【作者】 李明芬；

【导师】 罗殿中；

【作者基本信息】 广西医科大学 ， 病理学与病理生理学， 2014， 博士

【摘要】 干细胞指一类具有无限增殖能力和多向分化潜能的细胞。按其分化程度和分化能力，可分为胚胎干细胞(ESC)、诱导性多能干细胞（IPSC）和成体干细胞（ASC）。ASC主要包括造血干细胞（HSCs）和间充质干细胞（MSCs）等。MSCs可来源于多种组织或器官，如骨髓、脂肪、胎盘、脐带和牙髓等。其中，骨髓间充质干细胞(BMSCs)因来源相对丰富，且取材方便，体外分离和培养方法成熟，因而研究最深入，临床应用最广泛。MSCs主要有四个方面的功能：支持HSCs重建造血、促进组织修复和再生、肿瘤的靶向治疗和免疫调节功能。其中，MSCs的免疫调节功能是目前研究的热点。MSCs为低免疫原性，生理状态下不能激活同种异体淋巴细胞反应。此外，MSCs还对多种免疫细胞，包括T淋巴细胞、自然杀伤细胞（NK）、树突状细胞（DC）和B淋巴细胞等具有免疫抑制作用。因此，MSCs在免疫性疾病的预防和治疗中有重要应用价值。尤其BMSCs用于治疗或预防骨髓造血干细胞移植（HSCT）产生的急慢性移植物抗宿主病（GVHD）的研究较多。尽管如此，关于MSCs的基础研究和临床试验还远远不足，许多问题尚未得到解答。目前存在的问题有：MSCs治疗免疫性疾病的机制，尤其对各种参与疾病的效应细胞的作用和机制；如何保证MSCs的临床应用效果；MSCs临床治疗的安全性等。CD8+T淋巴细胞及其激活后形成的细胞毒性T淋巴细胞（CTL）是参与移植物抗宿主病（GVHD）的主要效应性T淋巴细胞。由于MSCs在免疫性疾病的临床应用中，对GVHD的预防和治疗应用最多，效果也最明确，所以MSCs治疗GVHD的机制在MSCs对免疫性疾病的临床和实验研究中尤为重要，尤其MSCs对介导GVHD的主要效应性T淋巴细胞—CD8+T淋巴细胞的免疫调节作用和机制研究是其中的重要内容。目前国内外关于MSCs与CD8+T淋巴细胞相互作用的研究较少， MSCs调节CD8+T淋巴细胞功能的机制研究也很缺乏。为进一步探讨MSCs对CD8+T淋巴细胞的免疫调节功能，阐明其作用机制，本论文从以下三个方面进行研究。第一部分：人骨髓间充质干细胞的分离、培养和鉴定目的：体外分离、培养和扩增BMSCs并进行MSCs鉴定，为后续实验提供充足的细胞来源。方法：通过Ficoll密度梯度离心法分离骨髓单个核细胞，使用骨髓间充质干细胞培养基（BMSC-GM）进行体外培养和扩增；通过细胞定量接种和计数，制作细胞生长曲线；通过流式细胞术检测BMSCs的表面分子标记物；通过成骨和成脂诱导液体外培养考察BMSCs的多向分化能力；结合细胞形态、生长特性、表面分子标志物和多向分化能力等对所得细胞进行鉴定；比较各代细胞的特性。结果：体外培养的BMSCs呈纤维样，螺旋状生长；其生长曲线为“S”型，有典型潜伏期、生长期和平台期；表面分子标记物表现为CD29、CD105、CD166阳性，CD34、CD45阴性；BMSCs经体外诱导可分化为成骨细胞和脂肪细胞；结合细胞形态、生长特性、分子标志物和多向分化能力可鉴定所得细胞为MSC；P3-P6代BMSCs形态比较稳定一致，适用于后续实验。结论：通过体外分离、培养、扩增和鉴定，可获得足量的BMSCs，P3-P6代BMSCs适用于后续实验。第二部分：人骨髓间充质干细胞对CD8+T淋巴细胞的免疫调节功能研究目的：探讨BMSCs对CD8+T淋巴细胞的免疫调节作用，并初步探讨MSCs作用于CD8+T淋巴细胞的有效浓度范围。方法：通过免疫磁珠技术，阴性选择法分离纯化人外周血CD8+T淋巴细胞，流式细胞术检测CD3+CD8+T淋巴细胞的比例；以BMSCs为刺激细胞，与同种异体CD8+T淋巴细胞进行共培养，羟基荧光素乙酰乙酸琥珀酰亚胺酯（CFSE）法检测CD8+T淋巴细胞的增殖；以植物血凝素A（PHA）为多克隆T淋巴细胞刺激剂，将CD8+T淋巴细胞与不同浓度BMSCs进行共培养，检测CD8+T淋巴细胞的增殖；将BMSCs与CD8+T淋巴细胞进行共培养，以PHA为刺激剂，实时荧光定量-聚合酶链反应（real time-PCR）法检测CD8+T淋巴细胞内白细胞介素2（IL-2）, γ干扰素（IFN-γ）和颗粒酶B（GZMB）mRNA的表达。结果：免疫磁珠法分选所得细胞中，CD3+CD8+T淋巴细胞占88.1%；当CD8/BMSCs为1:1-100:1时，与BMSCs共培养后，CD8+T淋巴细胞的增殖率无明显升高；当CD8/BMSCs为1:1-20:1时，BMSCs可抑制PHA刺激的CD8+T淋巴细胞增殖，且该作用表现为对BMSCs的浓度依赖性，BMSCs浓度越高，抑制作用越明显；当CD8/BMSCs为50:1-100:1时，BMSCs不能抑制CD8+T淋巴细胞的增殖；此外，BMSCs可抑制CD8+T淋巴细胞内IL-2、IFN-γ和GZMB的表达。结论：BMSCs具有低免疫原性，不能刺激同种异体CD8+T淋巴细胞的增殖；BMSCs在一定浓度范围内对CD8+T淋巴细胞具有免疫抑制作用。第三部分:人骨髓间充质干细胞对CD8+T淋巴细胞的免疫调节功能机制研究目的：阐明BMSCs对CD8+T淋巴细胞的免疫调节机制，为临床应用奠定坚实基础。方法：建立transwell培养体系，避免BMSCs与CD8+T淋巴细胞的直接接触，检测CD8+T淋巴细胞的增殖；流式细胞术检测CD8+T淋巴细胞表面的NKG2D受体表达和BMSCs表面的MICA/B配体表达；将不同浓度BMSCs与CD8+T淋巴细胞共培养，检测CD8+T淋巴细胞的NKG2D受体表达；使用MIC A/B单克隆抗体封闭BMSCs表面的MIC A/B分子，再将BMSCs与CD8+T淋巴细胞进行共培养，检测CD8+T淋巴细胞的增殖和IL-2、IFN-γ和GZMB的表达；通过real time-PCR法检测与CD8+T淋巴细胞共培养后BMSCs内吲哚胺2、3-双加氧酶（IDO）和肝细胞生长因子（HGF）mRNA的表达；通过酶联免疫吸附试验（ELISA）检测前列腺素E2（PGE2）和转化生长因子β1（TGF-β1）在BMSCs与CD8+T淋巴细胞共培养体系上清液的含量；通过PGE2、IDO、TGF-β和HGF的阻断实验，检测分别或联合阻断PGE2、IDO、TGF-β和HGF后，BMSCs对CD8+T淋巴细胞增殖的抑制作用。