過表達CCR7的脂肪間充質干細胞對大鼠異體復合組織移植早期免疫調節(jié)作用的研究
發(fā)布時間：2019-05-18 14:14
【摘要】：研究背景和目的同種異體復合組織移植對特殊部位或大面積組織缺損的修復效果較理想,但為避免術后急性排斥反應,患者需長期大劑量服用免疫抑制劑,產(chǎn)生諸多副作用,阻礙其臨床開展。間充質干細胞(mesenchymal stem cells, MSCs)以其免疫調節(jié)作用成為同種異體復合組織移植免疫領域的研究熱點。MSCs通過旁分泌細胞因子和細胞直接接觸作用調節(jié)多種免疫細胞的活化和增殖,并有研究指出脂肪來源的MSCs免疫調節(jié)作用優(yōu)于其他組織來源的MSCs。同種異體復合組織移植急性排斥反應由T淋巴細胞介導,次級淋巴器官(secondary lymphoid organs, SLOs),包括脾和淋巴結等,是T細胞定居和啟動免疫反應的場所。然而,靜脈注射的MSCs在體內(nèi)各組織器官中隨機分布,體內(nèi)對T細胞的免疫調節(jié)作用不如體外理想。研究表明,利用含CCR7基因的慢病毒轉染可使MSCs過表達CC趨化因子受體-7 (CC chemokinereceptor7,CCR7)并有效歸巢于次級淋巴器官,有助于減輕T細胞介導的移植物抗宿主反應。因此,本課題旨在探索人脂肪間充質干細胞(human adipose-drived stem cells, hASCs)是否能過表達大鼠的CCR7并能有效歸巢大鼠的次級淋巴器官,從而減輕大鼠同種異體復合組織移植后的急性排斥反應,為臨床治療同種異體復合組織移植早期的免疫排斥反應提供新的策略。方法1.細胞準備。1)分離培養(yǎng)鑒定hASCs,RT-PCR和流式方法檢測炎性細胞因子刺激前后的hASCsCCR7水平;2)用含有大鼠CCR7-GFP基因及其對照GFP基因的慢病毒轉染 hASCs,使其成為 rCCR7-GFP-hASCs(C-A)和 GFP-hASCs(G-A),RT-PCR方法檢測轉染后細胞內(nèi)基因元件WPRE水平及CCR7水平,流式方法檢測膜CCR7表達水平;3)比較病毒轉染前后細胞的表面標志物(流式檢測),分化能力(誘導成骨、成脂、成軟骨分化)及細胞增殖能力(CCK-8法)。2.體外趨化能力和體內(nèi)歸巢能力檢驗。1)利用transwell檢測C-A和G-A向趨化因子CCL21的定向遷移能力;2)流式方法檢測比較靜脈注射C-A和G-A的大鼠脾和淋巴結中GFP陽性細胞所占比例;3)脾和淋巴結的冰凍切片免疫熒光染色檢測細胞是否歸巢次級淋巴器官及其與T細胞的空間分布關系。3.體內(nèi)外C-A對大鼠T細胞的免疫調節(jié)作用的檢測。1)體外,將hASCs、C-A和G-A各以兩個濃度梯度與混合淋巴反應共培養(yǎng),流式檢測T細胞分化情況(CD4+的輔助性T細胞所占比例及其中Th1、Th2、Th17、Treg所占比例)以及上清中的細胞因子IFN-γ、IL-2、IL-6、IL-17、IL-4、IL-10的含量;2)大鼠尾靜脈注射細胞24h后,行同種異體下腹部皮瓣游離移植,術后連續(xù)觀察皮瓣外觀變化,比較術后第3d、7d、14d皮瓣病理表現(xiàn),檢測脾和淋巴結中T細胞分化情況及血漿中細胞因子IFN-γ、IL-2、IL-6、IL-17、IL-4、IL-10的含量(方法同上)。結果1、hASCs成功分離,且炎性細胞因子刺激前后均檢測不到CCR7表達;含CCR7-GFP基因的慢病毒及其對照GFP病毒轉染均使hASCs表達綠色熒光蛋白;C-A和G-A胞內(nèi)WPRE水平明顯高于未轉染的hASCs細胞(P0.001),且C-A高表達大鼠CCR7, G-A不表達CCR7;轉染前后的細胞形態(tài)、表面標志物、分化能力和增殖能力無明顯差異。2、體外,C-A向趨化因子遷移量較G-A顯著增多(P0.001);大鼠體內(nèi),C-A注射組脾和淋巴結中GFP陽性細胞所占比例顯著高于G-A注射組(P0.01);脾和淋巴結冰凍切片免疫熒光染色中,C-A或G-A自發(fā)綠色熒光,T細胞被標記紅色熒光,C-A注射組脾和淋巴結中綠色熒光細胞明顯多于G-A注射組,且綠色熒光細胞聚集于發(fā)紅色熒光的細胞周圍。3、①體外混合淋巴反應結果:hASCs組細胞增殖的集落少于對照組,CD4+細胞比例及Th1/ Th2比值顯著低于對照組(P0.01),Treg/Th17比值高于對照組(P0.05);hASCs組上清中促炎細胞因子IFN-y、IL-2、IL-6、IL-17含量低于對照組(P0.05),而抗炎細胞因子IL-4、IL-10高于對照組(P0.05);不同濃度hASCs比較,差別有統(tǒng)計學意義。②皮瓣觀測結果:C-A組移植物存活時間較G-A組和單純移植組長(P0.01);外觀出現(xiàn)急性排斥反應的最初征象較其余兩組晚;同一取材時間點,C-A組的病理表現(xiàn)最輕。③體內(nèi)指標檢測結果:C-A組脾和淋巴結中Th1/Th2比值高峰出現(xiàn)較晚,之后迅速下降,Treg/Th17比值持續(xù)升高;當其余兩組血漿IFN-γ、IL-2、IL-4、IL-10較高時,C-A組相應指標較低。之后C-A組IFN-γ、IL-2先升高又迅速降低,IL-4、IL-10持續(xù)升高,IL-6、IL-17緩慢上升又下降。結論CCR7-hASCs能有效歸巢次級淋巴器官并聚集于T細胞分布區(qū),通過調節(jié)Th1/Th2和Treg/Th17平衡,減輕大鼠異體復合組織移植早期的急性排斥反應,為應用hASCs調節(jié)同種異體復合組織移植的免疫反應提供一定的依據(jù)。
[Abstract]:In order to avoid the postoperative acute rejection, the patients need to take long-term and long-term administration of the immunosuppressive agent, which can prevent the clinical development. Mesenchymal stem cells (MSCs) play an important role in the study of the immune regulation of the allogenic compound tissue. MSCs can regulate the activation and proliferation of a plurality of immune cells by direct contact of the paracrine cytokines and the cells, and the research indicates that the immunomodulatory effect of the fat-derived MSCs is superior to that of other tissue-derived MSCs. The acute rejection of the allogenic compound tissue is mediated by T-lymphocytes, secondary lymphoid organs (SLOs), including the spleen and the lymph nodes, and are the sites for the T-cell settlement and the initiation of the immune response. However, the injected MSCs are randomly distributed in various organs of the body, and the immunoregulation effect of the in vivo on T cells is not as ideal as in vitro. Studies have shown that the use of a lentiviral transfection containing a CCR7 gene can make it possible for MSCs to overexpress the CC chemokine receptor-7 (CC chemotactic factor 7, CCR7) and to effectively nest with the secondary lymphoid organs, contributing to the reduction of T cell-mediated graft-versus-host reactions. Therefore, the purpose of this study is to explore whether human adipose-derived stem cells (hASs) can express the CCR7 of the rat and can be used for the effective homing of the