人脐带来源间充质干细胞的免疫调节作用

发布时间：2018-02-17 00:30

本文关键词： 人脐带来源间充质干细胞人外周血单个核细胞前列腺素E2 免疫抑制人脐带来源间充质干细胞前列腺素E2 白介素1β 系统性红斑狼疮白介素17　出处：《中国协和医科大学》2010年博士论文　论文类型：学位论文

【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。
[Abstract]:Background: mesenchymal stem cells (Mesenchymal Stem cells, MSC) is a kind of self-renewal and multilineage differentiation potential (selfrenewal) (multi-lineage differentiation) of adult stem cells. It has a wide range of sources, supporting hematopoiesis and differentiation function of MSC; with the deeper understanding, people also found that MSC has low immunity the original and strong immune function. At present, most research is mainly from human bone marrow mesenchymal stem cells (hBM-MSC). Human umbilical cord derived mesenchymal stem cells (hUC-MSC) due to its more human bone marrow derived mesenchymal stem cells more easy preparation and low risk of viral infection, and umbilical cord mesenchymal stem cells can be easily cultured in vitro and amplification and other advantages has become human bone marrow derived mesenchymal stem cells are very good alternatives. However, human umbilical cord derived mesenchymal stem cells in regulating immune function is No further study.
Objective: To study the human umbilical cord derived mesenchymal stem cells play the role of immunological effect and the role of the mechanism. Methods: human peripheral blood mononuclear cells by different stimuli after stimulation with human umbilical cord derived mesenchymal stem cells co culture, proliferation and IFN- gamma through detection of human peripheral blood mononuclear cells the observation of secretion of human umbilical cord derived mesenchymal stem cells to study the immune regulation effect; cultured mesenchymal stem cells immune regulation whether the need for direct contact with Transwell cells cultivation plate; determining factor mechanism of human umbilical cord derived mesenchymal stem cells play the immunological effect of various inhibitors; in addition, by flow cytometry human peripheral blood mononuclear cells Annexin-V to observe the human umbilical cord derived mesenchymal stem cells play the effect and related mechanism.
Results: human umbilical cord derived mesenchymal stem cells can secrete IFN- effectively inhibited the proliferation and gamma of human peripheral blood mononuclear cells after stimulation; Transwell co culture system showed that the inhibition effect is not affected by cell contact, this is mainly through the inhibition of immune factor effect; inhibitor trials excluded TGF- beta IDO, and NO, and to determine the prostaglandin E2 (PGE2) is the human umbilical cord derived mesenchymal stem cells play the immunological effect mediated factor; inflammatory cytokines IFN- and IL-1 beta gamma can increase secretion of human umbilical cord derived mesenchymal stem cells PGE2; human umbilical cord derived mesenchymal stem cells to apoptosis the protection of human peripheral blood mononuclear cells after stimulation, and this protective effect is through the secretion of PGE2 play a role.
Conclusion: human umbilical cord derived mesenchymal stem cells (hUC-MSC) can also play a good immune regulatory role, and for the first time found that the immunological effect of the cells is mainly through the secretion of PGE2, this mechanism provides a theoretical basis for future clinical application of human umbilical cord derived mesenchymal stem cells.
Background: a recent concern factor IL-17, as a marker of the adaptive immune system inflammation, was first produced by a new T cell group 'Th17'. There has been much progress in research in recent years, including the standard molecular mechanism of Thl7 cell differentiation. These tend to lead to clear Th17 cells in host defense and immune function of great help. Recent studies have demonstrated that IL-17 produced by inflammatory cells stimulate the central link of many human autoimmune disease animal model of autoimmune disease development and disease. And the activation of IL-23 / IL-17 axis for systemic lupus erythematosus (SLE) disease is inflammation important. Mesenchymal stem cells are multipotent stem cells that can differentiate into tissues of mesodermal origin and isolated from various tissues by.MSCs has been very have on immune cells Extensive immunomodulatory effect has many regulatory effects on many effector functions. Recently, fetal bone marrow derived MSCs can promote the expansion of IL-17 secreting cells from normal donors. However, the effect of MSCs from umbilical cord on immune cells is not clear.
Objective: To study the umbilical cord derived mesenchymal stem cells (hUC-MSCs) regulating effect for immune from healthy donors and patients with T SLE lymphocytes, especially for Th17 cells. This can be extended to understand our regulatory function for MSCs immune cells as well as its dependence on treatment of immune disorders provide the potential for clinical use more insights.
Methods: we will hPBMCs (from healthy donors and SLE patients) or CD4+T (from healthy donors) and hUC-MSCs cell culture, and the culture system of cells called.HPBMCs cells induced by mitogen stimulation of PHA co cultured CD4+T cells by ati-CD3/CD28 Dynabeads stimulation. We use real-time quantitative PCR, ELISA comparison of adsorption from healthy donor or SLE in patients with hPBMCs. The expression levels of IL-17 secretion and detection of addition of IL-6, changes in the expression of PGE2 and IL-1 beta IL-17 and neutralization reagent. CD4+T cells were detected by flow cytometry in the interaction of hUC-MSCs and the expression of IL-17 secretion changes by enzyme-linked immunosorbent assay. The expression of IFN- gamma and TGF- beta was detected by ELISA, and the proportion of Thl cells and Treg cells by flow cytometry. We used enzyme-linked immunosorbent assay for IL-23 patients compared to healthy and SLE secretion level Change.
Results: normal donor derived CD4+T cells and human umbilical cord derived MSCs co culture than pure cultures of normal donor CD4+T cells can produce high levels of IL-17 secretion. The same results have also been confirmed in normal donor PBMCs cells. Inhibition test confirmed that the effect may be produced by PGE2 or IL-1 beta mediated, and IL-23 not involved. Then we will hUC-MSCs and sources of SLE in patients with PBMCs cells were co cultured in vitro, the ability of IL-17 and hUC-MSCs can promote SLE secretion of patients, but did not affect the secretion of IL-23. At the same time, hUC-MSCs reduced the number of Thl cells and increase the number of Treg cells.
Conclusion: in summary, our results are the first reported hUC-MSCs while promoting healthy donors and SLE patients. PBMCs and IL-1 beta IL-17.PGE2 may participate in hUC-MSCs this role. This finding may use for hUC-MSCs points in SLE patients the potential pathogenicity. Considering the correlation between IL-17 content and the activity of SLE in serum of patients with the disease. The clinical application of hUC-MSCs treatment of SLE may require more careful research.

【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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