人胎盘源间充质干细胞的生物学特性及其对T细胞负性协同抑制作用
发布时间：2018-09-03 16:39
【摘要】： 间充质干细胞(mesenchymal stem cell,MSCs)是来源于发育中胚层的一类多能干细胞,具有自我更新及多向分化潜能,它在特定诱导条件下可分化为多种类型的组织细胞,可形成骨、软骨、脂肪、心肌等多种组织。最初MSCs来源于骨髓,由于人骨髓源间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)在骨髓中含量极少,一般仅为骨髓细胞量的0.01%—0.001%,且取材较难,近年来已成功从胎盘、脐血、密致骨、脂肪、肌肉等多种组织中分离,而原被作为“废弃物”丢弃的胎盘,取材相对容易。本文旨在从成熟胎盘中获得胎盘源间充质干细胞(placenta derived mesenchymal stem cells,PMSCs),并研究比较了PMSCs与BMSCs的免疫生物学特性。 近年的研究表明,MSCs除能支持体外造血、促进体内造血重建,还具有低免疫原性和免疫调节作用抑制同种异体免疫排斥反应,在异基因干细胞移植时能降低宿主抗移植物反应(HVGR)和移植物抗宿主反应(GVHD)、可延长移植物生存时间,提高移植成功率,是一种较为理想的组织工程种子细胞,但具体的免疫调节的机制尚未完全阐明。研究表明,人PMSCs对T细胞活化、增殖有抑制作用。现有实验表明人PMSCs不表达HLA-DR,PMSCs具有低免疫原性,而对于胎盘源MSCs免疫调节作用的报道较少。本实验从人胎盘中分离获取MSCs,并对其生物学特性与BMSCs进一步比较,并观察了高表达负性协同刺激分子PD-L1的PMSCs在体外对T细胞周期和活化、对细胞因子的分泌等方面的调节作用,为临床应用和基础研究提供实验的理论依据。 第一部分人胎盘源与人骨髓源间充质干细胞的体外分离及生物学特性的比较 目的:比较人胎盘源间充质干细胞(HPMSCs)与人骨髓源间充质干细胞(HBMSCs)的体外分离的方法及两种细胞生物学特性。 方法:采取酶消化法分离人胎盘组织,用密度梯度离心法分离骨髓单个核细胞,分别进行贴壁分离和传代培养,通过倒置相差显微镜观察细胞形态,用流式细胞仪检测细胞表面标志的表达做比较性分析。并在诱导剂作用下进行细胞分化实验、鉴定干细胞,并用PHA刺激的T细胞与PMSCs或BMSCs共培养,观察T细胞与两者的相互作用。 结果:(1)两种来源的间充质干细胞均贴壁生长、呈成纤维细胞样形态、表达CD29、CD44、CD73、CD90、CD105、CD106、CD166,但不表达CD34、CD45、HLA-DR分子;(2)两种来源的间充质干细胞均能在体外向成脂肪细胞、成骨细胞方向诱导分化,(3);两种来源的间充质干细胞在体外对T细胞的活化增殖有明显的抑制作用。 结论:人PMSCs与人BMSCs具有相似的细胞体外生物学特性、表型和多向分化的潜能及其负性免疫调节作用,是一种较为理想的组织工程种子细胞。 第二部分负性协同刺激分子PD-L1在人胎盘源MSCs的表达及其负性免疫调节作用 目的:探讨人胎盘间充质干细胞(PMSCs)对负性协同刺激分子PD-L1的表达以及对T细胞体外的负性协同作用。 方法:用体外扩增三代后的PMSCs和HBMSCs,采用流式细胞仪和免疫荧光方法来分析两种细胞上协同刺激分子的表达情况,~3H-TdR掺入法检测PMSCs对PHA刺激后的T细胞增殖的影响以及PD-L1单克隆抗体部分阻断PMSCs抑制T细胞增殖的影响;流式细胞术分析PMSCs对PHA作用下T细胞周期和早期活化分子CD69表达的影响,ELISA法检测细胞因子水平,以及阻断PD-L1作用后的变化。 结果:(1)人PMSCs和HBMSCs均不表达CD80、CD83、CD86等正性协同刺激分子,而PMSCs高表达PD-L1,HBMSCs则低水平表达PD-L1。(2)PMSCs可抑制T细胞体外增殖,并使PHA刺激下的T细胞滞留于细胞周期的G0/G1期,下调活化T细胞早期表面分子CD69的表达,以及调节IL-2、IFN-γ、IL-10的分泌。由此表明,PMSCs介导的负性免疫调节作用部分是通过高表达PD-L1分子,PD-L1是PMSCs表达的主要负性免疫调节分子。 结论:人PMSCs在体外具有T细胞活化和增殖的抑制作用,其表达的PD-L1分子介导重要的免疫调节作用。
[Abstract]:Mesenchymal stem cells (MSCs) are a class of pluripotent stem cells derived from the developing mesoderm. They have the potential of self-renewal and multi-directional differentiation. They can differentiate into various types of tissue cells under specific induction conditions and can form bone, cartilage, fat, myocardium and other tissues. The content of bone marrow mesenchymal stem cells (BMSCs) in bone marrow is very small, generally only 0.01%-0.001% of the bone marrow cells, and it is difficult to obtain materials. In recent years, BMSCs have been successfully isolated from placenta, umbilical cord blood, compact bone, fat, muscle and other tissues, but the placenta discarded as a "waste" is relatively easy to obtain materials. The aim of this study was to obtain placenta derived mesenchymal stem cells (PMSCs) from mature placentas and compare the immunobiological characteristics of PMSCs and BMSCs.
