滇南小耳猪骨髓间质干细胞诱导移植免疫耐受的研究
发布时间：2018-08-23 19:39
【摘要】： 【背景】同种异体原位心肺移植已经成为治疗终末期心肺疾病的有效方法,为医学科学开辟了一个新的领域。目前,供体心肺保存、外科手术技术、术后感染已不再是阻碍心肺移植成功的最主要因素。但是,移植术后的免疫排斥反应严重威胁着移植患者的长期生存,供体短缺也困扰着外科医生。诱导对供体器官特异性免疫耐受(donor-specific graft tolerance)能有效的解决这一难题,而且某些耐受方案已经在小动物模型中取得了较好的效果,但是尚缺乏大动物模型的验证。实验动物小型猪在器官移植免疫耐受的研究中具有显著的优势。骨髓间质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)是存在于骨髓中的一种成体干细胞,具有支持造血、组织修复、多向分化潜能以及潜在的免疫学特性。随着对小型猪的研究不断深入,滇南小耳猪已经成为研究人类疾病模型最具潜力的实验动物,尤其是在心肺移植方面的研究,其更具有优势。最近研究表明人类和啮齿类动物BMSCs在体外可抑制T淋巴细胞的增殖,治疗移植物抗宿主病;在狒狒体内可延长移植皮肤的存活时间。但是其在体内的迁移变化,免疫调节的分子机制尚不清楚,而且尚未见到关于滇南小耳猪骨髓间质干细胞(porcine bonemarrow-derived mesenchymal stem cells,pBMSCs)免疫学特性的研究报到。因此,本实验拟通过建立pBMSCs体外分离、培养扩增体系研究pBMSCs的生物学特性;通过体外混合培养体系及将pBMSCs输入实验猪体内研究pBMSCs的免疫学特性及其机制。 【方法】(1)采用全骨髓贴壁法,进行pBMSCs的分离培养。采用倒置显微镜观察pBMSCs的生长情况和透射电镜观察pBMSCs的超微结构对pBMSCs进行形态学鉴定;采用流式细胞术对pBMSCs的分子表型进行鉴定;在体外,对pBMSCs向成骨细胞和脂肪细胞诱导分化鉴定pBMSCs的多向分化潜能。(2)建立混合淋巴细胞培养体系。通过体外混合淋巴细胞培养(Mixed Lymphocyte Culture Test,MLC)检测pBMSCs对外周血淋巴细胞(peripheral blood lymphocytes,PBLs)增殖的抑制效应,通过实时定量聚合酶链反应(Real Time-Polymerase Chain Reaction,RealTime -PCR)检测混合培养后的淋巴细胞Foxp3基因及细胞因子干扰素γ(interferon-gamma,IFN-γ)、转化生长因子β(transforming growth factor beta,TGF-β)的变化,探讨pBMSCs体外免疫抑制效应及其机制。(3)将同种异体pBMSCs经外周静脉输入滇南小耳猪体内,流式细胞术检测不同时间内实验猪外周血T淋巴细胞亚群的变化,Real Time-PCR检测外周血淋巴细胞Foxp3基因及细胞因子IFN-γ、TGF-β随输入时间的变化,探讨pBMSCs的低免疫原性。 【结果】(1)通过全骨髓贴壁法,获得了高纯度、高活性的原代pBMSCs。对所得到的pBMSCs体外培养扩增20代次,其生物学性状稳定。形态学鉴定pBMSCs为成纤维样细胞;分子表型鉴定pBMSCs稳定表达CD29、CD44、CD105,不表达CD45、SLA-DR;多向分化潜能鉴定pBMSCs能够分化为成骨细胞及脂肪细胞。本研究建立了滇南小耳猪BMSCs体外分离纯化、培养扩增的方法体系,获得了滇南小耳猪BMSCs细胞系并大量超低温冷冻保存,为下一步滇南小耳猪BMSCs免疫学特性的研究与诱导滇南小耳猪心肺移植免疫耐受的研究提供了种子细胞。(2)建立滇南小耳猪BMSCs和淋巴细胞共培养体系。pBMSCs对外周血淋巴细胞及PHA刺激的外周血淋巴细胞的增殖有显著的抑制作用,且呈剂量依赖性。然而pBMSCs培养上清对外周血淋巴细胞的增殖无抑制作用,甚至是刺激外周血淋巴细胞的增殖。混合培养后的PBLs高表达Foxp3、TGF-β及IFN-γ;IFN-γ表达水平明显低于Foxp3和TGF-β。(3)将pBMSCs输入滇南小耳猪体内后,外周血淋巴细胞Foxp3、TGF-β及IFN-γ在输注pBMSCs 24h后表达均显著增高,且随时间的延长而升高,到72h达高峰,96h时开始下降(P＜0.05);外周血T淋巴细胞亚群的变化无统计学差异。 【结论】(1)采用全骨髓贴壁法,可获得高纯度、高活性的原代pBMSCs。通过体外传代培养扩增获得了pBMSCs细胞系。对获得的pBMScs体外培养扩增20代次,并进行形态学,分子表型及多向分化潜能鉴定,其生物学性状稳定。为下一步滇南小耳猪pBMSCs免疫调节特性及机制的研究与pBMSCs诱导心肺移植免疫耐受的研究提供了种子细胞。(2)在体外混合淋巴细胞反应中,pBMSCs能够抑制外周血淋巴细胞的增殖。在体内研究中,pBMSCs不刺激同种免疫应答的产生,能够调节免疫系统。(3)pBMSCs具有低免疫原性及免疫调节特性。其机制与可溶性细胞因子间的相互作用,诱导Foxp3基因高表达的调节性T细胞产生以及细胞间的直接接触有关。为下一步同种异体pBMSCs诱导滇南小耳猪心肺移植免疫耐受的研究奠定基础。(4)pBMSCs有望在滇南小耳猪心肺移植模型中诱导出稳定、持久的免疫耐受。
