人胎盤源間充質(zhì)干細胞的生物學(xué)特性及其對T細胞負性協(xié)同抑制作用
發(fā)布時間：2018-09-03 16:39
【摘要】： 間充質(zhì)干細胞(mesenchymal stem cell,MSCs)是來源于發(fā)育中胚層的一類多能干細胞,具有自我更新及多向分化潛能,它在特定誘導(dǎo)條件下可分化為多種類型的組織細胞,可形成骨、軟骨、脂肪、心肌等多種組織。最初MSCs來源于骨髓,由于人骨髓源間充質(zhì)干細胞(bone marrow mesenchymal stem cells,BMSCs)在骨髓中含量極少,一般僅為骨髓細胞量的0.01%—0.001%,且取材較難,近年來已成功從胎盤、臍血、密致骨、脂肪、肌肉等多種組織中分離,而原被作為“廢棄物”丟棄的胎盤,取材相對容易。本文旨在從成熟胎盤中獲得胎盤源間充質(zhì)干細胞(placenta derived mesenchymal stem cells,PMSCs),并研究比較了PMSCs與BMSCs的免疫生物學(xué)特性。 近年的研究表明,MSCs除能支持體外造血、促進體內(nèi)造血重建,還具有低免疫原性和免疫調(diào)節(jié)作用抑制同種異體免疫排斥反應(yīng),在異基因干細胞移植時能降低宿主抗移植物反應(yīng)(HVGR)和移植物抗宿主反應(yīng)(GVHD)、可延長移植物生存時間,提高移植成功率,是一種較為理想的組織工程種子細胞,但具體的免疫調(diào)節(jié)的機制尚未完全闡明。研究表明,人PMSCs對T細胞活化、增殖有抑制作用�，F(xiàn)有實驗表明人PMSCs不表達HLA-DR,PMSCs具有低免疫原性,而對于胎盤源MSCs免疫調(diào)節(jié)作用的報道較少。本實驗從人胎盤中分離獲取MSCs,并對其生物學(xué)特性與BMSCs進一步比較,并觀察了高表達負性協(xié)同刺激分子PD-L1的PMSCs在體外對T細胞周期和活化、對細胞因子的分泌等方面的調(diào)節(jié)作用,為臨床應(yīng)用和基礎(chǔ)研究提供實驗的理論依據(jù)。 第一部分人胎盤源與人骨髓源間充質(zhì)干細胞的體外分離及生物學(xué)特性的比較 目的:比較人胎盤源間充質(zhì)干細胞(HPMSCs)與人骨髓源間充質(zhì)干細胞(HBMSCs)的體外分離的方法及兩種細胞生物學(xué)特性。 方法:采取酶消化法分離人胎盤組織,用密度梯度離心法分離骨髓單個核細胞,分別進行貼壁分離和傳代培養(yǎng),通過倒置相差顯微鏡觀察細胞形態(tài),用流式細胞儀檢測細胞表面標(biāo)志的表達做比較性分析。并在誘導(dǎo)劑作用下進行細胞分化實驗、鑒定干細胞,并用PHA刺激的T細胞與PMSCs或BMSCs共培養(yǎng),觀察T細胞與兩者的相互作用。 結(jié)果:(1)兩種來源的間充質(zhì)干細胞均貼壁生長、呈成纖維細胞樣形態(tài)、表達CD29、CD44、CD73、CD90、CD105、CD106、CD166,但不表達CD34、CD45、HLA-DR分子;(2)兩種來源的間充質(zhì)干細胞均能在體外向成脂肪細胞、成骨細胞方向誘導(dǎo)分化,(3);兩種來源的間充質(zhì)干細胞在體外對T細胞的活化增殖有明顯的抑制作用。 結(jié)論:人PMSCs與人BMSCs具有相似的細胞體外生物學(xué)特性、表型和多向分化的潛能及其負性免疫調(diào)節(jié)作用,是一種較為理想的組織工程種子細胞。 第二部分負性協(xié)同刺激分子PD-L1在人胎盤源MSCs的表達及其負性免疫調(diào)節(jié)作用 目的:探討人胎盤間充質(zhì)干細胞(PMSCs)對負性協(xié)同刺激分子PD-L1的表達以及對T細胞體外的負性協(xié)同作用。 方法:用體外擴增三代后的PMSCs和HBMSCs,采用流式細胞儀和免疫熒光方法來分析兩種細胞上協(xié)同刺激分子的表達情況,~3H-TdR摻入法檢測PMSCs對PHA刺激后的T細胞增殖的影響以及PD-L1單克隆抗體部分阻斷PMSCs抑制T細胞增殖的影響;流式細胞術(shù)分析PMSCs對PHA作用下T細胞周期和早期活化分子CD69表達的影響,ELISA法檢測細胞因子水平,以及阻斷PD-L1作用后的變化。 結(jié)果:(1)人PMSCs和HBMSCs均不表達CD80、CD83、CD86等正性協(xié)同刺激分子,而PMSCs高表達PD-L1,HBMSCs則低水平表達PD-L1。(2)PMSCs可抑制T細胞體外增殖,并使PHA刺激下的T細胞滯留于細胞周期的G0/G1期,下調(diào)活化T細胞早期表面分子CD69的表達,以及調(diào)節(jié)IL-2、IFN-γ、IL-10的分泌。由此表明,PMSCs介導(dǎo)的負性免疫調(diào)節(jié)作用部分是通過高表達PD-L1分子,PD-L1是PMSCs表達的主要負性免疫調(diào)節(jié)分子。 結(jié)論:人PMSCs在體外具有T細胞活化和增殖的抑制作用,其表達的PD-L1分子介導(dǎo)重要的免疫調(diào)節(jié)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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329;R392
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