大鼠骨髓间充质干细胞诱导同种异体心脏移植免疫耐受

发布时间：2018-04-28 23:24

本文选题：大鼠 + 骨髓　；参考：《复旦大学》2007年博士论文

【摘要】： 器官移植的治疗目的是向人体引入异体的细胞、组织或器官，从而代替丧失相应功能的细胞、组织或器官，并使受体接受，不被机体免疫系统排斥，而且移植物也不排斥宿主。目前器官移植术后，受体多须终身使用免疫抑制剂，降低受体的全身免疫功能，减少或抑制受体对移植物的免疫排斥反应以达到维持移植物长期存活的目的。但是长期使用用免疫抑制剂导致感染和肿瘤高发，而且各种免疫抑制剂药物的毒副作用影响受体的生存质量，严重者甚至导致慢性移植物功能衰竭的发生。此外，即使受体长期服用并能耐受免疫抑制剂，移植物长期存活率也并不理想。诱导受体对供者器官特异性免疫耐受是解决排斥反应最理想的措施。也就是说在长期不使用免疫抑制剂的情况下，移植物既不被受体排斥，发生宿主抗移植物反应；同时也不致发生移植物抗宿主反应，但是仍保留对除移植物以外的正常免疫应答。间充质干细胞是中胚层来源的具有多向分化能力的干细胞，主要存在于全身结缔组织和器官间质中，以骨髓组织中含量最丰富。间充质干细胞具有多向分化能力，可以分化为骨、软骨、脂肪等；具有较低的免疫原性，在体内可以逃避免疫系统的攻击；同时发现具有免疫调节作用，可以抑制同种异体T细胞增殖，研究表明间充质干细胞可以延长皮肤移植物的存活时间，减少移植物抗宿主病的发生。近来对间充质干细胞免疫调控功能的研究，提示其在诱导器官移植免疫耐受中的应用前景。本研究从大鼠骨髓中分离出间充质干细胞，建立大鼠同种异位心脏移植模型，旨在研究骨髓间充质干细胞在心脏移植中诱导免疫耐受，减少免疫排斥的作用，并探讨其可能机制。本研究分为四部分。第一部分：大鼠骨髓间充质干细胞的分离培养和生物学鉴定第二部分大鼠骨髓间充质干细胞对同种T细胞的作用及其机制第三部分大鼠同种异体心脏移植模型的建立第四部分大鼠骨髓间充质干细胞对同种异体心脏移植的作用及其机制第一部分大鼠骨髓间充质干细胞的分离培养和生物学鉴定【目的】探讨体外分离、纯化大鼠骨髓间充质干细胞的方法，并分析其表型特点；评价大鼠骨髓间充质干细胞进行同种异体移植的安全性。【方法】用密度梯度离心法分离纯化大鼠骨髓MSCs，传代扩增，，形态学观察，流式细胞仪分析测定细胞表面抗原，DAPI标记。20只Wistar大鼠，体重200—250g。随机分为2组：A空白对照组，B MSCs移植组。观察移植术后大鼠一般情况，术后7天处死取骨髓涂片，免疫荧光镜下观察。【结果】MSCs是骨髓细胞中的单个核细胞，密度梯度离心法能有效分离纯化大鼠骨髓MSCs，MSCs在含10％胎牛血清的L-DMEM中生长性状相对稳定，细胞呈均一的成纤维细胞样，均一表达CD90，不表达CD34、CD45。MSCs移植组可以长期存活，术后7天骨髓涂片可见存活的MSCs。【结论】本实验建立了一种体外分离纯化、培养扩增大鼠骨髓MSCs的方法，MSCs稳定表达CD90，不表达CD45、CD34；同种异体来源的MSCs可在宿主体内存活、定居，并未发生免疫排斥。第二部分大鼠骨髓间充质干细胞对同种T细胞的作用及其机制【目的】探讨骨髓间充质干细胞(MSCs)在混和淋巴细胞反应中对同种异体T淋巴细胞免疫应答反应的影响，并初步探讨其作用机制。【方法】建立MSCs和同种异体淋巴细胞共培养体系，反应体系总量250μl。以SD大鼠的脾T淋巴细胞为刺激细胞，以Wistar大鼠的脾T淋巴细胞为反应细胞，分为6组。组Ⅰ：对照组，1×10~5／50μl刺激细胞和1×10~5／50μl反应细胞共同培养；组Ⅱ：1×10~5／50μl反应细胞与1×10~4／50μl SD大鼠的MSCs共同培养；组Ⅲ：1×10~5／50μl刺激细胞和1×10~5／50μl反应细胞并加入1×10~4／100μl SD大鼠的MSCs共同培养；组Ⅳ：细胞种类及数量同第三组，，另加1-甲基色氨酸(1—MT)(终浓度1mmol／ml)；组Ⅴ：细胞种类及数量同第三组，另加植物刺激素(终浓度2μg／ml)；组Ⅵ：每孔加入反应细胞和刺激细胞各50μl(各含细胞数1×10~5个)及MSC0.01ml(含细胞数1×10~3个)。混合培养120小时，结束培养前13小时，每孔加入~3H-TdR 20ul，以液闪测定仪测定各组的CPM值。反相高效液相色谱法检测MSC和MLR共培养体系中色氨酸含量。【结果】MSCs可以抑制MLC体系中T淋巴细胞增殖，并呈现出剂量依赖关系；同时MSCs和MLR共培养体系中色氨酸含量明显降低。1-MT可以阻断这一作用。【结论】MSCs在体外可抑制同种异体T淋巴细胞的免疫应答，IDO参与了这种免疫抑制作用。第三部分同种异体大鼠异位心脏移植模型的建立【目的】以改良Ono法进行大鼠腹部心脏移植，建立动物模型。【方法】以体重200～250gSD大鼠为供体，体重200～250g的Wistar大鼠为受体，进行大鼠腹部异位心脏移植手术。SD大鼠经麻醉、肝素化后，打开胸腔，阻断主动脉，经主动脉根部灌注心肌保护液，将供体的心脏取下。Wistar大鼠麻醉后，打开腹腔，将供体的主动脉与受体的腹主动脉行端侧吻合，将供体的肺动脉和受体的下腔静脉行端侧吻合。