secondary lymphoid organs of the rat, so as to reduce the acute rejection after the transplantation of the allogenic compound tissue of the rat. And provides a new strategy for the clinical treatment of the early immune rejection reaction of the allogenic compound tissue transplantation. Method 1. Cell preparation.1) Isolation and identification of hASCs, RT-PCR and flow methods for the detection of the level of hASCCCR7 before and after the inflammatory cytokine stimulation;2) transfecting hASCs with lentiviruses containing the rat CCR7-GFP gene and its control GFP gene to be rCCR7-GFP-hASs (C-A) and GFP-hASCs (G-A), in that RT-PCR method, the WPRE level and the CCR7 level of the gene element in the transfected cell are detected, the expression level of the membrane CCR7 is detected by the flow method, Chondrogenic differentiation) and cell proliferation ability (CCK-8 method). in vitro chemotactic ability and in vivo homing capacity test.1) the directional migration ability of C-A and G-A to the chemokine CCL21 was detected by transwell;2) the proportion of GFP positive cells in the spleen and the lymph nodes of the rats compared with the intravenous C-A and G-A was detected by the flow method; 3) The immunofluorescence staining of the frozen sections of the spleen and the lymph nodes was used to detect whether the cells were in the secondary lymphoid organs of the nest and their spatial distribution with the T cells. 1) In vitro, hASCs, C-A and G-A were co-cultured with mixed lymph reaction with two concentration gradients, and the proportion of T-cell differentiation (CD4 + helper T-cells and the ratio of Th1, Th2, Th17, The ratio of Treg and the content of IL-2, IL-6, IL-17, IL-4 and IL-10 in the supernatant and the content of IL-6, IL-17, IL-4 and IL-10 in the supernatant of the rat were observed. The changes of T cell in the spleen and lymph nodes and the levels of IL-2, IL-6, IL-17, IL-4 and IL-10 in the plasma were detected by the 14-d skin flap. Results 1. The expression of CCR7 was not detected before and after the successful separation of hASCs, and the expression of CCR7 was not detected before and after the stimulation of the inflammatory cytokines. The expression of the green fluorescent protein was made by the transfection of the lentivirus containing the CCR7-GFP gene and the control GFP virus. The level of the WPRE in the C-A and G-A cells was significantly higher than that of the untransfected hASs cells (P0.001). The expression of CCR7 and G-A in C-A showed no significant difference in the cell morphology, surface marker, differentiation ability and proliferation ability before and after transfection. In vitro, the migration of C-A to chemokines increased significantly (P 0.001); in rats, The proportion of GFP positive cells in the spleen and lymph nodes of the C-A injection group was significantly higher than that of the G-A group (P0.01); in the immunofluorescence staining of the frozen sections of the spleen and the lymph nodes, the C-A or G-A spontaneous green fluorescence and the T cells were labeled with red fluorescence, In that C-A group, the green fluorescent cells in the spleen and lymph nodes were significantly more than that of the G-A injection group, and the green fluorescent cells were clustered around the cells emitting red fluorescence. The ratio of CD4 + cells and the ratio of Th1/ Th2 was significantly lower than that in the control group (P0.01), and the ratio of Treg/ Th17 was higher than that of the control group (P0.05). The content of the pro-inflammatory cytokines, IFN-y, IL-2, IL-6 and IL-17 in the group of hASs was lower than that of the control group (P0.05), while the anti-inflammatory cytokine IL-4 and IL-10 were higher than that in the control group (P0.05). The difference of hASCs in different concentrations was statistically significant. The results showed that the survival time of C-A group was less than that of G-A group