混合皮肤移植中自体角朊细胞表达B7-H1分子在诱导免疫耐受中的作用
发布时间：2018-07-25 16:18
【摘要】： 目的:探讨自体角朊细胞在自体皮岛抑制效应中的作用及其机制。方法: 1.分离外周血淋巴细胞和表皮细胞,建立MELR试验体系,通过引入附加的自体表皮细胞(并以等量的异体表皮细胞为对照),在体外模拟自体皮岛效应。 2.分析加入和不加入自体角朊细胞的MELR体系中,IFN-γ、IL-2、IL-4及IL-10等几种细胞因子分泌的动力学曲线。根据以上动力学曲线,在加入和不加入自体角朊细胞的MELR体系中,引入相应的中和性抗体,观察反应格局的变化。 3.通过IL-10基因敲除小鼠实验,来观察IL-10在诱导免疫抑制中T细胞和角朊细胞各自的重要作用。 4.对角朊细胞进行B7-1的转染,以及负性调节分子CTLA-4的阻断实验,分析这些经典的共刺激分子与角朊细胞诱导抑制的关系。5.观察MELR体系中角朊细胞B7-H1的表达情况,并通过B7-H1 单抗的阻断实验,来探讨这一新发现的B7分子在自体皮岛抑制效应中的作用。 结果: 1.在La+Eb+Kcb的实验组中,8小时开始出现IL-2,并逐渐增高,16小时开始有IFN-γ的出现,也逐渐增高。但始终没有检测到IL-4和IL-10的存在。在La+Eb+Kca的实验组中,起初的一段时间里,也出现了较低浓度的IL-2和IFN-γ,到了32小时后开始下降并消失。以此同时开始出现IL-10和IL-4,并开始持续增高,特别是IL-10在48小时升高尤为明显。单独加入抗IL-10时,在32小时以前,可明显缓解抑制,这可能与系统早期只出现IL-10有关。而在32小时以后或单独加入抗IL-4时,都没有很大的缓解作用。 2.在同种异体增殖抑制作用中起主要作用的是淋巴细胞来源的IL-10而并非角朊细胞分泌的IL-10,但后者有部分协同作用。 3.自体角朊细胞在转染B7-1基因后,对同种异体增殖反应的抑制诱导能力消失,但加入抗B7-1单抗阻断B7-1的作用后,其诱导抑制的能力又恢复。而CTLA-4在自体角朊细胞诱导的抑制中并不发挥相关作用。 4. B7-H1在MELR的体系中4小时开始有微弱的表达,而8小时之后,就有较强的表达;加入B7-H1单抗或PD-1单抗来阻断实验后,自体角朊细胞的抑制作用缓解或消失。 结论: 1.自体角朊细胞是通过激活T细胞分泌IL-10激活Th2亚群而抑制Th1亚群来诱发局部免疫抑制。 2.自体角朊细胞通过B7-1、B7-2以外的其他分子提供共刺激信号,而且其负向调节作用与CTLA-4无关。 3.自体角朊细胞是通过B7-H1提供共刺激信号,进而激活分泌IL-10的Th2细胞,最终遏制Th1功能诱导局部免疫抑制的。
[Abstract]:Aim: to investigate the role and mechanism of autologous keratinocytes in the inhibition of autologous skin island. Methods: 1. The peripheral blood lymphocytes and epidermal cells were isolated, and the MELR test system was established. The autologous skin island effect was simulated in vitro by introducing additional autologous epidermal cells (and using allogeneic epidermal cells as control). 2. The kinetics curves of cytokines secretion such as IL-2IL-4 and IL-10 in MELR system with or without autologous keratinocytes were analyzed. According to the above kinetic curves, the neutralizing antibody was introduced into the MELR system with or without autologous keratinocytes, and the change of reaction pattern was observed. IL-10 gene knockout mice were used to observe the important roles of IL-10 in inducing immunosuppression of T cells and keratinocytes. 4. The relationship between these classical costimulatory molecules and the induction inhibition of keratinocytes was analyzed by B7-1 transfection and CTLA-4 blocking assay. The expression of B7-H1 in keratinocytes in MELR system was observed and the role of B7 molecule in the inhibition of autologous skin island was investigated by blocking the B7-H1 monoclonal antibody. Results: 1. In the experimental group of La Eb Kcb, IL-2 began to appear at 8 hours, and increased gradually at 16 hours. However, the presence of IL-4 and IL-10 was never detected. Lower concentrations of IL-2 and IFN- 纬 also appeared in the La Eb Kca experimental group at first, and began to decrease and disappear after 32 hours. At the same time, IL-10 and IL-4 began to appear, and began to increase continuously, especially at 48 hours after the increase of IL-10. When anti IL-10 was added alone, the inhibition was significantly alleviated before 32 hours, which may be related to the presence of only IL-10 in the early stage of the system. However, after 32 hours or after the addition of anti-IL-4 alone, there was no significant relief. 2. 2. IL-10 from lymphocytes rather than IL-10 secreted by keratinocytes may play a major role in the inhibition of allogeneic proliferation, but the latter has some synergistic effects. After transfection of B7-1 gene, the inhibitory effect of autologous keratinocytes on allogeneic proliferation disappeared, but the ability of inducing inhibition of B7-1 was restored after the addition of anti-B7-1 McAb to block the effect of B7-1. However, CTLA-4 did not play a related role in the inhibition induced by autologous keratinocytes. 4. The expression of B7-H1 in MELR system began to be weak at 4 hours, but after 8 hours, there was strong expression, and the inhibitory effect of autologous keratinocytes was alleviated or disappeared after the addition of B7-H1 McAb or PD-1 McAb to block the experiment. Conclusion: 1. Autologous keratinocytes induce local immunosuppression by activating T cells secreting IL-10 to activate Th2 subsets and inhibiting Th1 subsets. 2. Autologous keratinocytes provide costimulatory signals through molecules other than B7-1 and B7-2, and their negative regulation is not related to CTLA-4. Autologous keratinocytes provide costimulatory signals through B7-H1, and then activate Th2 cells secreting IL-10, and finally inhibit the function of Th1 to induce local immunosuppression.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.4
