人脐带间充质干细胞与环孢素相互作用的研究

发布时间：2018-11-25 14:39

【摘要】：目的:随着对间充质干细胞(Mesenchymal stem cell, MSC)多向分化潜能及免疫调节特性的深入研究，间充质干细胞已成为组织工程和再生医学细胞治疗的主要候选干细胞之一，同时也被越来越多的应用到异基因造血干细胞移植（allogeneic haematopoietic stem cell transplantation, Allo-HSCT）中，用来防治移植物抗宿主疾病(graft-versus-host disease, GVHD)的发生。环孢素（CyclosporineA, CsA）作为免疫抑制剂的代表，在肾移植、骨髓移植等器官移植中有广泛的使用，明显降低了移植物排异的发生，使器官移植学能够进一步的发展；但是环孢素用药时间长，肝肾、中枢神经系统等重要脏器的毒副作用大，需要频繁监测血药浓度来调整剂量，所以环孢素与其它免疫抑制剂联合应用受到推荐，，联合用药在不影响免疫抑制作用的同时，降低了环孢素的使用剂量，减少了不良反应的发生。本实验旨在研究脐带间充质干细胞（umbilical cord mesenchymal stem cell， UC-MSC）对T淋巴胞的活化增殖的免疫调节效应,以及与环孢素之间的相互作用，特别是二者在免疫抑制方面是否有协同效应，为指导临床用药提供一定的理论基础。方法:用MTT的方法检测环孢素对MSC增殖的影响；用流式细胞仪检测环孢素对MSC凋亡及细胞周期影响；PHA刺激下的人外周血单个核细胞(peripheral blood mononuclear cell, PBMC)与人脐带MSC共培养，实验组加入不同浓度的环孢素，通过ELISA的方法检测人外周血单个核细胞IFN-γ的分泌情况来观察脐带间充质干细胞或/和环孢素的免疫调节效应。结果:单独应用不同浓度的环孢素（30ng/ml、60ng/ml、120ng/ml、240ng/ml、480ng/ml）对脐带MSC增殖的抑制率与无药对照组之间无显著差异（P0.05）。小剂量环孢素（30ng/ml、60ng/ml、120ng/ml）作用脐带间充质干细胞72h后，MSC的凋亡率分别为13.26%、14.76%、18.59%，各组之间无显著差异（P0.05），各组凋亡率均显著高于无药物作用下的MSC凋亡率（0.27%）；大剂量环孢素（240ng/ml、480ng/ml）作用脐带间充质干细胞72h后，MSC的凋亡率分别为37.58%和57.14%，均显著高于无药对照组及小剂量组。脐带MSC经过含有环孢素的培养基培养72h后，随环孢素浓度的增加，各实验组G0/G1期细胞比值逐渐降低，而S期细胞比值逐渐升高，G2期变化不明显；且G0/G1期及S期与对照组相比，差异均具有统计学意义（P0.05）；提示环孢素可明显促进脐带MSC增殖，促进细胞由G0/G1期进入S期。在MSC与PBMC共培养体系中，MSC组IFN-γ的分泌显著低于无MSC组（P0.05），且高浓度MSC组IFN-γ的分泌显著低于低浓度MSC组，即脐带MSC能够有效抑制PHA刺激下PBMCIFN-γ的分泌，并与MSC浓度呈正相关；等量的PBMC在PHA刺激下，MSC组、CsA组、MSC+CsA组的IFN-γ分泌分别为506.5pg/ml、213pg/ml、150pg/ml，均显著低于对照组（PBMC+PHA）的1337pg/ml，差异具有统计学意义（P0.01），且各实验组间IFN-γ分泌为：MSC组 CsA组MSC+CsA组，差异具有统计学意义（P0.01）。结论:脐带MSC能有效抑制PHA刺激下T淋巴细胞的IFN-γ分泌，并呈现出MSC浓度依赖性，提示其对T淋巴细胞的活化增殖具有一定的免疫抑制作用，这可能是MSC减轻GVHD的机制之一。另外脐带MSC与CsA在免疫抑制方面具有协同作用；环孢素对MSC增殖无抑制作用，提示CsA不会影响MSC的细胞活性和功能。本实验的结果为脐带MSC治疗GVHD以及与CsA联合应用的安全有效性提供一定的理论依据。
[Abstract]:Objective: To study the multi-directional differentiation potential and the immunoregulation of mesenchymal stem cells (MSC), the mesenchymal stem cells have become one of the main candidate stem cells for tissue engineering and regenerative medicine cell therapy. At the same time, it is also applied to allogenic hematopoietic stem cell transplanation (Allo-HSCT) to control the occurrence of graft-versus-host disease (GVHD). Cyclosporine A (CsA), as a representative of the immunosuppressive agent, is widely used in the organ transplantation such as kidney transplantation, bone marrow transplantation and the like, so that the occurrence of graft rejection is obviously reduced, and the organ transplantation learning can be further developed; but the administration time of the ciclosporin is long, the liver and the kidney, the toxic and side effect of the important organs such as the central nervous system is large, the blood concentration is frequently monitored to adjust the dose, and the occurrence of the adverse reaction is reduced. The purpose of this study is to study the immunomodulatory effect of the umbilical cord mesenchymal stem cells (UC-MSC) on the activation and proliferation of T-Lymphatic cells, as well as the interaction with the ciclosporin, in particular whether they have a synergistic effect in terms of immunosuppression, and provides a certain theoretical basis for guiding