【作者】 王丁；

【导师】 韩忠朝；

【作者基本信息】 中国协和医科大学 ， 内科学， 2010， 博士

【摘要】 背景：间充质干细胞(mesenchymal stem cells, MSC)是存在于多种组织中的一种具有自我更新和多向分化潜能的多能干细胞。因其具有易于分离扩增、多向分化、造血支持、低免疫原性和免疫调节等特性,而成为组织工程和再生医学中理想的种子细胞。目前,试验研究和治疗性临床应用均以成体骨髓来源的间充质干细胞(bone marrow mesenchymal stem cell, BM-MSC)作为研究对象。但成人骨髓间充质干细胞(hBM-MSC)存在来源有限、取样时要进行侵袭性操作以及间充质干细胞的绝对数量和增殖分化能力会随着供者年龄增加而下降等不足,急需科研人员寻找一种新的更具优势的替代细胞。大量的实验研究表明,人脐带间充质干细胞(human umbilical cord matrix stem cell, hUC-MSC)具有以下不可比拟的优势：第一,利用胎儿分娩后的废弃物,组织来源广泛,无伦理学限制；第二,具有更强的增殖能力,易于长期培养,经体外扩增可获得丰富的细胞；第三,具有成体干细胞和胚胎干细胞(embryonic stem cell, ESC)的双重表面标记；第四,连续多次传代后仍然维持干细胞特性。因此,人脐带间充质干细胞作为细胞治疗中一种非常有希望的干细胞替代来源,具有无限的潜力和广泛的临床应用前景,而其免疫学特性正是细胞治疗成功与否的决定性因素。目的：淋巴细胞(lymphocyte)和单核细胞(monocyte)是外周血单个核细胞(Peripheral blood mononuclear cells, PBMC)重要的组成部分。本研究旨在揭示人脐带间充质干细胞、T淋巴细胞以及单核细胞三者之间的相互作用关系及其机制,为人脐带间充质干细胞的临床应用提供必要的理论基础。方法：(1)应用流式细胞技术检测成人骨髓间充质干细胞和人脐带间充质干细胞的免疫表型以及胚胎干细胞的全能标志物。(2)在诱导体系中,定向诱导人脐带间充质干细胞向脂肪细胞、成骨细胞和软骨细胞分化,用油红0染色、Von Kossa染色、茜素红染色以及二型胶原组化染色观测脂滴形成、钙盐沉积和二型胶原合成情况以鉴定人脐带间充质干细胞的多向分化潜能。(3)体外建立人脐带间充质干细胞和CD4+／CD8+T淋巴细胞的共培养体系以及人脐带间充质干细胞、CD4+／CD8+T淋巴细胞和CD14+单核细胞的三者共培养体系。利用流式细胞技术(Brdu掺入试验)检测T淋巴细胞的增殖情况,并且用酶联免疫吸附试验试剂盒检测其IFN-γ(interfern-γ)的分泌水平,从而反映人脐带间充质干细胞对活化后的T淋巴细胞的免疫抑制作用以及CD14+单核细胞对于这一作用的影响。(4)用transwell培养板培养人脐带间充质干细胞和CD4+／CD8+T淋巴细胞以研究人脐带间充质干细胞发挥免疫抑制作用是否需要细胞直接接触。(5)外源添加TGF-β(transforming growth factor-β)单克隆中和抗体、吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase, IDO)特异性抑制剂1-M-Trp、一氧化氮(N0)合酶竞争性抑制剂L-NMA和前列腺素E2(prostaglandin E2, PGE2)合成抑制剂NS-398(环氧化酶-2选择性抑制剂)和吲哚美辛(环氧化酶非选择性抑制剂),以IFN-γ的分泌水平作为评价指标,鉴定介导人脐带间充质干细胞免疫抑制作用的可溶性因子。(6)通过添加外源性PGE2并且应用ELISA试剂盒检测人脐带间充质干细胞和CD4+／CD8+T淋巴细胞共培养体系中PGE2的水平佐证PGE2是否中介人脐带间充质干细胞的免疫抑制作用。(7)制备CD3／CD28抗体交联磁珠(anti-CD3/CD28 Dynabeads)活化的CD4+T淋巴细胞、CD8+T淋巴细胞和外周血单个核细胞三种条件培养基,用ELISA试剂盒检测条件培养基中IFN-γ、TNF-α(tumor necrosis factor-a)、IL-1β(interleukin-1β)和IL-6(inter-leukin-6)的水平。同时应用ELISA方法对以上三种条件培养基以及四种因子的重组蛋白刺激前后,人脐带间充质干细胞分泌PGE2的水平进行定量检测,以明确人脐带间充质干细胞产生PGE2的驱动因子。(8)外源重组IL-1β刺激人脐带间充质干细胞,应用Real-time PCR方法检测环氧化酶-2(cyclooxygenase, COX-2)的表达并用ELISA试剂盒检测PGE2的分泌水平。(9)利用几种特异性的信号通路蛋白抑制剂：PD98059为细胞外信号调节激酶(extra-cellular signal-regulated kinase, ERK)特异性抑制剂、SB203580为p38丝裂原活化蛋白激酶(p38mitogen-activated protein kinase, p38 MAPK)特异性抑制剂、SP600125为c-Jun氨基末端激酶(c-jun-N-terminal kinase, JNK)特异性抑制剂、H89为蛋白激酶A(protein kinase A, PKA)特异性抑制剂、Wortmannin为磷脂酰肌醇3-激酶(phosphatidylnsitol 3-kinase, PI3K)特异性抑制剂、IKK-NBD肽为NF-κB特异性抑制剂、Go6983和Go6976为蛋白激酶C(protein kinase C, PKC)特异性抑制剂来初步确定IL-1β激发人脐带间充质干细胞产生PGE2的信号通路相关蛋白。(10)应用ELISA试剂盒检测CD14+单核细胞加入人脐带间充质干细胞与CD4+／CD8+T淋巴细胞共培养体系前后,上清中IL-1β和PGE2的含量。(11)利用流式细胞技术(Brdu掺入试验)和ELISA试剂盒检测IL-1受体拮抗剂(IL-1 receptor antagonist,IL-1RA)加入人脐带间充质干细胞、CD4+／CD8+T淋巴细胞和CD14+单核细胞的三者共培养体系前后,T淋巴细胞的增殖情况、IFN-γ的分泌水平以及PGE2的含量,以进一步研究单核细胞是否通过分泌IL-1β促进人脐带间充质干细胞产生PGE2,从而增强人脐带间充质干细胞的免疫抑制作用。