结果：与直接接触的培养组比较，transwell培养组CD8+T淋巴细胞的增殖率升高，但未完全恢复；CD8+T淋巴细胞表达NKG2D受体，BMSCs表达MICA/B配体；当CD8/BMSCs为1:1-20:1时，BMSCs可抑制CD8+T淋巴细胞的NKG2D受体表达，且该作用表现为对BMSCs的浓度依赖性，BMSCs浓度越高，抑制作用越明显；当CD8/BMSCs为50:1-100:1时，BMSCs不能抑制CD8+T淋巴细胞的NKG2D表达；封闭BMSCs表面的MIC A/B配体后，共培养体系CD8+T淋巴细胞的增殖率和细胞因子表达量升高；共培养体系BMSCs的IDO表达增高，HGF表达无明显变化；共培养体系上清液PGE2和TGF-β1的含量增高；分别阻断PGE2、IDO和TGF-β后，CD8+T淋巴细胞的增殖率升高，而阻断HGF无明显改变；同时阻断PGE2、IDO和TGF-β后，CD8+T淋巴细胞的增殖率升高，但明显低于三者单独作用时的理论叠加水平。结论：BMSCs对CD8+T淋巴细胞的免疫抑制作用需要细胞间的直接接触，并在一定程度上有可溶性因子参与；CD8+T淋巴细胞表达NKG2D受体，BMSCs表达MIC A/B配体；BMSCs可在一定浓度范围内以浓度依赖的形式下调CD8+T淋巴细胞表面的NKG2D受体，从而抑制CD8+T淋巴细胞的增殖；BMSCs表面的MIC A/B配体参与BMSCs对CD8+T淋巴细胞的免疫抑制作用，NKG2D受体与MIC A/B配体的相互作用是BMSCs对CD8+T淋巴细胞免疫调节作用的机制之一；可溶性因子PGE2、IDO和TGF-β参与BMSCs对CD8+T淋巴细胞的免疫调节功能，但三者间无协同作用。总而言之，本研究揭示了BMSCs对CD8+T淋巴细胞的免疫调节功能及其机制。证明CD8+T淋巴细胞表面的NKG2D受体和BMSCs表面的MIC A/B配体与BMSCs对CD8+T淋巴细胞免疫抑制作用有关。提出BMSCs通过其表面的MIC A/B下调CD8+T淋巴细胞表面的NKG2D表达，从而抑制CD8+T淋巴细胞的增殖和功能。NKG2D和MICA/B的相互作用介导了两细胞间的直接接触，是BMSCs对CD8+T淋巴细胞免疫抑制作用的重要机制之一。此外，本研究证明PGE2、IDO和TGF-β参与BMSCs对CD8+T淋巴细胞的免疫调节作用。提出可溶性因子PGE2、IDO和TGF-β是BMSCs对CD8+T淋巴细胞免疫调节作用的机制之一，但三者的作用无叠加效应。本研究结果进一步丰富了BMSCs在免疫性疾病临床应用的分子机理研究，为BMSCs的临床应用奠定了坚实的基础，也为提高BMSCs在免疫性疾病的临床应用效果提供了新的思路和启迪，具有重要的理论价值。更多还原

【Abstract】 Stem cells (SC) have unlimited proliferation and multi-directionaldifferentiation potential. They can be divided into embryonic stem cells (ESC),induced pluripotent stem cells (IPSC）and adult stem cells (ASC) according tothe differentiation ability. ASC include hematopoietic stem cells（HSCs）andmesenchymal stem cells (MSCs). MSCs can be derived from a variety of tissuesor organs include bone marrow, fat, placenta, umbilical cord and dental pulp.Bone marrow mesenchymal stem cells (BMSCs) have been studied deeply andapplied widly due to its relatively abundant sources and matured isolation andculture methods.MSCs have four major functions: supporting the hematopoieticreconstruction of HSCs, promoting the tissue repair and regeneration, targetingtherapy of tumor and immune modulation. Immune modulation is a hotspot ofMSCs research. MSCs cannot activate the reaction of allogeneic lymphocytesbecause of their low immunogenicity. In addition, MSCs can inhibit theproliferation, activation and effect of a variety of immune cells include T cells,natural killer cells (NK), dendritic cells (DC) and B lymphocytes. Therefore,MSCs have important application value in the prevention and treatment ofimmune disease. BMSCs are widely used in the prevention or treatment of graftversus host disease (GVHD). However, the basic research and clinical trialsabout MSCs are far from enough and many problems have not been answered.The problems existed are: The mechanism of the treatment of immune diseaseby MSCs, especially the effect and mechanism of MSCs on all kinds of effector cells involved in the immune disease; How to ensure the effect of the clinicalapplication of MSCs; The safety of the clinical application of MSCs.CD8+T lymphocytes and activated cytotoxic T lymphocytes (CTL) are themain effector lymphocytes in GVHD. The mechanism of the treatment ofGVHD