【作者】 陈可；

【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)是一类具有自我更新(selfrenewal)和多向分化潜能(multi-lineage differentiation)的成体干细胞。它来源广泛,具有支持造血和多向分化功能；随着对MSC认识的不断加深,人们还发现MSC具有低免疫原性和较强的免疫调节功能。目前研究最多的主要是人骨髓来源的间充质干细胞(hBM-MSC).人脐带来源的间充质干细胞(hUC-MSC)由于其较人骨髓来源间充质干细胞更易于制备以及低风险病毒感染,并且脐带来源间充质干细胞易于体外培养及扩增等优势已经成为人骨髓来源间充质干细胞非常好的替代品。但是,人脐带来源间充质干细胞在免疫调节方面功能尚未有深入研究。目的：研究人脐带来源间充质干细胞发挥免疫学效应的作用及发挥作用的机制。方法：人外周血单个核细胞由不同的刺激剂刺激后与人脐带来源间充质干细胞共培养,通过检测人外周血单个核细胞的增殖及IFN-γ分泌情况观察人脐带来源间充质干细胞免疫调节效应；用transwell培育板培养来研究间充质干细胞的免疫调节作用是否需要细胞直接接触；用各种抑制剂确定人脐带来源间充质干细胞发挥免疫学效应的因子机制；另外,通过流式检测人外周血单个核细胞Annexin-V的情况来观察人脐带来源间充质干细胞发挥效应的情况及相关机制。结果：人脐带来源间充质干细胞能够有效有效抑制人外周血单个核细胞刺激后的增殖及IFN-γ的分泌；Transwell系统共培养结果显示此抑制效果不受细胞接触的影响,既是这种抑制作用主要通过因子发挥免疫效应；抑制剂试验排除了TGF-β,IDO和NO的可能,并确定了前列腺素E2(PGE2)是人脐带来源间充质干细胞发挥免疫学效应的介导因子；炎症因子IFN-γ和IL-1β能显著上调人脐带来源间充质干细胞PGE2的分泌；人脐带来源间充质干细胞能保护人外周血单个核细胞刺激后的凋亡发生,并且此保护效应也是通过分泌PGE2发挥的作用。结论：人脐带来源间充质干细胞(hUC-MSC)同样能够发挥很好的免疫调节作用,并且第一次发现这种细胞的免疫学效应主要是通过PGE2的分泌实现,这一机制对于将来人脐带来源间充质干细胞的临床应用提供了理论基础。背景：一种最近受人关注的因子IL-17,作为适应性免疫应答系统的标志炎症因子,最初由一群新T细胞群‘Th17’产生。在最近几年的研究中取得了许多相关成果,包括Thl7细胞趋导分化的分子机制的统一标准。这些对于弄清Th17细胞在宿主防御和自身免疫中的作用有很大帮助。最近的研究证实由IL-17产生细胞激发的炎症是许多人类自身免疫病动物自身免疫病模型的发展和致病的中心环节。并且激活的IL-23／IL-17轴对于系统性红斑狼疮(SLE)病免疫炎症非常重要。间充质干细胞是一种多能干细胞,可以向中胚层来源组织分化并且从各种组织分离得到。MSCs已经被认为在免疫细胞上拥有非常广泛的免疫调节作用,对许多效应功能有调节作用。最近报道胎儿骨髓来源MSCs能够促进正常供者来源的IL-17分泌细胞的扩增。而脐带来源MSCs对于免疫细胞这一效应尚不清楚。目的：本文主要研究脐带来源间充质干细胞(hUC-MSCs)对于来自于正常供者或者SLE患者T淋巴细胞的免疫调节效应作用,特别是对于Th17细胞。这样可以扩展我们对于MSCs免疫调节功能的理解以及其作为细胞依赖治疗免疫紊乱疾病的临床运用潜力提供更多见解。方法：我们将hPBMCs(来自于健康供者和SLE病人)或者CD4+T(来自于健康供者)细胞与hUC-MSCs培养,并将这个培养体系称为共培养细胞。hPBMCs细胞由丝裂原PHA刺激,而CD4+T细胞由ati-CD3/CD28 Dynabeads刺激。①我们用实时定量PCR,酶联免疫吸附法对比建康供者或者SLE患者来源hPBMCs表达IL-17分泌的水平。并检测加入另IL-6,PGE2和IL-1β中和试剂的IL-17表达改变。用酶联免疫吸附法和流式细胞仪检测CD4+T细胞在与hUC-MSCs作用后IL-17分泌表达量的改变。③IFN-γ和TGF-β的表达用酶联免疫吸附法检测,而Thl细胞和Treg细胞比例由流式细胞仪技术检测。④我们用酶联免疫吸附法比较健康供者和SLE患者IL-23的分泌水平改变。结果：正常供者来源CD4+T细胞与脐带来源MSCs共培养比单纯培养正常供者CD4+T细胞能够产生高的IL-17分泌水平。相同的结果在正常供者PBMCs细胞中也得到证实。抑制试验证实这个效应可能部分是通过PGE2或者IL-1β介导产生的,并且IL-23不参与其中。接下来我们将hUC-MSCs与SLE患者来源PBMCs细胞共培养,体外诱导IL-17的能力,hUC-MSCs能够更促进SLE病人分泌,但是并不能影响IL-23的分泌。同时,hUC-MSCs降低了Thl细胞数量而增加Treg细胞数量。结论：综上所述,我们的结果第一次报道了hUC-MSCs同时促进健康供者和SLE病人来源PBMCs产生IL-17。PGE2和IL-1β可能部分参与hUC-MSCs的这个促进作用。这个发现可能对于hUC-MSCs运用于SLE患者潜在致病作用提出观点。考虑到血清中IL-17含量与SLE患者疾病活性相关性,临床运用hUC-MSCs治疗SLE可能需要更多仔细的研究。更多还原

【Abstract】 BACKGROUND:Mesenchymal stem cells(MSCs), the nonhematopoietic progenitor cells found in various adult tissues, are characterized by their ease of isolation and their rapid growth in vitro while maintaining their differentiation potential, allowing for extensive culture expansion to obtain large quantities suitable for therapeutic use. Mesenchymal stem cells isolated from human bone marrow, which are poorly immunogenic and have potent immunosuppressive activities, have emerged as a promising candidate for cellular therapeutics for the treatment of disorders caused by abnormal immune responses. Human umbilical cord