结果：(1)人脐带间充质干细胞与成人骨髓间充质干细胞免疫表型相似,但人脐带间充质干细胞高表达胚胎干细胞标志物OCT4和SSEA-4。(2)在特定的诱导条件下,人脐带间充质干细胞具有向脂肪细胞、骨细胞和软骨细胞三系分化的能力。(3)人脐带间充质干细胞能够抑制CD3／CD28抗体交联磁珠活化的CD4+／CD8+T淋巴细胞的增殖以及IFN-γ的分泌,而且CD14+单核细胞能够增强人脐带间充质干细胞的这一抑制作用。(4)人脐带间充质干细胞发挥免疫抑制作用并不依赖于细胞间的直接接触。PGE2合成抑制剂能够显著反转人脐带间充质干细胞的免疫抑制作用；而外源性PGE2能够模拟人脐带间充质干细胞的免疫抑制作用。(5)炎症因子IFN-γ和IL-1β均能驱动人脐带间充质干细胞产生PGE2。ERK、p38-MAPK和JNK的特异性抑制剂能够显著下调IL-1β诱导的PGE2的分泌。(6)CD14+单核细胞能够显著上调人脐带间充质干细胞与CD4+／CD8+T淋巴细胞共培养上清中IL-1β以及PGE2的水平,并且上调的幅度与单核细胞的数量呈正相关。(7)IL-1RA能够下调人脐带间充质干细胞、CD4+／CD8+T淋巴细胞和CD14+单核细胞三者共培养上清中PGE2的水平,同时显著逆转T淋巴细胞的增殖活力以及IFN-γ的分泌水平。结论：CD14+单核细胞通过分泌炎症因子IL-1β,经由MAPK信号通路显著上调人脐带间充质干细胞中PGE2的表达,从而增强人脐带间充质干细胞对活化CD4+／CD8+T淋巴细胞的免疫抑制作用。背景：间充质干细胞(mesenchymal stem cells, MSC)以其卓越的免疫调节能力,日益成为广大科研人员关注的热点。经体内外大量研究证实,间充质干细胞能够调控T淋巴细胞、B淋巴细胞、NK细胞、树突状细胞、巨噬细胞等多种免疫细胞的功能,为治疗免疫相关疾病以及自身免疫性疾病带来了曙光。人胎儿脐带基质来源的间充质干细胞(human umbilical cord matrix stem cell, hUC-MSC)不仅具有许多独特的优势,而且像骨髓间充质干细胞(bone marrow mesenchymal stem cell, BM-MSC)一样,能够高效调节T淋巴细胞的活性和功能。目的：人脐带间充质干细胞能够分泌多种生长因子和细胞因子。本研究旨在揭示人脐带间充质干细胞产生IL-6的机制,以及IL-6在人脐带间充质干细胞影响肿瘤细胞增殖过程中的作用。方法：建立人外周血单个核细胞(hPBMC)和人脐带间充质干细胞的共培养体系以观察两种细胞之间的相互作用。制备多种条件培养基并建立人脐带间充质干细胞的单独培养体系,以研究可溶性因子对IL-6分泌的影响。用ELISA定量检测培养上清中IL-6、IL-1β和PGE2的水平。应用工L-1受体抑制剂和多种信号通路抑制剂确定CD14+单核细胞是否通过分泌IL-1β而上调IL-6的表达以及相关的信号通路。Western blot用以检测NF-KB的活化。用流式细胞仪检测肿瘤细胞Brdu的掺入情况,以证实人脐带间充质干细胞是否能够影响肿瘤细胞的增殖,同时评估IL-6在这一过程中的作用。结果：(1)共培养体系中IL-6的浓度与人脐带间充质干细胞的数量呈正相关,并随着共培养时间的延长而呈进行性富集。(2)IL-6主要由人脐带间充质干细胞分泌。(3)CD14+单核细胞通过释放IL-1β诱导人脐带间充质干细胞产生IL-6。(4)PGE2并不参与诱导人脐带间充质干细胞分泌IL-6。(5)c-Jun氨基末端激酶(JNK)信号通路及转录因子NF-κB均与IL-1β诱导人脐带间充质干细胞表达IL-6紧密相关。(6)由IL-1β刺激人脐带间充质干细胞所制备的条件培养基能够促进MCF-7细胞的增殖,但IL-6中和抗体部分逆转了这一促进效应。(7)木犀草素以剂量依赖性的方式显著抑制人脐带间充质干细胞IL-6的分泌。结论：(1)CD14+单核细胞释放的IL-1β经由c-Jun氨基末端激酶(JNK)信号通路活化转录因子NF-κB,从而诱导人脐带间充质干细胞产生IL-6。(2)IL-6中介人脐带间充质干细胞对MCF-7细胞增殖的促进作用。(3)木犀草素以剂量依赖性方式显著抑制人脐带间充质干细胞IL-6的分泌。更多还原

【Abstract】 Background:Mesenchymal stem cells (MSC), isolated from many tissues, are multipotent stem cells with the capacity for self-renewal and multilineage differentiation. They can be isolated with ease and expanded in large quantities, their multipotency, supporting hematopoiesis. Their low immunogenicity and immunoregulation properties present them as a promising stem cell candidate for tissue engineering and regenerative medicine. Up to date, most experimental research and therapeutic applications are based on MSC derived from adult bone marrow (ABM-MSC). But human bone marrow MSC (hBM-MSC) have many drawbacks, including the limited volume, the invasive procedures of their harvest and the age-dependent