【结果】大鼠异位心脏移植模型成功率达100％。总手术时间约60～75min，平均(70.1±4.9min)；供心摘取时间约8～12min，平均(10.5±2.3min)；受体准备时间约11～15min，平均(13.7±2.0min) 【结论】改良Ono法进行大鼠腹部心脏移植模型可靠易行，可重复率高第四部分大鼠骨髓间充质干细胞对同种异体心脏移植的作用及其机制【目的】研究静脉输注骨髓间充质干细胞(MSCs)对心脏移植术后急性排斥反应的影响及其机制。【方法】64只Wistar大鼠随机分成4组，每组16只，作为受体。组Ⅰ：空白对照组经腰静脉注射PBS；组Ⅱ术中经腰静脉注射供体大鼠骨髓间充质干细胞1×10~6／ml；组Ⅲ术后7天每天使用环胞霉素5mg／Kg；组Ⅳ术中供心经腰静脉注射供体大鼠骨髓间充质干细胞1×10~6／ml，术后7天每天使用环胞霉素5mg／Kg。观察各组供心存活时间；术后七天取供心，观察病理改变，并取脾脏细胞悬液流式细胞仪检测CD4~+、CD8~+的百分比，计算并比较各组CD4~+／CD8~+的比值，ELISA法检测外周血细胞因子干扰素-γ(Interferon-γ，IFN-γ)、白细胞介素—10(interleukin-10，IL-10)水平。【结果】移植心脏的存活时间为组Ⅰ：7.5±0.9天；组Ⅱ：15.5±1.6天；组Ⅲ：17.3±1.3天；组Ⅳ：33.4±3.2天。统计学分析显示，移植心脏存活时间组Ⅱ比组Ⅰ明显延长(p＜0.05)，组Ⅳ比其它三组明显延长(P＜0.01)。组Ⅳ移植心脏病理改变较其它三组为轻。受体外周血CD4／CD8比例，组Ⅱ、Ⅲ和Ⅳ均高于组Ⅰ(P＜0.05)；组Ⅳ与其它三组相比亦明显增高(P＜0.05)。受体血清IFN-γ水平，组Ⅱ、Ⅲ明显低于组Ⅰ，组Ⅳ低于其他三组(P＜0.05)。血清IL-10水平，组Ⅱ、Ⅲ和Ⅳ均高于组Ⅰ(p＜0.05)，组Ⅳ高于组Ⅰ和组Ⅱ(P＜0.05) 【结论】骨髓间充质干细胞可减轻大鼠同种异体移植心脏的免疫排斥反应并延长移植心脏的存活时间。其机制可能与调节同种异体T细胞功能和细胞因子分泌有关。环胞霉素A与骨髓间充质干细胞联合应用抑制免疫排斥反应有协同作用。
[Abstract]:The purpose of organ transplantation is to introduce allogenic cells, tissues or organs to the human body instead of the cells, tissues or organs that lose the corresponding function, and to make receptors accept, not be rejected by the immune system, and the grafts do not repel the host. The systemic immune function reduces or inhibits the rejection of the receptor to the graft for the purpose of maintaining the long-term survival of the graft. However, the long-term use of immunosuppressive agents leads to high incidence of infection and tumor, and the toxic side effects of various immunosuppressive drugs affect the quality of the receptor, and the serious may even lead to chronic graft work. In addition, the long-term survival rate of the graft is not ideal even if the receptor is long taken and can tolerate immunosuppressive agents. The most ideal measure to induce the recipient's organ specific immune tolerance is to solve the rejection. That is to say, the graft is not rejected by the receptor in the long term without the use of immunosuppressive agents. The host is resistant to graft reaction, and does not cause graft versus host reaction, but it still maintains normal immune response except for allografts.