by MSCs is particularly important because MSCs were mostly used inthe prevention and treatment of GVHD. The immune modulation andmechanism of MSCs on CD8+T lymphocytes which is the main effector cells inGVHD is important. Nowadays, the reports about MSCs-CD8+T lymphocytesinteraction is less, and the mechanism research about MSCs-mediatedmodulation on CD8+T lymphocytes is lacking. In order to further clarify theimmune rmodulation and mechanism of MSCs on CD8+T lymphocytes, wefinished this study with three parts as follows.Part1: Isolation, culture and identification of human bone marrowmesenchymal stem cellsObject: To isolate, culture, amplify and identify BMSCs in vitro, andprovide sufficient cell source for subsequent experiments.Method: First, we separate the bone marrow mononuclear cells throughficoll density gradient centrifugation, cultured and amplified the cells useing thebone marrow mesenchymal stem cells cultured medium (BMSC-GM). Then, weget the cell growth curve through quantitative inoculation and counting. Inaddition, we detect the BMSCs surface molecular markers through flowcytometry detection. Moreover, we investigate the multi-directionaldifferentiation of BMSCs by cultured BMSCs with osteoblast and adipocyteinduced medium. Finally, we identify the cells by the combining use of cellmorphology, growth characteristics, surface molecular markers and multi- directional differentiation ability.Result: The BMSCs cultured in vitro were fibrous cells and have spiralgrowth performance. The growth curve of BMSCs was "S" type which hastypical incubation period, growth period and platform period. The surfacemolecular markers of BMSCs were positive with CD29, CD105and CD166,negative with CD34and CD45. BMSCs can be differentiated into osteoblast andadipocyte by inducing cultured in vitro. The combining use of cell morphology,growth characteristics, surface molecular markers and multi-directionaldifferentiation ability can determine the cells as MSCs. The cells of P3-P6generation were suitable for subsequent experiments because of their relativelystable characteristics.Conclution: We can obtain plenty of BMSCs through isolation, culture,amplification and identification in vitro. The cells of P3-P6generation weresuitable for subsequent experimentsPart2: Research of the immune modulation of BMSCs on CD8+TlymphocytesObject: To study the immune modulation of BMSCs on CD8+Tlymphocytes and preliminary discuss the effective concentration range ofBMSCs in modulating CD8+T cells.Method: First, we purified the peripheral blood CD8+T lymphocytes byimmune magnetic beads technology and negative selection method, and detectthe proportion of CD3+CD8+T lymphocytes by flow cytometry detection. Then,we observe the ability of BMSCs to activated CD8+T lymphocytes byco-cultured BMSCs with CD8+T lymphocytes and then detected