decline in the absolute number, differentiation and proliferation capacity, which has prompted researchers to look for alternative sources of MSC. Increasing evidence confirms that MSC derived from human fetal umbilical cord matrix (hUC-MSC) have the incomparable advantages:(1) Take full advantage of discarded materials from the new born baby, which are easily accessible and less troublesome in ethical controversy. (2) hUC-MSC are readily long-time preparation and can be scaled up in large numbers because of short doubling time. (3) hUC-MSC contain both human embryonic stem cells and human mesenchymal stem cells markers. (4) hUC-MSC maintain "stemness " for several serial passages. Above all, hUC-MSC offer tremendous promise in cellular therapeutics, while the key to the success is the immunological properties of hUC-MSC.Objective:Lymphocyte and monocyte are the important subsets of human peripheral blood mononuclear cells (PBMC). This study was designed to illustrate the interaction among hUC-MSC, T lymphocyte and CD14+monocyte, which will offer us new strategy for the clinical application of hUC-MSC. Methods:(1) To compare hUC-MSC phenotypic markers with hBM-MSC, specific cell-surface and intracellular marker were examined by flow cytometry. (2) hUC-MSC were induced for adipocytes, osteoblasts and chondrocytes in the specific-induction medium. Oil red 0 staining, Von Kossa staining, alizarin red staining and collagen II histochemistry assays were performed to examine lipid droplet, mineralization and the production of collagen II. (3) hUC-MSC and CD4+/CD8+T lymphocyte or hUC-MSC, CD4+/CD8+T lymphocyte and CD14+monocyte were cocultured in vitro. T lymphocyte proliferation was assessed by flow cytometry(Brdu incorporation assay).And IFN-γsecretion was detected by ELISA kits. (4)Transwell plates were used to block the direct contact between hUC-MSC and T lymphocyte. (5) Coculture experiments were performed in the presence or absence of specific inhibitors for the possible soluble mediators, including a neutralizing anti-TGF-βmonoclonal antibody; 1-M-Trp, an inhibitor of IDO enzymatic activity; L-NMA, a competitive inhibitor of NO synthases; NS-398, selective cyclooxygenase (COX)-2 inhibitor and indomethacin, non-selective COX inhibitor. (6) hUC-MSC were cocultured with activated T lymphocyte, and cell-free supernatants were detected by Prostaglandin E2 (PGE2) ELISA kits. Additionally, exogenous PGE2 was added to T lymphocyte culture system in some experiment. (7) Cell-free condition medium (CM) from CD4+T lymphocyte, CD8+T lymphocyte or hPBMC stimulated by