Mesenchymal stem cells are the multidirectional stem cells derived from mesoderm, mainly in the connective tissue and interstitial tissue of the whole body, and are the most abundant in the bone marrow tissue. Mesenchymal stem cells have the ability to differentiate into bone, cartilage and fat, which can be differentiated into bone, cartilage and fat. It has a low immunogenicity and can escape the immune system in the body. At the same time, it is found that the immune regulation can inhibit the proliferation of allogenic T cells. The study shows that mesenchymal stem cells can prolong the survival time of the skin graft and reduce the occurrence of graft versus host disease. Recent studies on the immune regulation function of mesenchymal stem cells suggest that it can induce immune tolerance in organ transplantation. In this study, mesenchymal stem cells were isolated from rat bone marrow, and a rat model of heterotopic heart transplantation was established. The aim of this study was to study the immune tolerance induced by bone marrow mesenchymal stem cells in heart transplantation, reduce immune rejection, and explore its possible mechanism. This study was divided into four parts.
Part one: isolation, culture and biological identification of rat bone marrow mesenchymal stem cells
The second part is the effect and mechanism of rat bone marrow mesenchymal stem cells on homologous T cells.
The third part is the establishment of rat cardiac allograft model.
The effect and mechanism of fourth parts of rat bone marrow mesenchymal stem cells on allograft heart transplantation
Part one: isolation, culture and biological identification of rat bone marrow mesenchymal stem cells
[Objective] to investigate the method of isolation and purification of rat bone marrow mesenchymal stem cells in vitro, and to analyze its phenotypic characteristics, and to evaluate the safety of allograft transplantation of bone marrow mesenchymal stem cells in rats.
[Methods] the density gradient centrifugation was used to separate and purify the rat bone marrow MSCs and be amplified, and the morphological observation and flow cytometry were used to determine the cell surface antigen. The DAPI labeled.20 rats were only Wistar rats, and the weight 200 to 250g. were randomly divided into 2 groups: A blank control group and B MSCs transplantation group. The general condition of the rats after the transplantation was observed and the bone was killed 7 days after the operation. Medullary smears were observed under immunofluorescence.
[results] MSCs is a mononuclear cell in bone marrow cells. Density gradient centrifugation can effectively separate and purify rat bone marrow MSCs. The growth character of MSCs in L-DMEM containing 10% fetal bovine serum is relatively stable, the cells are homogeneous fibroblast like, CD90 is expressed as CD90, and CD34 is not expressed. The CD45.MSCs transplantation group can survive for a long time and the bone marrow is 7 days after the operation. The smear can be seen in the surviving MSCs.
[Conclusion] in this experiment, a method of isolation and purification in vitro was established to culture and amplify the MSCs of rat bone marrow. MSCs expressed CD90 steadily and did not express CD45, CD34. The MSCs of allogeneic origin could survive in the host and settled without immune rejection.
The second part is the effect and mechanism of rat bone marrow mesenchymal stem cells on homologous T cells.
[Objective] to investigate the effect of bone marrow mesenchymal stem cells (MSCs) on the immune response of allogeneic T lymphocyte in mixed lymphocyte reaction and to explore its mechanism.
[Methods] to establish a co culture system of MSCs and allogeneic lymphocyte, the total amount of the reaction system was 250 Mu L., the spleen T lymphocyte of SD rats was used as the stimulating cell, and the spleen T lymphocyte of Wistar rats was divided into 6 groups. Group I: the control group, 1 x 10~5 / 50 micron stimulus cells and 1 x 10~5 / 50 micron l reactive cells co culture; group II: 1 10~5 / 50 mu l reactive cells were co cultured with MSCs of 1 x 10~4 / 50 mu L SD rats; group III: 1 x 10~5 / 50 micron l irritation cells and 1 x 10~5 / 50 micron l reactive cells and 1 x 10~4 / 100 micron l rats. V: the type and number of cells in the same third groups, plus plant stimulating hormone (terminal concentration 2 g / ml), group VI: 50 mu L (each cell number 1 x 10~5) and MSC0.01ml (the number of cells with 1 x 10~3) per pore, mixed culture for 120 hours, 13 hours before culture, ~3H-TdR 20ul per pore, and measured by liquid flash meter The CPM value of each group was determined by RP HPLC. The tryptophan content in MSC and MLR co culture system was detected.