the CD8+Tlymphocytes proliferation by carboxyfluorescein diacetate succinimidyl ester (CFSE) method. In addition, we established the co-cultred system of BMSCsand CD8+T lymphocytes, use the phytohaemagglutinin A (PHA) as polyclonalT lymphocytes stimulator, and detected the proliferation of CD8+Tlymphocytes. Finally, we detected the mRNA expression of interleukin2(IL-2),interferon-γ (IFN-γ) and particle enzyme B (GZMB) of CD8+T lymphocytes inthe co-cultured system by real time-polymerase chain reaction(real time-PCR)method.Result: The propotion of CD3+CD8+T lymphocytes in the cells obtainedfrom magnetic bead separation was88.1%. When CD8/BMSCs ratio is1:1-100:1, the CD8+T lymphocytes proliferation rate has no obvious rise afterco-cultured with BMSCs. BMSCs could inhibit the CD8+T lymphocytesproliferation in a dose-dependent form when the CD8/BMSCs ratio is1:1-20:1and the effect is more obvious when the concentration of BMSCs is higher.When CD8/BMSCs ratio is50:1-100:1, the CD8+T lymphocytes proliferationwas not inhibited by BMSCs. BMSCs can inhibit the IL-2, IFN-γ and GZMBexpression in CD8+T lymphocytes.Conclution: BMSCs lack the stimulatory capacity toward CD8+Tlymphocytes because of their low immunogenicity. BMSCs can suppress theCD8+T lymphocty activation in a certain concentration range.Part3: Research of the mechanism of BMSCs-mediated immunemodulation on CD8+T lymphocytesObject: To clarify the BMSCs-mediated immune modulation mechanismand provide a massy foundation for clinical application.Method: First, we detect the CD8+T lymphocytes proliferation in atranswell system which can avoid the direct contact between BMSCs and CD8+ T lymphocytes. Then we detect the NKG2D expression on CD8+T lymphocytesand the MIC A/B expression on BMSCs by flow cytometry detection. Inadditiom, we detect the NKG2D expression on CD8+T lymphocytes in theco-cultured system seted up by CD8+T lymphocytes and BMSCs with differentCD8/BMSCs ratios. Moreover, we blocked the MIC A/B molecule on BMSCswith MIC A/B monoclonal antibody before co-cultured the BMSCs with CD8+T lymphocytes and then detect the proliferation and the IL-2, IFN-γ and GZMBexpression of CD8+T lymphocytes. We also detected the mRNA expression ofIDO and HGF in BMSCs which was co-cultured with CD8+T lymphocytes byreal time-PCR method and detected the PGE2and TGF-β1content in thesupernatant of co-cultured system. Finally, we detect the inhibition of BMSCson CD8+T lymphocytes proliferation after blocked the soluble factors PGE2,IDO, TGF-β and HGF respectively and simultaneously.Result: The CD8+T lymphocytes proliferation was higher but did notcompletely recovered in the transwell system when compared with the directcontact system. CD8+T lymphocytes express NKG2D receptor and BMSCsexpress MIC A/B ligand. BMSCs can suppress the NKG2D expression on CD8+T lymphocytes in a