecting the phosphorylation of STAT5 tyrosine protein kinase in JaK3/STAT5 signal transduction pathway. And does no play a role in the Mixedlymphocyte reaction of Mixedlymphocyte reactionation in which allogeneic lymphocytes act as stimulus factors in the process of inhibiting the proliferation of peripheral blood lymphocytes by human MSCs (hMSCs)? Based on this, we isolated and cultured hMSCs and established MLRs with allogeneic lymphocytes as stimulus factors to investigate the production of no in the reaction system and its role in the effects of hMSC on lymphocyte proliferation, apoptosis and lymphocyte FOXP3 mRNA expression. Bone marrow suspension was prepared from healthy volunteers by sterile extraction. Bone marrow-derived mesenchymal stem cells were isolated by whole bone marrow adherent culture method. The MSCs were subcultured after P4/P5 digestion. Peripheral blood T lymphocytes were used as reaction cells, Mitomycin C inactivated allogeneic lymphocytes were used as stimulators to establish a mixed lymphocyte culture system (MLR). MSC-MLR) in which the ratio of MSC to reactive T lymphocytes was 1: 10. In the presence or absence of inducible nitric oxide synthase (iNOS) inhibitor (L-NAME), the ratio of MSC to reactive T lymphocytes was 1: 10. The content of no in supernatant of MLR system was detected by CCK-8 method, and the expression of FOXP3mRNA in MLR system was detected by real-time PCR and flow cytometry. The results showed that 1 hMSC could inhibit the reactive proliferation of lymphocytes, and the OD value of lymphocyte proliferation activity was 0.49 卤0.03. After the addition of L-NAME, the OD value was 0.49 卤0.03. The ratio of apoptosis in co-culture system was (20.83 卤1.69) under the intervention of 0.92 卤0.03.2 hMSC. After the addition of L-NAME, the percentage of lymphocyte apoptosis was (9.56 卤0.96) in the reaction system. Compared with the hMSC intervention group, the expression of Foxp3 mRNA in the co-cultured system was significantly higher than that in the control group (0.74 卤0.15)% and (1.56 卤0.34)%, compared with that in the hMSC intervention group (P < 0. 05). There was significant difference (P < 0. 05). The expression level of Foxp3 mRNA was (0. 81 卤0. 18) after adding L-NAME. It is concluded that no plays an important role in the inhibition of lymphocyte proliferation induced by allogeneic antigens by hMSC. The mechanism may be to promote apoptosis, affect the expression of FOXP3 and thus affect the function of regulatory T cells.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R457;R392

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