[results] MSCs can inhibit the proliferation of T lymphocytes in the MLC system, and present a dose dependent relationship. At the same time, the content of tryptophan in the co culture system of MSCs and MLR can be significantly reduced by.1-MT to block this effect.
[Conclusion] MSCs can inhibit the immune response of allogeneic T lymphocytes in vitro, and IDO is involved in this immunosuppressive effect.
The third part is the establishment of heterotopic heart transplantation model in rats.
[Objective] to establish an animal model of abdominal heart transplantation in rats by modified Ono method.
[Methods] the Wistar rats of 200 ~ 250gSD body weight and 200 to 250g were used as receptors, and the rats were anesthetized with.SD rats with abdominal ectopic heart transplantation. After heparinized, the thoracic cavity was opened, the aorta was blocked and the myocardial protective solution was perfused through the aorta root. The donor heart was removed from the.Wistar rats and the abdominal cavity was opened. The abdominal cavity was opened and will be opened. The aorta of the aorta and the recipient's abdominal aorta end to side anastomosis, and the donor pulmonary artery and the recipient's inferior vena cava end to side anastomosis.
[results] the total operation time of the model of heterotopic heart transplantation in rats was about 60 ~ 75min, average (70.1 + 4.9min), and the time of donor heart extraction was about 8 ~ 12min, average (10.5 + 2.3min), and the preparation time of the receptor was 11 ~ 15min, average (13.7 + 2.0min).
[Conclusion] the modified Ono method is reliable and highly repeatable for abdominal heart transplantation in rats.
The effect and mechanism of fourth parts of rat bone marrow mesenchymal stem cells on allograft heart transplantation
[Objective] to study the effect and mechanism of intravenous infusion of bone marrow mesenchymal stem cells (MSCs) on acute rejection after heart transplantation.
[Methods] 64 Wistar rats were randomly divided into 4 groups, with 16 rats in each group. Group I: the blank control group was injected with PBS in the lumbar vein, and the bone marrow mesenchymal stem cells of the donor rats were injected into the lumbar vein during the second operation 1 x 10~6 / ml, and the group used cyclosporin 5mg / Kg every day after the third operation; the group IV was injected into the donor rat via the lumbar vein. Bone marrow mesenchymal stem cells were 1 x 10~6 / ml, 7 days after operation, the donor heart survival time was observed by cyclosporin 5mg / Kg.. The donor heart was observed on the seven day after operation, and the pathological changes were observed and the percentage of CD4~+ and CD8~+ was detected by the splenic cell suspension flow cytometry. The ratio of CD4~+ / CD8~+ in each group was calculated and compared. ELISA method was used to detect the peripheral blood cell cause. The levels of interferon - gamma (Interferon- gamma, IFN- gamma) and interleukin - 10 (interleukin-10, IL-10).
[results] the survival time of the transplanted heart was group I: 7.5 + 0.9 days, group II: 15.5 + 1.6 days, group III: 17.3 + 1.3 days, group IV: 33.4 + 3.2 days. Statistical analysis showed that the survival time group of the transplanted heart group was significantly longer than group I (P < 0.05), group IV was significantly longer than the other three groups (P < 0.01). Group IV transplantation heart pathological changes were compared with those in group IV The three groups were light. Group II, III and IV were higher than group I (P < 0.05). Group IV was also significantly higher than the other three groups (P < 0.05). The level of IFN- gamma in the receptor serum, group II, III was significantly lower than group I, group IV was lower than the other three groups (P < 0.05). Serum IL-10 level, group II, III and IV were higher than group I (P < 0.05), group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV, group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV, group IV group IV, group IV group IV (P < 0.05), group IV Group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV, group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV Higher than group I and group II (P < 0.05)
[Conclusion] bone marrow mesenchymal stem cells can relieve the immune rejection of allograft heart and prolong the survival time of transplanted heart. The mechanism may be related to the regulation of the function of allogenic T cells and the secretion of cytokine. The combination of cytosamycin A and bone marrow mesenchymal stem cells combined with the inhibition of immune rejection has a synergistic effect. Use.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R654.2;R392.4

【参考文献】