anti-CD3/CD28 Dynabeads were prepared. IFN-γ、TNF-α、IL-1βand IL-6 were quantified in the CM by ELISA kit. These three CM and the recombinant protein of the four inflammatory cytokines were added individually to the hUC-MSC culture system to test their effect on PGE2 production. (8) Exogenous IL-1βwas added in the culture of hUC-MSC, then COX-2 mRNA expression was detected by Real-time PCR and PGE2 production was assessed by ELISA kit. (9) To define the signaling pathway involved in IL-1β-induced up-regulation of PGE2, hUC-MSC were pre-treated with some specific inhibitors of different signaling proteins:PD98059(a specific inhibitor of ERK), SB203580(a specific inhibitor of p38MAPK), SP600125(a specific inhibitor of JNK), H89 (a specific inhibitor of PKA),Wortmannin (a specific inhibitor of PI3K),IKK-NBD(a specific inhibitor of NF-κB), Go6983 and Go6976 (a specific inhibitor of PKC). (10) IL-1βand PGE2 were quantified by ELISA kit in the coculture of hUC-MSC and CD4+/CD8+T lymphocyte in the presence or absence of CD14+monocyte. (11) IL-1RA was added in the coculture of CD4+/CD8+T lymphocyte, CD14+monocyte and hUC-MSC, then T lymphocyte proliferation was assessed by flow cytometry (Brdu incorporation assay). Meanwhile, IFN-γand PGE2 were quantified by ELISA kit.Result:(1) The expression profile of phenotypic markers for hUC-MSC was similar to that of hBM-MSC. Moreover, hUC-MSC expressed the embryonic stem cell specific markers, including 0CT4 and SSEA-4. (2) hUC-MSC were able to differentiate toward adipogenic, osteogenic and chondrogenic lineages in lineage-specific inductive condition, indicating that hUC-MSC possess multilineage differentiation potential. (3) hUC-MSC exhibited a dose-dependent inhibition on both cell proliferation and IFN-γproduction when either CD4+T lymphocyte or CD8+T lymphocyte activated with anti-CD3/CD28 Dynabeads. Inclusion of CD14+monocyte in the coculture enhanced this inhibition. (4)Transwell experiments, inhibition of PGE2 synthesis experiments, exogenous PGE2 experiments and quantification of PGE2 revealed that PGE2 was an important soluble factor for hUC-MSC-mediated immuno-inhibition. (5)Proinflammatory cytokines IFN-γand IL-1βsignificantly induced the production of PGE2 in hUC-MSC. ERK, p38 kinase, and JNK specific inhibitor significantly decreased IL-1β-induced PGE2 production. (6) Inclusion of CD14+monocyte in the CD4+/CD8+T lymphocyte and hUC-MSC cocultures resulted in simultaneously increased expression of IL-1βand PGE2, and the more monocyte were added the larger amounts of IL-1βand PGE2 were produced. (7) IL-1RA not only