and the group of pure transplantation (P0.01). The first sign of acute rejection in appearance was later than that of the other two groups. At the same time point, the pathological behavior of C-A group was the lightest. The results showed that the ratio of Th1/ Th2 in the spleen and lymph nodes of the C-A group was late and then decreased rapidly, and the ratio of Treg/ Th17 continued to increase; when the other two groups of plasma IFN-1, IL-2, IL-4 and IL-10 were higher, the corresponding index of C-A group was lower. The increase of IL-4, IL-10 and IL-6 and IL-17 in group C-A and IL-6, IL-6 and IL-17 increased and decreased. Conclusion CCR7-hASs can be used for the effective homing of the secondary lymphoid organs and aggregation in the T cell distribution area. By adjusting the balance of Th1/ Th2 and Treg/ Th17, the acute rejection in the early stage of the transplantation of the allogenic compound tissue of the rat is reduced, and a certain basis is provided for the application of hASs to regulate the immune response of the allogenic compound tissue transplantation.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R622
本文編號：2480073
[Abstract]:In order to avoid the postoperative acute rejection, the patients need to take long-term and long-term administration of the immunosuppressive agent, which can prevent the clinical development. Mesenchymal stem cells (MSCs) play an important role in the study of the immune regulation of the allogenic compound tissue. MSCs can regulate the activation and proliferation of a plurality of immune cells by direct contact of the paracrine cytokines and the cells, and the research indicates that the immunomodulatory effect of the fat-derived MSCs is superior to that of other tissue-derived MSCs. The acute rejection of the allogenic compound tissue is mediated by T-lymphocytes, secondary lymphoid organs (SLOs), including the spleen and the lymph nodes, and are the sites for the T-cell settlement and the initiation of the immune response. However, the injected MSCs are randomly distributed in various organs of the body, and the immunoregulation effect of the in vivo on T cells is not as ideal as in vitro. Studies have shown that the use of a lentiviral transfection containing a CCR7 gene can make it possible for MSCs to overexpress the CC chemokine receptor-7 (CC chemotactic factor 7, CCR7) and to effectively nest with the secondary lymphoid organs, contributing to the reduction of T cell-mediated graft-versus-host reactions. Therefore, the purpose of this study is to explore whether human adipose-derived stem cells (hASs) can express the CCR7 of the rat and can be used for the effective homing of the secondary lymphoid organs of the rat, so as to reduce the acute rejection after the transplantation of the allogenic compound tissue of the rat. And provides a new strategy for the clinical treatment of the early immune rejection reaction of the allogenic compound tissue transplantation. Method 1. Cell preparation.1) Isolation and identification of hASCs, RT-PCR and flow methods for the detection of the level of hASCCCR7 before and after the inflammatory cytokine stimulation;2) transfecting hASCs with lentiviruses containing the rat CCR7-GFP gene and its control GFP gene to be rCCR7-GFP-hASs (C-A) and GFP-hASCs (G-A), in that RT-PCR method, the WPRE level and the CCR7 level of the gene element in the transfected cell are detected, the expression level of the membrane CCR7 is detected by the flow method, Chondrogenic differentiation) and cell proliferation ability (CCK-8 method). in vitro chemotactic ability and in vivo homing capacity test.1) the directional migration ability of C-A and G-A to the chemokine CCL21 was detected by transwell;2) the proportion of GFP positive cells in the spleen and the lymph nodes of the rats compared with the intravenous C-A and G-A was detected by the flow method; 3) The immunofluorescence staining of the frozen sections of the spleen and the lymph nodes was used to detect whether the cells were in the secondary lymphoid organs of the nest and their spatial distribution with the T cells. 