[Abstract]:[BACKGROUND] Orthotopic heart-lung transplantation has become an effective method for the treatment of end-stage cardiopulmonary diseases, and has opened up a new field for medical science. At present, donor heart-lung preservation, surgical techniques and postoperative infection are no longer the main factors that hinder the success of heart-lung transplantation. However, the immunological rejection after transplantation is serious. Induction of donor-specific graft tolerance can effectively solve this problem, and some tolerance schemes have achieved good results in small animal models, but there is no validation of large animal models. * bone marrow-derived mesenchymal stem cells (BMSCs) is a kind of adult stem cell that exists in bone marrow. It has the support of hematopoietic, tissue repair, multipotential differentiation and potential immunological characteristics. With the development of miniature pigs, * Further research has been carried out. * Yunnan ear pig has become the most potential laboratory animal for studying human disease models, especially in the study of heart lung transplantation. It has more advantages. Recent studies have shown that BMSCs and human rodents can inhibit T lymphocyte proliferation and treat graft-versus-host disease in vitro, and extend in baboon. The survival time of transplanted skin, but its migration and change in the body, the molecular mechanism of immune regulation is not clear, and the immunological characteristics of porcine BoneMarrow-Derived mesenchymal stem * cells (pBMSCs) have not been reported. Therefore, this experiment is to establish pBMSCs in vitro. In addition, the biological characteristics of pBMSCs were studied by culture amplification system. * the immunological characteristics and mechanism of pBMSCs were studied through in vitro mixed culture system and pBMSCs in pigs.
[Methods] The whole bone marrow adherence method was used to isolate and culture pBMSCs. The growth of pBMSCs was observed by inverted microscope and the ultrastructure of pBMSCs was observed by transmission electron microscope. (2) Establish a mixed lymphocyte culture system. The inhibitory effect of pBMSCs on the proliferation of peripheral blood lymphocytes (PBLs) was detected by Mixed Lymphocyte Culture Test (MLC) and real-time quantitative polymerase chain reaction (Real Time-Polym). The changes of Foxp3 gene, interferon-gamma (IFN-gamma) and transforming growth factor beta (TGF-beta) in mixed cultured lymphocytes were detected by erase Chain Reaction (RealTime-PCR), and the immunosuppressive effect and mechanism of pBMSCs in vitro were investigated. (3) Allogeneic pBMSCs were transfused into peripheral vein. In the South Yunnan * * ear pig, flow cytometry was used to detect the changes of peripheral blood T lymphocyte subsets in experimental pigs. Real Time-PCR was used to detect the changes of Foxp3 gene and cytokines IFN- IFN- and TGF- beta in peripheral blood lymphocytes with the change of input time, and to explore the low immunogenicity of pBMSCs.
