本文選題：人羊膜間充質(zhì)干細(xì)胞 + 人骨髓間充質(zhì)干細(xì)胞��； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究目的本實(shí)驗(yàn)通過在體外分別建立人羊膜間充質(zhì)干細(xì)胞(human amniotic mesenchymal stem cell,hAMSC)、骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cell,hBMSC)與PHA刺激的外周血單個核細(xì)胞(PMBC)共培養(yǎng)體系的方法,比較hAMSC與hBMSC對異體外周血淋巴細(xì)胞的免疫調(diào)節(jié)功能,希望為未來hAMSC治療植物抗宿主病(graft-versus-hostdiease,GVHD)的臨床應(yīng)用以及解決hBMSC來源有限的問題進(jìn)一步提供實(shí)驗(yàn)依據(jù)及新思路。實(shí)驗(yàn)方法(1)選用酶消化法從新鮮胎盤羊膜組織中分離得到人羊膜間充質(zhì)干細(xì)胞并利用傳代純化方法篩選出在體外能穩(wěn)定傳代培養(yǎng)的hAMSC;采用Ficoll-Hypaque密度梯度離心法從骨髓中分離得到間充質(zhì)干細(xì)胞,在體外傳代培養(yǎng)得到純化的hBMSC。(2)采取Ficoll-Hypaque密度梯度離心法分離獲取人外周血單個核細(xì)胞(peripheral blood mononuclear cell,PBMC)。(3)取經(jīng)絲裂霉素(mitomvcin C,MC)處理的第4代hAMSC和hBMSC分別與PHA刺激的PBMC共培養(yǎng)72小時(shí),將兩種來源的MSC對外周血淋巴細(xì)胞免疫調(diào)節(jié)功能的比較分為實(shí)驗(yàn)組(hAMSC+PBMC+PHA組和hBMSC+PBMC+PHA組),陽性對照組(PBMC+PHA組),陰性對照組(單獨(dú)培養(yǎng)的PBMC組、經(jīng)絲裂霉素處理后單獨(dú)培養(yǎng)的hAMSC和hBMSC組)。(4)采取流式細(xì)胞術(shù)檢測各實(shí)驗(yàn)組和陽性對照組中Treg、Th1、Th2、Tc1、Tc2細(xì)胞比例。(5)采取酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測實(shí)驗(yàn)組和對照組中上清液中白介素-2(IL-2)、白介素-10(IL-10)的含量。I實(shí)驗(yàn)結(jié)果(1)與 PBMC+PHA 組比較,hAMSC+PBMC+PHA 組、hBMSC+ PBMC+PHA 組中 Treg、Th2、Tc2 的比例均明顯上升(P0.05),hAMSC+PBMC+PHA 組、hBMSC+PBMC+PHA 組中 Th1、Tc1 的比例明顯下降(P0.05),hAMSC+PBMC+PHA、hBMSC+ PBMC+PHA共培養(yǎng)組T細(xì)胞亞群的變化差異無統(tǒng)計(jì)學(xué)意義(P0.05)。(2)ELISA檢測結(jié)果顯示,與PBMC+PHA組比較,hAMSC+PBMC+PHA組、hBMSC+PBMC+PHA組上清液中IL-2的含量明顯下降(P0.05),hAMSC+PBMC+PHA組、hBMSC+PBMC+PHA組上清液中IL-10的含量明顯上升(P0.05),hAMSC+PBMC+PHA、hBMSC+PBMC+PHA共培養(yǎng)組上清液中 IL-2、IL-10含量的差異無統(tǒng)計(jì)學(xué)意義(P0.05)。實(shí)驗(yàn)結(jié)論hAMSC、hBMSC在體外均能使經(jīng)PHA刺激的異體淋巴細(xì)胞中Treg、Th2、Tc2細(xì)胞亞群的比例上升并促進(jìn)其IL-10的分泌,均能下調(diào)Th1、Tc1細(xì)胞亞群的比例并抑制其IL-2的分泌。hAMSC、hBMSC具有相似的免疫調(diào)節(jié)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R392

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