本文编号：2144367
[Abstract]:Aim: to investigate the role and mechanism of autologous keratinocytes in the inhibition of autologous skin island. Methods: 1. The peripheral blood lymphocytes and epidermal cells were isolated, and the MELR test system was established. The autologous skin island effect was simulated in vitro by introducing additional autologous epidermal cells (and using allogeneic epidermal cells as control). 2. The kinetics curves of cytokines secretion such as IL-2IL-4 and IL-10 in MELR system with or without autologous keratinocytes were analyzed. According to the above kinetic curves, the neutralizing antibody was introduced into the MELR system with or without autologous keratinocytes, and the change of reaction pattern was observed. IL-10 gene knockout mice were used to observe the important roles of IL-10 in inducing immunosuppression of T cells and keratinocytes. 4. The relationship between these classical costimulatory molecules and the induction inhibition of keratinocytes was analyzed by B7-1 transfection and CTLA-4 blocking assay. The expression of B7-H1 in keratinocytes in MELR system was observed and the role of B7 molecule in the inhibition of autologous skin island was investigated by blocking the B7-H1 monoclonal antibody. Results: 1. In the experimental group of La Eb Kcb, IL-2 began to appear at 8 hours, and increased gradually at 16 hours. However, the presence of IL-4 and IL-10 was never detected. Lower concentrations of IL-2 and IFN- 纬 also appeared in the La Eb Kca experimental group at first, and began to decrease and disappear after 32 hours. At the same time, IL-10 and IL-4 began to appear, and began to increase continuously, especially at 48 hours after the increase of IL-10. When anti IL-10 was added alone, the inhibition was significantly alleviated before 32 hours, which may be related to the presence of only IL-10 in the early stage of the system. However, after 32 hours or after the addition of anti-IL-4 alone, there was no significant relief. 2. 2. IL-10 from lymphocytes rather than IL-10 secreted by keratinocytes may play a major role in the inhibition of allogeneic proliferation, but the latter has some synergistic effects. After transfection of B7-1 gene, the inhibitory effect of autologous keratinocytes on allogeneic proliferation disappeared, but the ability of inducing inhibition of B7-1 was restored after the addition of anti-B7-1 McAb to block the effect of B7-1. However, CTLA-4 did not play a related role in the inhibition induced by autologous keratinocytes. 4. The expression of B7-H1 in MELR system began to be weak at 4 hours, but after 8 hours, there was strong expression, and the inhibitory effect of autologous keratinocytes was alleviated or disappeared after the addition of B7-H1 McAb or PD-1 McAb to block the experiment. Conclusion: 1. Autologous keratinocytes induce local immunosuppression by activating T cells secreting IL-10 to activate Th2 subsets and inhibiting Th1 subsets. 2. Autologous keratinocytes provide costimulatory signals through molecules other than B7-1 and B7-2, and their negative regulation is not related to CTLA-4. Autologous keratinocytes provide costimulatory signals through B7-H1, and then activate Th2 cells secreting IL-10, and finally inhibit the function of Th1 to induce local immunosuppression.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.4
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