[Results] Primary pBMSCs with high purity and activity were obtained by whole bone marrow adherence method. The obtained pBMSCs were cultured and amplified 20 times in vitro, and their biological characteristics were stable. MSCs can differentiate into osteoblasts and adipocytes. In this study, we established a method for isolation, purification, culture and amplification of BMSCs from South Yunnan * * ear pig, and obtained the BMSCs cell line of Yunnan Yunnan small ear pig * and a large number of cryopreservation, so as to further study the immunological characteristics of BMSCs and induce the lung transplantation immunity of Yunnan small ear pig. Tolerance studies provided seed cells. (2) the establishment of BMSCs and * lymphocyte co culture system in Dian Nan ear pig.PBMSCs inhibited the proliferation of peripheral blood lymphocytes and PHA stimulated peripheral lymphocytes in a dose-dependent manner. However, pBMSCs culture supernatants had no inhibitory effect on the proliferation of peripheral blood lymphocytes. It is to stimulate the proliferation of peripheral blood lymphocytes. After mixed culture, PBLs overexpressed Foxp3, TGF- beta and IFN- gamma; the expression level of IFN- gamma was significantly lower than that of Foxp3 and TGF- *. (3) after pBMSCs was imported into Yunnan Province, the expression of Foxp3, TGF- beta and IFN- gamma in peripheral blood lymphocytes increased significantly, and increased with time. It reached the peak at 72 hours and began to decrease at 96 hours (P < 0.05).
[Conclusion] (1) using the whole bone marrow adherence method, a high purity and high activity primary pBMSCs. can be obtained through in vitro propagation and culture. The pBMSCs cell line was obtained. The pBMScs obtained in vitro was amplified for 20 generations, and the morphology, molecular phenotype and multipotential differentiation potential were identified. Its biological characteristics were stable. * it is the next step to pBMS in Yunnan Province. In vivo, pBMSCs can regulate the immune system without stimulating the production of alloimmune responses. (3) pBMSCs can regulate the immune system. Hypoimmunogenicity and immunomodulatory properties. The mechanism is related to the interaction between soluble cytokines, the production of regulatory T cells with high Foxp3 gene expression and direct contact between cells. It will lay a foundation for the further study of allogeneic pBMSCs inducing cardiopulmonary transplantation immune tolerance in southern Yunnan pigs. (4) pBMSCs are expected to be used in southern Yunnan. Stable and sustained immune tolerance was induced in a small ear pig heart lung transplantation model.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1
本文编号：2199711
[Abstract]:[BACKGROUND] Orthotopic heart-lung transplantation has become an effective method for the treatment of end-stage cardiopulmonary diseases, and has opened up a new field for medical science. At present, donor heart-lung preservation, surgical techniques and postoperative infection are no longer the main factors that hinder the success of heart-lung transplantation. However, the immunological rejection after transplantation is serious. Induction of donor-specific graft tolerance can effectively solve this problem, and some tolerance schemes have achieved good results in small animal models, but there is no validation of large animal models. * bone marrow-derived mesenchymal stem cells (BMSCs) is a kind of adult stem cell that exists in bone marrow. It has the support of hematopoietic, tissue repair, multipotential differentiation and potential immunological characteristics. With the development of miniature pigs, * Further research has been carried out. * Yunnan ear pig has become the most potential laboratory animal for studying human disease models, especially in the study of heart lung transplantation. It has more advantages. Recent studies have shown that BMSCs and human rodents can inhibit T lymphocyte proliferation and treat graft-versus-host disease in vitro, and extend in baboon. The survival time of transplanted skin, but its migration and change in the body, the molecular mechanism of immune regulation is not clear, and the immunological characteristics of porcine BoneMarrow-Derived mesenchymal stem * cells (pBMSCs) have not been reported. Therefore, this experiment is to establish pBMSCs in vitro. In addition, the biological characteristics of pBMSCs were studied by culture amplification system. * the immunological characteristics and mechanism of pBMSCs were studied through in vitro mixed culture system and pBMSCs in pigs.