Recent studies have shown that MSCs can not only support hematopoiesis in vitro and promote hematopoietic reconstruction in vivo, but also inhibit allograft rejection with low immunogenicity and immunomodulation. MSCs can reduce host-versus-graft response (HVGR) and graft-versus-host reaction (GVHD) in allogeneic stem cell transplantation, prolong the survival time of transplants and improve the survival rate of transplants. The success rate of transplantation is an ideal seed cell for tissue engineering, but the specific mechanism of immune regulation has not been fully elucidated. Studies have shown that human PMSCs inhibit the activation and proliferation of T cells. In this study, MSCs were isolated from human placenta and their biological characteristics were compared with those of BMSCs. The regulatory effects of PMSCs with high expression of negative costimulatory molecule PD-L1 on T cell cycle, activation and cytokine secretion were observed in vitro, which provided experimental theoretical basis for clinical application and basic research.
Part one: isolation and biological characteristics of mesenchymal stem cells from human placenta and human bone marrow
AIM: To compare the methods of isolating human placental derived mesenchymal stem cells (HPMSCs) and human bone marrow derived mesenchymal stem cells (HBMSCs) in vitro and the two cell biological characteristics.
Methods: Human placenta tissue was isolated by enzymatic digestion, bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in vitro. Cell morphology was observed by inverted phase contrast microscope, and the expression of cell surface markers was detected by flow cytometry. In the experiment, stem cells were identified and co-cultured with PMSCs or BMSCs by PHA-stimulated T cells.
Results: (1) MSCs from both sources adhered to the wall and grew in fibroblast-like morphology, expressing CD29, CD44, CD73, CD90, CD105, CD106 and CD166, but not expressing CD34, CD45 and HLA-DR; (2) MSCs from both sources could differentiate into adipocytes and osteoblasts in vitro; (3) MSCs from both sources could differentiate into adipocytes and osteoblasts; (3) MSCs from both sources could differentiate into adipocytes and osteoblasts in vitro. In vitro, the cells significantly inhibited the activation and proliferation of T cells.
CONCLUSION: Human PMSCs and human BMSCs have similar biological characteristics in vitro, phenotypic and multidirectional differentiation potentials and negative immunomodulatory effects. They are ideal seed cells for tissue engineering.
The second part is the negative co stimulatory molecule PD-L1 expression and its negative immunomodulatory effect on human placental MSCs.
AIM: To investigate the effect of human placental mesenchymal stem cells (PMSCs) on the expression of negative costimulatory molecule PD-L1 and the negative synergistic effect on T cells in vitro.
Methods: Three generations of PMSCs and HBMSCs were amplified in vitro. The expression of co-stimulatory molecules was analyzed by flow cytometry and immunofluorescence. The effects of PMSCs on the proliferation of PHA-stimulated T cells were detected by ~3H-TdR incorporation assay, and the effects of PD-L1 monoclonal antibody partially blocked the proliferation of PMSCs. The effects of PMSCs on the expression of CD69, a T cell cycle and early activating molecule, were analyzed by cytometry. The levels of cytokines were detected by ELISA and the changes after blocking the action of PD-L1 were observed.
Results: (1) Human PMSCs and HBMSCs did not express positive co-stimulatory molecules such as CD80, CD83 and CD86, while PMSCs overexpressed PD-L1 and HBMSCs underexpressed PD-L1. (2) PMSCs inhibited the proliferation of T cells in vitro, and caused PHA-stimulated T cells to remain in the G0/G1 phase of cell cycle, down-regulated the expression of CD69 on the early surface of activated T cells, and regulated IL-2. This suggests that PMSCs mediate negative immune regulation by overexpressing PD-L1, which is the main negative immunoregulatory molecule expressed by PMSCs.