derived mesenchymal stem cells (hUC-MSCs) resemble bone marrow MSCs (BM-MSCs) in many respects, but its immunosuppression are still lack in the literature.OBJECTION:This study was designed to investigat the immunosuppressive action by MSCs derived from human umbilical cord, and in the meantime to analyze the potential mechanism(s).METHODS:hUC-MSCs were co-cultured with MHC-mismatched allogeneic hPBMCs which in response to mitogenic or allogeneic stimulus. Both proliferation of human peripheral blood mononuclear cells (hPBMCs) and their IFN-γproduction were used to determine the immunosuppressive action of hUC-MSCs. And the transwell experiments were carried out for analyzing the contact necessary; Blocking experiments were used to identify which soluble factor(s) mediated the immunosuppressive effects; In addition, the data of Annexin-V showed the ability of hUC-MSCs on the apotosis of activated-hPBMCs.RESULTS:In this study, both proliferation of human peripheral blood mononuclear cells (hPBMCs) and their IFN-γproduction in response to mitogenic or allogeneic stimulus were effectively inhibited by hUC-MSCs. Co-culture experiments in transwell systems indicated that the suppression was largely mediated by soluble factor(s). Blocking experiments identified prostaglandin E2 (PGE2) as the major factor, because inhibition of PGE2 synthesis almost completely mitigated the immunosuppressive effects, whereas neutralization of TGF-β, IDO and NO activities had little effects. Moreover, the inflammatory cytokines, IFN-γand IL-1βproduced by hPBMCs upon activation notably up-regulated the expression of cyclooxygenase-2 (COX-2) and the production of PGE2 by hUC-MSCs. In addition, PGE2 was also found able to protect hPBMCs from apoptosis.CONCLUSION:In conclusion, our data have demonstrated for the first time that the PGE2 mediated mechanism by which hUC-MSCs exert their immunomodulatory effects. These mechanisms can render the theory basic to clinical usage of hUC-MSCs. Background:The emerging role of interleukin(IL)-17 as a hallmark proinflammatory cytokine of the adaptive immune system, producted primarily by a new T helper cell subset termed’Th17’, has received considerable attention. There has been much progress in the past year, leading to identification of the molecular mechanisms that drive differentiation of Th17 T cells. This has helped to clarify many aspects of their role in host defense as well as in autoimmunity. Recent studies have shown that inflammation instigated by IL-17-producing cells is central to the development and pathogenesis of several human autoimmune diseases and animal models of autoimmunity. And the activated IL-23/IL-17 axis is important for the inflammatory immunity in SLE. Mesenchymal stem cells (MSC) are multipotent stem cells that can differentiate into tissues of mesodermal origin and can be isolated from various