滇南小耳豬骨髓間質(zhì)干細(xì)胞誘導(dǎo)移植免疫耐受的研究
發(fā)布時(shí)間：2018-08-23 19:39
【摘要】： 【背景】同種異體原位心肺移植已經(jīng)成為治療終末期心肺疾病的有效方法,為醫(yī)學(xué)科學(xué)開辟了一個(gè)新的領(lǐng)域。目前,供體心肺保存、外科手術(shù)技術(shù)、術(shù)后感染已不再是阻礙心肺移植成功的最主要因素。但是,移植術(shù)后的免疫排斥反應(yīng)嚴(yán)重威脅著移植患者的長期生存,供體短缺也困擾著外科醫(yī)生。誘導(dǎo)對供體器官特異性免疫耐受(donor-specific graft tolerance)能有效的解決這一難題,而且某些耐受方案已經(jīng)在小動物模型中取得了較好的效果,但是尚缺乏大動物模型的驗(yàn)證。實(shí)驗(yàn)動物小型豬在器官移植免疫耐受的研究中具有顯著的優(yōu)勢。骨髓間質(zhì)干細(xì)胞(bone marrow-derived mesenchymal stem cells,BMSCs)是存在于骨髓中的一種成體干細(xì)胞,具有支持造血、組織修復(fù)、多向分化潛能以及潛在的免疫學(xué)特性。隨著對小型豬的研究不斷深入,滇南小耳豬已經(jīng)成為研究人類疾病模型最具潛力的實(shí)驗(yàn)動物,尤其是在心肺移植方面的研究,其更具有優(yōu)勢。最近研究表明人類和嚙齒類動物BMSCs在體外可抑制T淋巴細(xì)胞的增殖,治療移植物抗宿主病;在狒狒體內(nèi)可延長移植皮膚的存活時(shí)間。但是其在體內(nèi)的遷移變化,免疫調(diào)節(jié)的分子機(jī)制尚不清楚,而且尚未見到關(guān)于滇南小耳豬骨髓間質(zhì)干細(xì)胞(porcine bonemarrow-derived mesenchymal stem cells,pBMSCs)免疫學(xué)特性的研究報(bào)到。因此,本實(shí)驗(yàn)擬通過建立pBMSCs體外分離、培養(yǎng)擴(kuò)增體系研究pBMSCs的生物學(xué)特性;通過體外混合培養(yǎng)體系及將pBMSCs輸入實(shí)驗(yàn)豬體內(nèi)研究pBMSCs的免疫學(xué)特性及其機(jī)制。 【方法】(1)采用全骨髓貼壁法,進(jìn)行pBMSCs的分離培養(yǎng)。采用倒置顯微鏡觀察pBMSCs的生長情況和透射電鏡觀察pBMSCs的超微結(jié)構(gòu)對pBMSCs進(jìn)行形態(tài)學(xué)鑒定;采用流式細(xì)胞術(shù)對pBMSCs的分子表型進(jìn)行鑒定;在體外,對pBMSCs向成骨細(xì)胞和脂肪細(xì)胞誘導(dǎo)分化鑒定pBMSCs的多向分化潛能。(2)建立混合淋巴細(xì)胞培養(yǎng)體系。通過體外混合淋巴細(xì)胞培養(yǎng)(Mixed Lymphocyte Culture Test,MLC)檢測pBMSCs對外周血淋巴細(xì)胞(peripheral blood lymphocytes,PBLs)增殖的抑制效應(yīng),通過實(shí)時(shí)定量聚合酶鏈反應(yīng)(Real Time-Polymerase Chain Reaction,RealTime -PCR)檢測混合培養(yǎng)后的淋巴細(xì)胞Foxp3基因及細(xì)胞因子干擾素γ(interferon-gamma,IFN-γ)、轉(zhuǎn)化生長因子β(transforming growth factor beta,TGF-β)的變化,探討pBMSCs體外免疫抑制效應(yīng)及其機(jī)制。(3)將同種異體pBMSCs經(jīng)外周靜脈輸入滇南小耳豬體內(nèi),流式細(xì)胞術(shù)檢測不同時(shí)間內(nèi)實(shí)驗(yàn)豬外周血T淋巴細(xì)胞亞群的變化,Real Time-PCR檢測外周血淋巴細(xì)胞Foxp3基因及細(xì)胞因子IFN-γ、TGF-β隨輸入時(shí)間的變化,探討pBMSCs的低免疫原性。 