人臍帶間充質(zhì)干細(xì)胞與環(huán)孢素相互作用的研究
發(fā)布時(shí)間：2018-11-25 14:39
【摘要】：目的:隨著對(duì)間充質(zhì)干細(xì)胞(Mesenchymal stem cell, MSC)多向分化潛能及免疫調(diào)節(jié)特性的深入研究，間充質(zhì)干細(xì)胞已成為組織工程和再生醫(yī)學(xué)細(xì)胞治療的主要候選干細(xì)胞之一，同時(shí)也被越來越多的應(yīng)用到異基因造血干細(xì)胞移植（allogeneic haematopoietic stem cell transplantation, Allo-HSCT）中，用來防治移植物抗宿主疾病(graft-versus-host disease, GVHD)的發(fā)生。環(huán)孢素（CyclosporineA, CsA）作為免疫抑制劑的代表，在腎移植、骨髓移植等器官移植中有廣泛的使用，明顯降低了移植物排異的發(fā)生，使器官移植學(xué)能夠進(jìn)一步的發(fā)展；但是環(huán)孢素用藥時(shí)間長，肝腎、中樞神經(jīng)系統(tǒng)等重要臟器的毒副作用大，需要頻繁監(jiān)測(cè)血藥濃度來調(diào)整劑量，所以環(huán)孢素與其它免疫抑制劑聯(lián)合應(yīng)用受到推薦，，聯(lián)合用藥在不影響免疫抑制作用的同時(shí)，降低了環(huán)孢素的使用劑量，減少了不良反應(yīng)的發(fā)生。本實(shí)驗(yàn)旨在研究臍帶間充質(zhì)干細(xì)胞（umbilical cord mesenchymal stem cell， UC-MSC）對(duì)T淋巴胞的活化增殖的免疫調(diào)節(jié)效應(yīng),以及與環(huán)孢素之間的相互作用，特別是二者在免疫抑制方面是否有協(xié)同效應(yīng)，為指導(dǎo)臨床用藥提供一定的理論基礎(chǔ)。 方法:用MTT的方法檢測(cè)環(huán)孢素對(duì)MSC增殖的影響；用流式細(xì)胞儀檢測(cè)環(huán)孢素對(duì)MSC凋亡及細(xì)胞周期影響；PHA刺激下的人外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cell, PBMC)與人臍帶MSC共培養(yǎng)，實(shí)驗(yàn)組加入不同濃度的環(huán)孢素，通過ELISA的方法檢測(cè)人外周血單個(gè)核細(xì)胞IFN-γ的分泌情況來觀察臍帶間充質(zhì)干細(xì)胞或/和環(huán)孢素的免疫調(diào)節(jié)效應(yīng)。 結(jié)果:單獨(dú)應(yīng)用不同濃度的環(huán)孢素（30ng/ml、60ng/ml、120ng/ml、240ng/ml、480ng/ml）對(duì)臍帶MSC增殖的抑制率與無藥對(duì)照組之間無顯著差異（P0.05）。小劑量環(huán)孢素（30ng/ml、60ng/ml、120ng/ml）作用臍帶間充質(zhì)干細(xì)胞72h后，MSC的凋亡率分別為13.26%、14.76%、18.59%，各組之間無顯著差異（P0.05），各組凋亡率均顯著高于無藥物作用下的MSC凋亡率（0.27%）；大劑量環(huán)孢素（240ng/ml、480ng/ml）作用臍帶間充質(zhì)干細(xì)胞72h后，MSC的凋亡率分別為37.58%和57.14%，均顯著高于無藥對(duì)照組及小劑量組。臍帶MSC經(jīng)過含有環(huán)孢素的培養(yǎng)基培養(yǎng)72h后，隨環(huán)孢素濃度的增加，各實(shí)驗(yàn)組G0/G1期細(xì)胞比值逐漸降低，而S期細(xì)胞比值逐漸升高，G2期變化不明顯；且G0/G1期及S期與對(duì)照組相比，差異均具有統(tǒng)計(jì)學(xué)意義（P0.05）；提示環(huán)孢素可明顯促進(jìn)臍帶MSC增殖，促進(jìn)細(xì)胞由G0/G1期進(jìn)入S期。在MSC與PBMC共培養(yǎng)體系中，MSC組IFN-γ的分泌顯著低于無MSC組（P0.05），且高濃度MSC組IFN-γ的分泌顯著低于低濃度MSC組，即臍帶MSC能夠有效抑制PHA刺激下PBMCIFN-γ的分泌，并與MSC濃度呈正相關(guān)；等量的PBMC在PHA刺激下，MSC組、CsA組、MSC+CsA組的IFN-γ分泌分別為506.5pg/ml、213pg/ml、150pg/ml，均顯著低于對(duì)照組（PBMC+PHA）的1337pg/ml，差異具有統(tǒng)計(jì)學(xué)意義（P0.01），且各實(shí)驗(yàn)組間IFN-γ分泌為：MSC組 CsA組MSC+CsA組，差異具有統(tǒng)計(jì)學(xué)意義（P0.01）。 結(jié)論:臍帶MSC能有效抑制PHA刺激下T淋巴細(xì)胞的IFN-γ分泌，并呈現(xiàn)出MSC濃度依賴性，提示其對(duì)T淋巴細(xì)胞的活化增殖具有一定的免疫抑制作用，這可能是MSC減輕GVHD的機(jī)制之一。另外臍帶MSC與CsA在免疫抑制方面具有協(xié)同作用；環(huán)孢素對(duì)MSC增殖無抑制作用，提示CsA不會(huì)影響MSC的細(xì)胞活性和功能。本實(shí)驗(yàn)的結(jié)果為臍帶MSC治療GVHD以及與CsA聯(lián)合應(yīng)用的安全有效性提供一定的理論依據(jù)。
[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.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329
本文編號(hào)：2356466
[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.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329
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