dose-dependent from when the CD8/BMSCs ratio is1:1-20:1and the effect is more obvious when the concentration of BMSCs ishigher. When the CD8/BMSCs ratio is50:1-100:1, BMSCs can not suppress theNKG2D expression. The proliferation and cytokine expression of CD8+Tlymphocytes were increased when co-cultured CD8+T lymphocytes withBMSCs which was blocked the MIC A/B molecule. The IDO expression wasobviously increased and the HGF expression was not changed in BMSCs whenco-cultured with CD8+T lymphocytes. The content of PGE2and TGF-β1wasincreased in the supernatant of co-cultured system. The proliferation of CD8+T lymphocytes was increased when blocked the PGE2, IDO and TGF-βrespectively, while was not changed when blocked the HGF alone. Whenblocked the PGE2, IDO and TGF-β simultaneously, the proliferation wasobviously increased but significantly lower then the mean expected valuecalculated for an additive model.Conclution: The immune inhibition of BMSCs on CD8+T lymphocyteswas mediated by cell-cell contact. Soluble factors were involved in a certaindegree. CD8+T lymphocytes expree NKG2D receptor and BMSCs expressedMIC A/B ligand. BMSCs can down-modulate the NKG2D expression on CD8+T lymphocytes in a dose-dependent from. The MIC A/B of BMSCs wasinvolved in the immune modulation of BMSCs on CD8+T lymphocytes. Theinteraction of NKG2D and MIC A/B was one of the mechanisms ofBMSCs-mediated immune modulation on CD8+T lymphocytes. The solublefactors PGE2, IDO and TGF-β were involved in the immune modulation ofBMSCs on CD8+T lymphocytes, but they did not have synergistic effect.In conclution, our study reveals the immune modulation and mechanism ofBMSCs on CD8+T lymphocytes. First, we proved that the NKG2D receptor onCD8+T lymphocytes and the MIC A/B ligand on BMSCs were involved in theimmune modulation of BMSCs on CD8+T lymphocytes. We indicate that theBMSCs suppress the NKG2D expression on CD8+T lymphocytes through theMIC A/B and so they can inhibit the proliferation and founction of CD8+Tlymphocytes. The interaction of NKG2D and MIC A/B which mediated thecell-cell contacte of BMSCs and CD8+T lymphocytes was one of the importantmechanisms of BMSCs-mediate immune modulation on CD8+T lymphocytes.Second, we proved that the PGE2, IDO and TGF-β were involved in the immune modulation of BMSCs on CD8+T lymphocytes. We indicate that thePGE2, IDO and TGF-β were one of the mechanisms of BMSCs-mediateimmune modulation on CD8+T lymphocytes, but they did not have synergisticeffect. These results further enriched the molecular mechanism research of theclinical application with BMSCs in immune disease. In addition, these resultsprovide a massy foundation for the clinical application of BMSCs. Furthermore,these results provide a new thinking and enlightenment to improve the clinicalapplication effect of BMSCs in autoimmune disease.更多还原