间充质干细胞对树突状细胞体外分化成熟影响的实验研究

发布时间：2018-04-29 13:02

  本文选题：小鼠间充质干细胞株 + 胎盘源间充质干细胞　； 参考：《苏州大学》2009年硕士论文

【摘要】： 树突状细胞(dendritic cells, DCs)是功能最强大的抗原提呈细胞(antigen presenting cells, APCs)。由于表达丰富的协同刺激分子以及分泌多种具有免疫活性的细胞因子,DCs可以有效刺激初始T细胞活化。DCs的分化发育过程包括未成熟和成熟两个阶段,而DCs的生物学功能也与其成熟程度密切相关。在免疫稳态时,未成熟DCs(immature DCs, iDCs)通过不断摄取死细胞相关产物维持细胞正常的更新换代。当缺乏炎症因子时,这些iDCs表达较低水平的协同刺激分子,从而诱导引流淋巴结内T细胞无能、凋亡或调节性T细胞的产生。当感染发生时,iDCs在抗原和炎症因子的刺激下成熟,进入淋巴组织刺激初始或静息T细胞活化,从而介导效应T细胞应答。总之,DCs的功能主要依赖于细胞活化成熟的程度,不同亚群的iDCs参与诱导和维持外周耐受,而最终发育成熟的DCs可有效地促进效应T细胞的产生。 由于基因缺损、移植等原因,DCs的成熟过程常常被打乱,从而可能产生自身免疫性疾病或移植物抗宿主病(graft versus host disease, GVHD)。有报道显示,在系统性红斑狼疮(systemic lupus erythematosus, SLE)患者体内存在IFN-α刺激DCs缓慢活化。另外,在小鼠疾病模型中应用未活化DCs可显著改善急性GVHD的恶性程度。因此,调节DCs的活化程度有可能成为治愈这些疾病的有效手段。 间充质干细胞(mesenchymal stem cells, MSCs)具有强大的调节功能,在疾病治疗方面的应用前景让人期待。目前研究发现,MSCs不但可以抑制CD8+细胞毒性T细胞(cytotoxic T lymphocytes, CTLs)和自然杀伤(nature killer, NK)细胞的活化,也能抑制细胞或非特异性丝裂原介导的T细胞增殖。虽然MSCs对许多免疫细胞都具有直接的负调控效应,但MSCs对免疫应答的起始过程——DCs的成熟——是否也具有免疫调节功能还不清楚。 本实验分为两个部分,分别研究小鼠和人MSCs对DCs分化成熟的影响。在第一部分中,小鼠骨髓(bone marrow, BM)细胞经mGM-CSF和mIL-4刺激5 d可得到iDCs,与小鼠间充质干细胞株C3H10T1/2体外共培养,同时加入LPS刺激4 d。结果发现,对照组DCs悬浮生长并大量聚集成团,细胞表面出现树枝样突起,呈典型的成熟DCs形态。而共培养组DCs散在分布,细胞呈圆形,无树枝样突起;细胞表型检测发现,共培养组DCs表面CD11c、MHC-Ⅱ、CD86、CD40的表达均受到抑制;共培养组DCs细胞数低于对照组,提示C3H10T1/2细胞还可以抑制DCs的扩增;此外,共培养组DCs刺激脾细胞增殖的能力也明显下降,且伴随IFN-γ和IL-10分泌的减少。 在第二部分中,首先从人胎盘分离得到MSCs并进行鉴定,随后分别研究了胎盘源MSCs(placenta derived MSCs, PMSCs)对外周血(peripheral blood, PB)和脐血(umbilical cord blood, UCB)单核源DCs分化成熟的影响。胎盘组织经Ⅳ型胶原酶消化、贴壁和传代培养可获得人PMSCs,运用倒置显微镜和流式细胞仪进行细胞形态和表型的鉴定,证实已成功培养得到MSCs。同时应用hGM-CSF和hIL-4诱导获得人PB和UCB单核源DCs,然后与PMSCs进行共培养。结果显示,即使在LPS刺激下,共培养组DCs表面CD80、CD86、CD83、CD40的表达仍受到明显抑制,而CD14的表达则被上调。另外,PB单核源DCs刺激淋巴细胞增殖的能力也受到抑制。 综上所述,本实验表明MSCs可以在细胞形态、表型、刺激淋巴细胞增殖以及细胞因子分泌四个方面抑制DCs的成熟。本结果为进一步研究MSCs免疫调节DCs的分子机制提供了实验基础,也为临床治疗各种疾病带来启示和帮助。
[Abstract]:Dendritic cells (DCs) is the most powerful antigen presenting cell (antigen presenting cells, APCs). DCs can effectively stimulate the differentiation and development of the initial T cell activation.DCs, including the two stages of immature and mature stages due to the rich expression of synergistic stimulators and the secreting of many immune active cytokines. The biological function of DCs is closely related to its maturity. In the immune homeostasis, the immature DCs (immature DCs, iDCs) maintains normal cell regeneration through the continuous intake of dead cell related products. When lack of inflammatory factors, these iDCs express lower levels of synergistic stimulators, thus inducing T cells in the drainage lymph nodes. Incompetence, the production of apoptosis or regulatory T cells. When infection occurs, iDCs matures under the stimulation of antigen and inflammatory factors and enters the lymphoid tissue to stimulate initial or resting T cell activation, which mediates the response of the effect of T cells. In a word, the function of DCs is mainly dependent on the degree of cell activation and maturation, and the iDCs of different subgroups is involved in the induction and maintenance of the cells. Zhou tolerance, and the final mature DCs can effectively promote the production of T cells.