down-regulated PGE2 but also significantly reversed the promotional effect of CD14+monocyte on hUC-MSC-mediated inhibition to CD4+/CD8+T lymphocyte proliferation and IFN-γgeneration. Conclusion:Taken together, these results indicate that CD14+monocyte may promote the immunosuppressive effect of hUC-MSC by enhancing PGE2 synthesis through up-regulation of IL-1βexpression. And MAPK signaling pathways involving ERK, p38 kinase and JNK are strongly relevant to IL-1β-induced PGE2 production. Background:Mesenchymal stem cells (MSC) possess outstanding immunoregulatory talent. Previous studies have confirmed that MSC can modulate the function of many immune cells, such as T cells, B cells, NK cells, macrophages and dendritic cells. Human umbilical cord matrix stem cells (hUC-MSC) have the incomparable advantages. And they are able to inhibit proliferation and IFN-γsecretion of the activated T cells.Objective:hUC-MSC secret a number of growth factor and cytokines. This study was designed to illustrate the mechanism of IL-6 production of hUC-MSC. And the effect of IL-6 secreted by hUC-MSC on the proliferation of tumor cells. Methods:hPBMC and hUC-MSC were cocultured in vitro to study the interaction of these cells. The condition mediums (CM) from the culture system of CD14+ monocyte, CD14 cells and hPBMC were used to stimulate hUC-MSC. IL-6, IL-1 3 and PGE2 were quantified by ELISA kit. IL-1RA and the specific inhibitors of signal proteins were used to identify the signal pathway of IL-6 production. Western blot was used to confirm the degradation of IκB. Brdu incorporation assay of flow cytometry was used to detect the effect of hUC-MSC on the proliferation of tumor cells.Result:(1) IL-6 was upregulated in a number-dependent manner of hUC-MSC and time-dependent way in the coculture supernatant. (2) IL-6 was secreted mainly by hUC-MSC. (3) CD14+monocyte-paracrined IL-1βwas responsible for the induction of IL-6 in hUC-MSC. (4) PGE2 was not involved in the production of IL-6. (5) JNK signal pathway and NF-κB were activated in IL-6 induction by IL-1β. (6) The condition medium from IL-1β-stimulated hUC-MSC was able to increase the proliferation of MCF-7 cells. (7) Luteolin significantly inhibited the production of IL-6 in a dose-dependent manner.Conclusion:(1) IL-1β,secreted by CD14+monocyte, can activated the JNK signal pathway and the transcription factor NF-κB, which are highly involved in the production of IL-6 in hUC-MSC. (2) IL-6 mediate the promotion effect of hUC-MSC on the proliferation of MCF-7 cells. (3)Luteolin suppresses IL-6 production in a dose-dependent manner.更多还原