【結(jié)果】(1)通過全骨髓貼壁法,獲得了高純度、高活性的原代pBMSCs。對所得到的pBMSCs體外培養(yǎng)擴(kuò)增20代次,其生物學(xué)性狀穩(wěn)定。形態(tài)學(xué)鑒定pBMSCs為成纖維樣細(xì)胞;分子表型鑒定pBMSCs穩(wěn)定表達(dá)CD29、CD44、CD105,不表達(dá)CD45、SLA-DR;多向分化潛能鑒定pBMSCs能夠分化為成骨細(xì)胞及脂肪細(xì)胞。本研究建立了滇南小耳豬BMSCs體外分離純化、培養(yǎng)擴(kuò)增的方法體系,獲得了滇南小耳豬BMSCs細(xì)胞系并大量超低溫冷凍保存,為下一步滇南小耳豬BMSCs免疫學(xué)特性的研究與誘導(dǎo)滇南小耳豬心肺移植免疫耐受的研究提供了種子細(xì)胞。(2)建立滇南小耳豬BMSCs和淋巴細(xì)胞共培養(yǎng)體系。pBMSCs對外周血淋巴細(xì)胞及PHA刺激的外周血淋巴細(xì)胞的增殖有顯著的抑制作用,且呈劑量依賴性。然而pBMSCs培養(yǎng)上清對外周血淋巴細(xì)胞的增殖無抑制作用,甚至是刺激外周血淋巴細(xì)胞的增殖。混合培養(yǎng)后的PBLs高表達(dá)Foxp3、TGF-β及IFN-γ;IFN-γ表達(dá)水平明顯低于Foxp3和TGF-β。(3)將pBMSCs輸入滇南小耳豬體內(nèi)后,外周血淋巴細(xì)胞Foxp3、TGF-β及IFN-γ在輸注pBMSCs 24h后表達(dá)均顯著增高,且隨時(shí)間的延長而升高,到72h達(dá)高峰,96h時(shí)開始下降(P＜0.05);外周血T淋巴細(xì)胞亞群的變化無統(tǒng)計(jì)學(xué)差異。 【結(jié)論】(1)采用全骨髓貼壁法,可獲得高純度、高活性的原代pBMSCs。通過體外傳代培養(yǎng)擴(kuò)增獲得了pBMSCs細(xì)胞系。對獲得的pBMScs體外培養(yǎng)擴(kuò)增20代次,并進(jìn)行形態(tài)學(xué),分子表型及多向分化潛能鑒定,其生物學(xué)性狀穩(wěn)定。為下一步滇南小耳豬pBMSCs免疫調(diào)節(jié)特性及機(jī)制的研究與pBMSCs誘導(dǎo)心肺移植免疫耐受的研究提供了種子細(xì)胞。(2)在體外混合淋巴細(xì)胞反應(yīng)中,pBMSCs能夠抑制外周血淋巴細(xì)胞的增殖。在體內(nèi)研究中,pBMSCs不刺激同種免疫應(yīng)答的產(chǎn)生,能夠調(diào)節(jié)免疫系統(tǒng)。(3)pBMSCs具有低免疫原性及免疫調(diào)節(jié)特性。其機(jī)制與可溶性細(xì)胞因子間的相互作用,誘導(dǎo)Foxp3基因高表達(dá)的調(diào)節(jié)性T細(xì)胞產(chǎn)生以及細(xì)胞間的直接接觸有關(guān)。為下一步同種異體pBMSCs誘導(dǎo)滇南小耳豬心肺移植免疫耐受的研究奠定基礎(chǔ)。(4)pBMSCs有望在滇南小耳豬心肺移植模型中誘導(dǎo)出穩(wěn)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.
【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392.1
本文編號：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.
【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392.1
【參考文獻(xiàn)】
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