tissues. MSC have also been noted to have profound immunomodulatory effects on immune cells, leading to the modulation of several effector functions. Recently, the expansion of IL-17 producing cells from healthy donors is reportedly promoted by mesenchymal stem cells derived from fetal bone marrow.Objective:This article aimed to investigate the immunoregulatory effects of human umbilical cords-derived MSC on human T lymphocytes from healthy donors and Systemic Lupus Erythematosus patients, especially Th17 cells, so as to broaden our understanding of the immunomodulatory properties of MSC and provide insights as to their potential for clinical use as a cell-based therapy for immune-mediated disorders.Methods:In the present study, We cultured hPBMCs(from healthy donor and SLE patients) and CD4+T cells with or without hUC-MSCs, and named the coculture of hPBMCs or CD+4 T cells with hUC-MSCs as coculture cells. hPBMCs were incubated with PHA, while CD4+T cells were incubated with ati-CD3/CD28 Dynabeads in the presence or absence of hUC-MSCs.①We used qRT-PCR, Elisa analyses to compare the expression levels of IL-17 secretion of hPBMCs from healthy donor or SLE patients. The change of IL-17 expression when supplemented with additional IL-6, PGE2 and IL-1βneutralizing reagent. ②The expression of IL-17 secretion from CD4+T cells was tested by Elisa and Th-17 cells was tested by cytometry analyses.③The expression of IFN-γand TGF-βwere tested by Elisa and the percentage of Thl cells and Treg cells were tested by cytometry analyses.④We used Elisa to compare the expression levels ofIL-23 secretion of hPBMCs from healthy donor or SLE patients.Results:Significantly higher levels of IL-17 were produced when CD4+T cells from healthy donors were cocultured with hUC-MSCs than when CD4+T cells from healthy donors were cultured alone. The similar data was obtained on hPBMCs. Blocking experiments identified that this effect might be partially mediated through prostaglandin E2 (PGE2) and IL-1βwithout IL-23 involvement. We then cocultured hUC-MSCs with human PBMCs from Systemic Lupus Erythematosus patients. Ex vivo inductions of IL-17 by hUC-MSCs in stimulated lymphocytes were significantly higher in SLE patients than in controls, but this effect was not seen for IL-23. Also, hUC-MSC decrease the production of Thl cells and increase the production of Treg cells. Conclusion:Taken together, our results represent the first report of promotion of IL-17 production by hUC MSCs in both healthy donor and SLE patients. PGE2 and IL-1βmight also be partially involved in the promotive effect of hUC-MSCs. This finding may provide a new insight into the potential pathological roles of hUC-MSCs in SLE. Given that serum abundance of IL-17 has been correlated with SLE disease activity, clinical treatments using hUC-MSCs in SLE may require more careful research.更多还原