The maturation process of DCs is often disrupted because of genetic defect and transplantation, which may lead to autoimmune disease or graft versus host disease (GVHD). It is reported that IFN- alpha stimulates DCs slowly in patients with systemic lupus erythematosus (systemic lupus erythematosus, SLE). The use of unactivated DCs in the mouse disease model can significantly improve the degree of malignancy of acute GVHD. Therefore, the regulation of the activation of DCs may be an effective means to cure these diseases.
Mesenchymal stem cells (MSCs) has a strong regulatory function and is expected to be expected in the treatment of disease. At present, MSCs can not only inhibit the activation of CD8+ cell toxic T cells (cytotoxic T lymphocytes, CTLs) and natural killer (nature), but also inhibit cell or nonspecific cells. Heterosexual mitogen mediated proliferation of T cells. Although MSCs has a direct negative regulatory effect on many immune cells, MSCs is not clear about the initiation of the immune response, the maturation of DCs, whether or not it has the function of immunomodulatory.
The experiment was divided into two parts. In the first part, the mice bone marrow (bone marrow, BM) cells stimulated 5 d by mGM-CSF and mIL-4, and the mice were co cultured with the mouse mesenchymal stem cell line C3H10T1/2 in vitro and added LPS stimulation 4 d.. The control group was suspended and grown in suspension. A large number of clustered clusters, the surface of the cell appeared on the surface of the cell, showing a typical mature DCs form. While the co culture group DCs scattered in the distribution, the cells were round and no dendritic. The cell phenotype detection showed that the expression of DCs surface CD11c, MHC- II, CD86 and CD40 in co culture group were suppressed, and the number of DCs cells in co culture group was lower than that of the control group, suggesting C3H10T1/2 finer. The cells also inhibited the amplification of DCs. In addition, the ability of DCs to stimulate splenocytes proliferation in the co culture group decreased significantly, and the secretion of IFN- and IL-10 decreased.
In the second part, MSCs was first isolated from human placenta and identified. Then, the effects of placental source MSCs (placenta derived MSCs, PMSCs) on the differentiation and maturation of peripheral blood (peripheral blood, PB) and umbilical cord blood (umbilical cord blood) were studied. PMSCs was obtained by using inverted microscope and flow cytometry to identify the cell morphology and phenotype. It was confirmed that MSCs. was successfully cultured with hGM-CSF and hIL-4 to induce PB and UCB mononuclear DCs, and then co culture with PMSCs. The results showed that the DCs surface of the co culture group was CD80, CD86, and, even under LPS stimulation. The expression of CD14 was upregulated, while the expression of PB was up-regulated. In addition, the ability of monocyte derived DCs to stimulate lymphocyte proliferation was also inhibited.
To sum up, this experiment shows that MSCs can inhibit the maturation of DCs in the four aspects of cell morphology, phenotype, stimulating lymphocyte proliferation and cytokine secretion. This result provides an experimental basis for further study of the molecular mechanism of MSCs immunomodulatory DCs, and also provides inspiration and help for the clinical treatment of various diseases.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1

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