[Methods] The whole bone marrow adherence method was used to isolate and culture pBMSCs. The growth of pBMSCs was observed by inverted microscope and the ultrastructure of pBMSCs was observed by transmission electron microscope. (2) Establish a mixed lymphocyte culture system. The inhibitory effect of pBMSCs on the proliferation of peripheral blood lymphocytes (PBLs) was detected by Mixed Lymphocyte Culture Test (MLC) and real-time quantitative polymerase chain reaction (Real Time-Polym). The changes of Foxp3 gene, interferon-gamma (IFN-gamma) and transforming growth factor beta (TGF-beta) in mixed cultured lymphocytes were detected by erase Chain Reaction (RealTime-PCR), and the immunosuppressive effect and mechanism of pBMSCs in vitro were investigated. (3) Allogeneic pBMSCs were transfused into peripheral vein. In the South Yunnan * * ear pig, flow cytometry was used to detect the changes of peripheral blood T lymphocyte subsets in experimental pigs. Real Time-PCR was used to detect the changes of Foxp3 gene and cytokines IFN- IFN- and TGF- beta in peripheral blood lymphocytes with the change of input time, and to explore the low immunogenicity of pBMSCs.
[Results] Primary pBMSCs with high purity and activity were obtained by whole bone marrow adherence method. The obtained pBMSCs were cultured and amplified 20 times in vitro, and their biological characteristics were stable. MSCs can differentiate into osteoblasts and adipocytes. In this study, we established a method for isolation, purification, culture and amplification of BMSCs from South Yunnan * * ear pig, and obtained the BMSCs cell line of Yunnan Yunnan small ear pig * and a large number of cryopreservation, so as to further study the immunological characteristics of BMSCs and induce the lung transplantation immunity of Yunnan small ear pig. Tolerance studies provided seed cells. (2) the establishment of BMSCs and * lymphocyte co culture system in Dian Nan ear pig.PBMSCs inhibited the proliferation of peripheral blood lymphocytes and PHA stimulated peripheral lymphocytes in a dose-dependent manner. However, pBMSCs culture supernatants had no inhibitory effect on the proliferation of peripheral blood lymphocytes. It is to stimulate the proliferation of peripheral blood lymphocytes. After mixed culture, PBLs overexpressed Foxp3, TGF- beta and IFN- gamma; the expression level of IFN- gamma was significantly lower than that of Foxp3 and TGF- *. (3) after pBMSCs was imported into Yunnan Province, the expression of Foxp3, TGF- beta and IFN- gamma in peripheral blood lymphocytes increased significantly, and increased with time. It reached the peak at 72 hours and began to decrease at 96 hours (P < 0.05).
[Conclusion] (1) using the whole bone marrow adherence method, a high purity and high activity primary pBMSCs. can be obtained through in vitro propagation and culture. The pBMSCs cell line was obtained. The pBMScs obtained in vitro was amplified for 20 generations, and the morphology, molecular phenotype and multipotential differentiation potential were identified. Its biological characteristics were stable. * it is the next step to pBMS in Yunnan Province. In vivo, pBMSCs can regulate the immune system without stimulating the production of alloimmune responses. (3) pBMSCs can regulate the immune system. Hypoimmunogenicity and immunomodulatory properties. The mechanism is related to the interaction between soluble cytokines, the production of regulatory T cells with high Foxp3 gene expression and direct contact between cells. It will lay a foundation for the further study of allogeneic pBMSCs inducing cardiopulmonary transplantation immune tolerance in southern Yunnan pigs. (4) pBMSCs are expected to be used in southern Yunnan. Stable and sustained immune tolerance was induced in a small ear pig heart lung transplantation model.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1
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