人外周血未成熟树突状细胞体外扩增、鉴定及抗成熟特性的研究

发布时间：2018-10-05 07:55

【摘要】：创面处理是成功救治大面积深度烧伤病人的关键问题之一,及时、有效的覆盖创面,将对后续治疗产生积极的影响。同种异体皮肤移植为创面的覆盖提供了一条良好的思路,但因其具有抗原性,移植后的排斥反应严重影响了治疗效果。免疫抑制剂虽可延长其存活时间,但对于大面积烧伤病人而言,本身就处于一种免疫功能明显低下状态,大量抑制机体免疫力的抗排斥药物的应用反而增加了感染的危险性。因此诱导受体对供体皮肤产生特异性的免疫低下或免疫耐受状态而同时保持受体正常的免疫功能至关重要。树突状细胞(dendritic cell,DC)是体内功能最强的抗原呈递细胞(antigen-presenting cell,APC),是天然免疫和获得性免疫的重要调节剂。因其来源、发育阶段和细胞表型等的不同而存在功能异质性。而未成熟DC 由于其表面缺乏共刺激分子,不能够提供T 细胞活化所必需的共刺激信号,在免疫应答过程中非但不能激活T 细胞,反而可诱导其无能,从而成为诱导移植免疫耐受的重要靶细胞。虽然DC 在体内分布广泛,但数量少且分散,而未成熟DC 主要分布在外周非淋巴组织内,直接分离、提纯十分困难,不能满足体内或体外研究及临床应用的需要。同时在大面积烧伤病人体内存在着大量的内毒素(lipopolysaccharide,LPS)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)等促进DC 成熟的物质,因而明显影响了其致耐效果。白介素-10(interleukin-10,IL-10)是一种抗炎性细胞因子,在同种异体混合淋巴细胞反应中能抑制T 细胞的活化、增殖,有研究表明移植术前给予IL-10 可延长移植物的存活时间。而DC 又是T 细胞活化的关键环节,推测其抑制作用可能是通过对APC 功能的影响来实现的。在本研究中,我们联合应用rhGM-CSF 和rhIL-4 体外诱导、扩增人外周血分离的单个核细胞,第7d 同时加入rhIL-10 联合培养,于第9d 收获细胞,从形态学、细胞表面标志物以及混合淋巴细胞反应(MLR)中对同种异体未致敏T 淋巴细胞刺激能力三个方面进行鉴定。并将培养的细胞分别用LPS、TNF-α继续刺激2 天,再进行MLR,观察IL-10 对外周血单个核细胞来源的未成熟DC 的影响,以期为体外获得足够数量
[Abstract]:Wound management is one of the key problems in the successful treatment of large area deep burn patients. Timely and effective covering of wounds will have a positive impact on subsequent treatment. Skin allograft provides a good idea for wound coverage, but the rejection after transplantation seriously affects the therapeutic effect because of its antigenicity. Although immunosuppressants can prolong their survival time, for large area burn patients, they are in a state of obviously low immune function, and the use of a large number of anti-rejection drugs to suppress the body's immunity increases the risk of infection. Therefore, it is important to induce the receptor to produce specific hypoimmunity or immune tolerance to the donor skin while maintaining the normal immune function of the receptor. Dendritic cells (dendritic cell,DC) are the most potent antigen-presenting cells (antigen-presenting cell,APC) and important modulators of innate and acquired immunity. Functional heterogeneity exists due to its origin, developmental stages and cell phenotypes. Due to the lack of costimulatory molecules on the surface of immature DC, it can not provide the necessary costimulatory signal for T cell activation. In the course of immune response, it can not activate T cell, but induce its incompetence. Thus, it becomes an important target cell to induce transplantation immune tolerance. Although DC is widely distributed in vivo, its quantity is small and dispersed, while immature DC is mainly distributed in peripheral non-lymphoid tissues. It is very difficult to separate and purify directly, and can not meet the needs of research and clinical application in vivo or in vitro. At the same time, there are a lot of endotoxin (lipopolysaccharide,LPS) and tumor necrosis factor- 伪 (TNF- 伪) in large area burn patients to promote the maturation of DC. Interleukin-10 (interleukin-10,IL-10) is an anti-inflammatory cytokine, which can inhibit the activation and proliferation of T cells in allogeneic mixed lymphocyte reaction. Some studies have shown that IL-10 can prolong the survival time of grafts before transplantation. DC is the key link of T cell activation. It is speculated that the inhibitory effect of DC may be through the effect on APC function. In this study, the mononuclear cells isolated from human peripheral blood were amplified by rhGM-CSF and rhIL-4 in vitro, then co-cultured with rhIL-10 on the 7th day. The cells were harvested on the 9th day. Cell surface markers and the stimulating ability of allogeneic unsensitized T lymphocytes in mixed lymphocyte reaction (MLR) were identified. The cultured cells were further stimulated with LPS,TNF- 伪 for 2 days, and then MLR, was performed to observe the effect of IL-10 on immature DC from peripheral blood mononuclear cells in order to obtain sufficient quantity in vitro.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R644;R392

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