the clinical medicine. Methods: The effect of ciclosporin on the proliferation of MSC was measured by MTT method. The apoptosis and cell cycle were detected by flow cytometry. The peripheral blood mononuclear cells (PBMC) from peripheral blood of human peripheral blood were co-cultured with human umbilical cord MSC. Detection of the secretion of IFN-1 in human peripheral blood mononuclear cells by ELISA in order to observe the immunoregulation of mesenchymal stem cells or/ and ciclosporin in human peripheral blood Results: There was no significant difference (P> 0.05) between the inhibition rate of the proliferation of the umbilical cord MSC and the drug-free control group with different concentrations of ciclosporin (30ng/ ml, 60ng/ ml, 120ng/ ml, 240ng/ ml, 480ng/ ml) alone. The apoptosis rate of MSC was 13. 26%, 14. 76% and 18. 59%, respectively. There was no significant difference between the groups (P0.05). The apoptosis rate of MSC was significantly higher than that of MSC without drug (0. 05). The apoptotic rate of MSC was 37. 58% and 57. 14%, which was significantly higher than that of the non-drug control group, after 7h of the umbilical cord between the umbilical cord and the umbilical cord of the high-dose of ciclosporin (240ng/ ml, 480ng/ ml). The cell ratio of G0/ G1 phase of each experimental group was gradually decreased with the increase of the concentration of the ciclosporin, while the ratio of S-phase cells was gradually increased, and the change of the G2 phase was not obvious; and the G0/ G1 phase and the S phase were the same. Compared with the control group, the difference was statistically significant (P0.05); it was suggested that the Cyclosporin could obviously promote the proliferation of the umbilical cord MSC and promote the cell to be composed of G0/ G1. In MSC and PBMC co-culture system, the secretion of IFN-1 in MSC group was significantly lower than that of non-MSC group (P0.05), and the secretion of IFN-1 in high-concentration MSC group was significantly lower than that of low-concentration MSC group. The levels of IFN-1 in the MSC group, the MSC group, the CsA group and the MSC + CsA group were 506.5pg/ ml, 213pg/ ml and 150pg/ ml, respectively, which were significantly lower than that of the control group (PBMCs + PHA). + CsA group, the difference was of statistical significance (P Conclusion: The umbilical cord MSC can effectively inhibit the release of IFN-1 from T-lymphocytes under the stimulation of PHA, and show the concentration-dependence of MSC, suggesting that it has a certain immunosuppression effect on the activation and proliferation of T-lymphocytes, which may be the reduction of G by the MSC. One of the mechanisms of the VHD. In addition, the umbilical cord MSC and the CsA have a synergistic effect on the immunosuppression, and the ciclosporin has no inhibitory effect on the proliferation of the MSC, and the CsA does not affect the MSC. The results of this experiment are the safety and efficacy of the umbilical cord MSC in the treatment of GVHD and with the application of CsA.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

【参考文献】