过表达CCR7的脂肪间充质干细胞对大鼠异体复合组织移植早期免疫调节作用的研究
发布时间：2019-05-18 14:14
【摘要】：研究背景和目的同种异体复合组织移植对特殊部位或大面积组织缺损的修复效果较理想,但为避免术后急性排斥反应,患者需长期大剂量服用免疫抑制剂,产生诸多副作用,阻碍其临床开展。间充质干细胞(mesenchymal stem cells, MSCs)以其免疫调节作用成为同种异体复合组织移植免疫领域的研究热点。MSCs通过旁分泌细胞因子和细胞直接接触作用调节多种免疫细胞的活化和增殖,并有研究指出脂肪来源的MSCs免疫调节作用优于其他组织来源的MSCs。同种异体复合组织移植急性排斥反应由T淋巴细胞介导,次级淋巴器官(secondary lymphoid organs, SLOs),包括脾和淋巴结等,是T细胞定居和启动免疫反应的场所。然而,静脉注射的MSCs在体内各组织器官中随机分布,体内对T细胞的免疫调节作用不如体外理想。研究表明,利用含CCR7基因的慢病毒转染可使MSCs过表达CC趋化因子受体-7 (CC chemokinereceptor7,CCR7)并有效归巢于次级淋巴器官,有助于减轻T细胞介导的移植物抗宿主反应。因此,本课题旨在探索人脂肪间充质干细胞(human adipose-drived stem cells, hASCs)是否能过表达大鼠的CCR7并能有效归巢大鼠的次级淋巴器官,从而减轻大鼠同种异体复合组织移植后的急性排斥反应,为临床治疗同种异体复合组织移植早期的免疫排斥反应提供新的策略。方法1.细胞准备。1)分离培养鉴定hASCs,RT-PCR和流式方法检测炎性细胞因子刺激前后的hASCsCCR7水平;2)用含有大鼠CCR7-GFP基因及其对照GFP基因的慢病毒转染 hASCs,使其成为 rCCR7-GFP-hASCs(C-A)和 GFP-hASCs(G-A),RT-PCR方法检测转染后细胞内基因元件WPRE水平及CCR7水平,流式方法检测膜CCR7表达水平;3)比较病毒转染前后细胞的表面标志物(流式检测),分化能力(诱导成骨、成脂、成软骨分化)及细胞增殖能力(CCK-8法)。2.体外趋化能力和体内归巢能力检验。1)利用transwell检测C-A和G-A向趋化因子CCL21的定向迁移能力;2)流式方法检测比较静脉注射C-A和G-A的大鼠脾和淋巴结中GFP阳性细胞所占比例;3)脾和淋巴结的冰冻切片免疫荧光染色检测细胞是否归巢次级淋巴器官及其与T细胞的空间分布关系。3.体内外C-A对大鼠T细胞的免疫调节作用的检测。1)体外,将hASCs、C-A和G-A各以两个浓度梯度与混合淋巴反应共培养,流式检测T细胞分化情况(CD4+的辅助性T细胞所占比例及其中Th1、Th2、Th17、Treg所占比例)以及上清中的细胞因子IFN-γ、IL-2、IL-6、IL-17、IL-4、IL-10的含量;2)大鼠尾静脉注射细胞24h后,行同种异体下腹部皮瓣游离移植,术后连续观察皮瓣外观变化,比较术后第3d、7d、14d皮瓣病理表现,检测脾和淋巴结中T细胞分化情况及血浆中细胞因子IFN-γ、IL-2、IL-6、IL-17、IL-4、IL-10的含量(方法同上)。结果1、hASCs成功分离,且炎性细胞因子刺激前后均检测不到CCR7表达;含CCR7-GFP基因的慢病毒及其对照GFP病毒转染均使hASCs表达绿色荧光蛋白;C-A和G-A胞内WPRE水平明显高于未转染的hASCs细胞(P0.001),且C-A高表达大鼠CCR7, G-A不表达CCR7;转染前后的细胞形态、表面标志物、分化能力和增殖能力无明显差异。2、体外,C-A向趋化因子迁移量较G-A显著增多(P0.001);大鼠体内,C-A注射组脾和淋巴结中GFP阳性细胞所占比例显著高于G-A注射组(P0.01);脾和淋巴结冰冻切片免疫荧光染色中,C-A或G-A自发绿色荧光,T细胞被标记红色荧光,C-A注射组脾和淋巴结中绿色荧光细胞明显多于G-A注射组,且绿色荧光细胞聚集于发红色荧光的细胞周围。3、①体外混合淋巴反应结果:hASCs组细胞增殖的集落少于对照组,CD4+细胞比例及Th1/ Th2比值显著低于对照组(P0.01),Treg/Th17比值高于对照组(P0.05);hASCs组上清中促炎细胞因子IFN-y、IL-2、IL-6、IL-17含量低于对照组(P0.05),而抗炎细胞因子IL-4、IL-10高于对照组(P0.05);不同浓度hASCs比较,差别有统计学意义。②皮瓣观测结果:C-A组移植物存活时间较G-A组和单纯移植组长(P0.01);外观出现急性排斥反应的最初征象较其余两组晚;同一取材时间点,C-A组的病理表现最轻。③体内指标检测结果:C-A组脾和淋巴结中Th1/Th2比值高峰出现较晚,之后迅速下降,Treg/Th17比值持续升高;当其余两组血浆IFN-γ、IL-2、IL-4、IL-10较高时,C-A组相应指标较低。之后C-A组IFN-γ、IL-2先升高又迅速降低,IL-4、IL-10持续升高,IL-6、IL-17缓慢上升又下降。结论CCR7-hASCs能有效归巢次级淋巴器官并聚集于T细胞分布区,通过调节Th1/Th2和Treg/Th17平衡,减轻大鼠异体复合组织移植早期的急性排斥反应,为应用hASCs调节同种异体复合组织移植的免疫反应提供一定的依据。
[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.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R622
本文编号：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.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R622
【参考文献】
相关期刊论文 前8条
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