人羊膜间充质干细胞与骨髓间充质干细胞对外周血淋巴细胞免疫调节作用的比较

发布时间：2018-05-09 07:43

本文选题：人羊膜间充质干细胞 + 人骨髓间充质干细胞　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究目的本实验通过在体外分别建立人羊膜间充质干细胞(human amniotic mesenchymal stem cell,hAMSC)、骨髓间充质干细胞(bone marrow mesenchymal stem cell,hBMSC)与PHA刺激的外周血单个核细胞(PMBC)共培养体系的方法,比较hAMSC与hBMSC对异体外周血淋巴细胞的免疫调节功能,希望为未来hAMSC治疗植物抗宿主病(graft-versus-hostdiease,GVHD)的临床应用以及解决hBMSC来源有限的问题进一步提供实验依据及新思路。实验方法(1)选用酶消化法从新鲜胎盘羊膜组织中分离得到人羊膜间充质干细胞并利用传代纯化方法筛选出在体外能稳定传代培养的hAMSC;采用Ficoll-Hypaque密度梯度离心法从骨髓中分离得到间充质干细胞,在体外传代培养得到纯化的hBMSC。(2)采取Ficoll-Hypaque密度梯度离心法分离获取人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)。(3)取经丝裂霉素(mitomvcin C,MC)处理的第4代hAMSC和hBMSC分别与PHA刺激的PBMC共培养72小时,将两种来源的MSC对外周血淋巴细胞免疫调节功能的比较分为实验组(hAMSC+PBMC+PHA组和hBMSC+PBMC+PHA组),阳性对照组(PBMC+PHA组),阴性对照组(单独培养的PBMC组、经丝裂霉素处理后单独培养的hAMSC和hBMSC组)。(4)采取流式细胞术检测各实验组和阳性对照组中Treg、Th1、Th2、Tc1、Tc2细胞比例。(5)采取酶联免疫吸附试验(ELISA)检测实验组和对照组中上清液中白介素-2(IL-2)、白介素-10(IL-10)的含量。I实验结果(1)与 PBMC+PHA 组比较,hAMSC+PBMC+PHA 组、hBMSC+ PBMC+PHA 组中 Treg、Th2、Tc2 的比例均明显上升(P0.05),hAMSC+PBMC+PHA 组、hBMSC+PBMC+PHA 组中 Th1、Tc1 的比例明显下降(P0.05),hAMSC+PBMC+PHA、hBMSC+ PBMC+PHA共培养组T细胞亚群的变化差异无统计学意义(P0.05)。(2)ELISA检测结果显示,与PBMC+PHA组比较,hAMSC+PBMC+PHA组、hBMSC+PBMC+PHA组上清液中IL-2的含量明显下降(P0.05),hAMSC+PBMC+PHA组、hBMSC+PBMC+PHA组上清液中IL-10的含量明显上升(P0.05),hAMSC+PBMC+PHA、hBMSC+PBMC+PHA共培养组上清液中 IL-2、IL-10含量的差异无统计学意义(P0.05)。实验结论hAMSC、hBMSC在体外均能使经PHA刺激的异体淋巴细胞中Treg、Th2、Tc2细胞亚群的比例上升并促进其IL-10的分泌,均能下调Th1、Tc1细胞亚群的比例并抑制其IL-2的分泌。hAMSC、hBMSC具有相似的免疫调节功能。
[Abstract]:Objective to establish a co-culture system of human amniotic mesenchymal stem cells hAMSCC (human amniotic mesenchymal stem cells), bone marrow mesenchymal stem cells (BMSCs) and PHA stimulated peripheral blood mononuclear cells (PBMC) in vitro. To compare the immunomodulatory function of hAMSC and hBMSC on peripheral blood lymphocytes, we hope to provide experimental basis and new ideas for the future application of hAMSC in the treatment of plant-versus-host disease graft-versus-host disease (GV) and to solve the problem of limited source of hBMSC. Method 1) Human amniotic mesenchymal stem cells were isolated from fresh placental amniotic membrane by enzyme digestion method, and hAMSCs, which could be cultured stably in vitro, were screened by passage purification, and bone was obtained by Ficoll-Hypaque density gradient centrifugation. Mesenchymal stem cells were isolated from the marrow. Human peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation. Human peripheral blood mononuclear cells (PBMC) were cultured with PHA stimulated PBMC for 72 hours. The immunomodulatory function of peripheral blood lymphocytes was compared between two kinds of MSC groups: hAMSC PBMC PHA group and hBMSC PBMC PHA group; positive control group; PHA group; negative control group (single cultured PBMC group); HAMSC and hBMSC treated with mitomycin) the proportion of TregTh1Th2Tc1Tc2 Tc2 cells in each experimental group and positive control group was detected by flow cytometry (FCM). Elisa was used to detect the white in supernatant of experimental group and control group by enzyme linked immunosorbent assay (Elisa). The ratio of Treg-Th2Tc2 in hAMSC PBMC PHA PBMC PHA group and hBMSC PBMC PHA group were significantly increased. The percentage of Th1Tc1 in PBMC PHA group of hBMSC PBMC PHA group decreased significantly compared with that of PBMC PHA group. The ratio of Th1Tc1 in hAMSC PBMC hBMSC PBMC PHA was significantly decreased compared with that in PBMC PHA group. (1) the percentage of Th1Tc1 in hAMSC PBMC PHA PBMC PHA group was significantly higher than that in hAMSC PBMC PHA group and hBMSC PBMC PHA group, and the percentage of Th1Tc1 in hBMSC PBMC PHA group was significantly decreased compared with that in PBMC PHA group. There was no significant difference in T cell subsets between the two groups. The results of Elisa showed that there was no significant difference between the two groups. Compared with the PBMC PHA group, the content of IL-2 in the supernatant of hBMSC PBMC PHA group decreased significantly. The content of IL-10 in the supernatant of hBMSC PBMC PHA group was significantly higher than that of hAMSC PBMC PHA group. There was no significant difference in IL-2IL-10 content in the supernatant of hAMSC PBMC PHAHBMSC PBMC PHA co-culture group. Conclusion in vitro, hAMSC-hBMSC can increase the proportion of Treg-Th2Tc2 cell subsets in allogeneic lymphocytes stimulated by PHA and promote the secretion of IL-10. Both of them can down-regulate the proportion of Th1Tc1 cell subsets and inhibit the secretion of IL-2 by hAMSC-hBMSC.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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