1) In vitro, hASCs, C-A and G-A were co-cultured with mixed lymph reaction with two concentration gradients, and the proportion of T-cell differentiation (CD4 + helper T-cells and the ratio of Th1, Th2, Th17, The ratio of Treg and the content of IL-2, IL-6, IL-17, IL-4 and IL-10 in the supernatant and the content of IL-6, IL-17, IL-4 and IL-10 in the supernatant of the rat were observed. The changes of T cell in the spleen and lymph nodes and the levels of IL-2, IL-6, IL-17, IL-4 and IL-10 in the plasma were detected by the 14-d skin flap. Results 1. The expression of CCR7 was not detected before and after the successful separation of hASCs, and the expression of CCR7 was not detected before and after the stimulation of the inflammatory cytokines. The expression of the green fluorescent protein was made by the transfection of the lentivirus containing the CCR7-GFP gene and the control GFP virus. The level of the WPRE in the C-A and G-A cells was significantly higher than that of the untransfected hASs cells (P0.001). The expression of CCR7 and G-A in C-A showed no significant difference in the cell morphology, surface marker, differentiation ability and proliferation ability before and after transfection. In vitro, the migration of C-A to chemokines increased significantly (P 0.001); in rats, The proportion of GFP positive cells in the spleen and lymph nodes of the C-A injection group was significantly higher than that of the G-A group (P0.01); in the immunofluorescence staining of the frozen sections of the spleen and the lymph nodes, the C-A or G-A spontaneous green fluorescence and the T cells were labeled with red fluorescence, In that C-A group, the green fluorescent cells in the spleen and lymph nodes were significantly more than that of the G-A injection group, and the green fluorescent cells were clustered around the cells emitting red fluorescence. The ratio of CD4 + cells and the ratio of Th1/ Th2 was significantly lower than that in the control group (P0.01), and the ratio of Treg/ Th17 was higher than that of the control group (P0.05). The content of the pro-inflammatory cytokines, IFN-y, IL-2, IL-6 and IL-17 in the group of hASs was lower than that of the control group (P0.05), while the anti-inflammatory cytokine IL-4 and IL-10 were higher than that in the control group (P0.05). The difference of hASCs in different concentrations was statistically significant. The results showed that the survival time of C-A group was less than that of G-A group and the group of pure transplantation (P0.01). The first sign of acute rejection in appearance was later than that of the other two groups. At the same time point, the pathological behavior of C-A group was the lightest. The results showed that the ratio of Th1/ Th2 in the spleen and lymph nodes of the C-A group was late and then decreased rapidly, and the ratio of Treg/ Th17 continued to increase; when the other two groups of plasma IFN-1, IL-2, IL-4 and IL-10 were higher, the corresponding index of C-A group was lower. The increase of IL-4, IL-10 and IL-6 and IL-17 in group C-A and IL-6, IL-6 and IL-17 increased and decreased. Conclusion CCR7-hASs can be used for the effective homing of the secondary lymphoid organs and aggregation in the T cell distribution area. By adjusting the balance of Th1/ Th2 and Treg/ Th17, the acute rejection in the early stage of the transplantation of the allogenic compound tissue of the rat is reduced, and a certain basis is provided for the application of hASs to regulate the immune response of the allogenic compound tissue transplantation.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R622
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相關期刊論文 前8條
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