CONCLUSION: Human PMSCs can inhibit T cell activation and proliferation in vitro, and the expression of PD-L1 molecule mediates important immunomodulatory effects.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329;R392
[Abstract]:Mesenchymal stem cells (MSCs) are a class of pluripotent stem cells derived from the developing mesoderm. They have the potential of self-renewal and multi-directional differentiation. They can differentiate into various types of tissue cells under specific induction conditions and can form bone, cartilage, fat, myocardium and other tissues. The content of bone marrow mesenchymal stem cells (BMSCs) in bone marrow is very small, generally only 0.01%-0.001% of the bone marrow cells, and it is difficult to obtain materials. In recent years, BMSCs have been successfully isolated from placenta, umbilical cord blood, compact bone, fat, muscle and other tissues, but the placenta discarded as a "waste" is relatively easy to obtain materials. The aim of this study was to obtain placenta derived mesenchymal stem cells (PMSCs) from mature placentas and compare the immunobiological characteristics of PMSCs and BMSCs.
Recent studies have shown that MSCs can not only support hematopoiesis in vitro and promote hematopoietic reconstruction in vivo, but also inhibit allograft rejection with low immunogenicity and immunomodulation. MSCs can reduce host-versus-graft response (HVGR) and graft-versus-host reaction (GVHD) in allogeneic stem cell transplantation, prolong the survival time of transplants and improve the survival rate of transplants. The success rate of transplantation is an ideal seed cell for tissue engineering, but the specific mechanism of immune regulation has not been fully elucidated. Studies have shown that human PMSCs inhibit the activation and proliferation of T cells. In this study, MSCs were isolated from human placenta and their biological characteristics were compared with those of BMSCs. The regulatory effects of PMSCs with high expression of negative costimulatory molecule PD-L1 on T cell cycle, activation and cytokine secretion were observed in vitro, which provided experimental theoretical basis for clinical application and basic research.
Part one: isolation and biological characteristics of mesenchymal stem cells from human placenta and human bone marrow
AIM: To compare the methods of isolating human placental derived mesenchymal stem cells (HPMSCs) and human bone marrow derived mesenchymal stem cells (HBMSCs) in vitro and the two cell biological characteristics.
Methods: Human placenta tissue was isolated by enzymatic digestion, bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in vitro. Cell morphology was observed by inverted phase contrast microscope, and the expression of cell surface markers was detected by flow cytometry. In the experiment, stem cells were identified and co-cultured with PMSCs or BMSCs by PHA-stimulated T cells.
Results: (1) MSCs from both sources adhered to the wall and grew in fibroblast-like morphology, expressing CD29, CD44, CD73, CD90, CD105, CD106 and CD166, but not expressing CD34, CD45 and HLA-DR; (2) MSCs from both sources could differentiate into adipocytes and osteoblasts in vitro; (3) MSCs from both sources could differentiate into adipocytes and osteoblasts; (3) MSCs from both sources could differentiate into adipocytes and osteoblasts in vitro. In vitro, the cells significantly inhibited the activation and proliferation of T cells.
CONCLUSION: Human PMSCs and human BMSCs have similar biological characteristics in vitro, phenotypic and multidirectional differentiation potentials and negative immunomodulatory effects. They are ideal seed cells for tissue engineering.
The second part is the negative co stimulatory molecule PD-L1 expression and its negative immunomodulatory effect on human placental MSCs.
AIM: To investigate the effect of human placental mesenchymal stem cells (PMSCs) on the expression of negative costimulatory molecule PD-L1 and the negative synergistic effect on T cells in vitro.
Methods: Three generations of PMSCs and HBMSCs were amplified in vitro. The expression of co-stimulatory molecules was analyzed by flow cytometry and immunofluorescence. The effects of PMSCs on the proliferation of PHA-stimulated T cells were detected by ~3H-TdR incorporation assay, and the effects of PD-L1 monoclonal antibody partially blocked the proliferation of PMSCs. The effects of PMSCs on the expression of CD69, a T cell cycle and early activating molecule, were analyzed by cytometry. The levels of cytokines were detected by ELISA and the changes after blocking the action of PD-L1 were observed.
Results: (1) Human PMSCs and HBMSCs did not express positive co-stimulatory molecules such as CD80, CD83 and CD86, while PMSCs overexpressed PD-L1 and HBMSCs underexpressed PD-L1. (2) PMSCs inhibited the proliferation of T cells in vitro, and caused PHA-stimulated T cells to remain in the G0/G1 phase of cell cycle, down-regulated the expression of CD69 on the early surface of activated T cells, and regulated IL-2. This suggests that PMSCs mediate negative immune regulation by overexpressing PD-L1, which is the main negative immunoregulatory molecule expressed by PMSCs.
CONCLUSION: Human PMSCs can inhibit T cell activation and proliferation in vitro, and the expression of PD-L1 molecule mediates important immunomodulatory effects.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329;R392
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