【摘要】： 目的:探討自體角朊細(xì)胞在自體皮島抑制效應(yīng)中的作用及其機(jī)制。方法: 1.分離外周血淋巴細(xì)胞和表皮細(xì)胞,建立MELR試驗(yàn)體系,通過引入附加的自體表皮細(xì)胞(并以等量的異體表皮細(xì)胞為對(duì)照),在體外模擬自體皮島效應(yīng)。 2.分析加入和不加入自體角朊細(xì)胞的MELR體系中,IFN-γ、IL-2、IL-4及IL-10等幾種細(xì)胞因子分泌的動(dòng)力學(xué)曲線。根據(jù)以上動(dòng)力學(xué)曲線,在加入和不加入自體角朊細(xì)胞的MELR體系中,引入相應(yīng)的中和性抗體,觀察反應(yīng)格局的變化。 3.通過IL-10基因敲除小鼠實(shí)驗(yàn),來觀察IL-10在誘導(dǎo)免疫抑制中T細(xì)胞和角朊細(xì)胞各自的重要作用。 4.對(duì)角朊細(xì)胞進(jìn)行B7-1的轉(zhuǎn)染,以及負(fù)性調(diào)節(jié)分子CTLA-4的阻斷實(shí)驗(yàn),分析這些經(jīng)典的共刺激分子與角朊細(xì)胞誘導(dǎo)抑制的關(guān)系。5.觀察MELR體系中角朊細(xì)胞B7-H1的表達(dá)情況,并通過B7-H1 單抗的阻斷實(shí)驗(yàn),來探討這一新發(fā)現(xiàn)的B7分子在自體皮島抑制效應(yīng)中的作用。 結(jié)果: 1.在La+Eb+Kcb的實(shí)驗(yàn)組中,8小時(shí)開始出現(xiàn)IL-2,并逐漸增高,16小時(shí)開始有IFN-γ的出現(xiàn),也逐漸增高。但始終沒有檢測(cè)到IL-4和IL-10的存在。在La+Eb+Kca的實(shí)驗(yàn)組中,起初的一段時(shí)間里,也出現(xiàn)了較低濃度的IL-2和IFN-γ,到了32小時(shí)后開始下降并消失。以此同時(shí)開始出現(xiàn)IL-10和IL-4,并開始持續(xù)增高,特別是IL-10在48小時(shí)升高尤為明顯。單獨(dú)加入抗IL-10時(shí),在32小時(shí)以前,可明顯緩解抑制,這可能與系統(tǒng)早期只出現(xiàn)IL-10有關(guān)。而在32小時(shí)以后或單獨(dú)加入抗IL-4時(shí),都沒有很大的緩解作用。 2.在同種異體增殖抑制作用中起主要作用的是淋巴細(xì)胞來源的IL-10而并非角朊細(xì)胞分泌的IL-10,但后者有部分協(xié)同作用。 3.自體角朊細(xì)胞在轉(zhuǎn)染B7-1基因后,對(duì)同種異體增殖反應(yīng)的抑制誘導(dǎo)能力消失,但加入抗B7-1單抗阻斷B7-1的作用后,其誘導(dǎo)抑制的能力又恢復(fù)。而CTLA-4在自體角朊細(xì)胞誘導(dǎo)的抑制中并不發(fā)揮相關(guān)作用。 4. B7-H1在MELR的體系中4小時(shí)開始有微弱的表達(dá),而8小時(shí)之后,就有較強(qiáng)的表達(dá);加入B7-H1單抗或PD-1單抗來阻斷實(shí)驗(yàn)后,自體角朊細(xì)胞的抑制作用緩解或消失。 結(jié)論: 1.自體角朊細(xì)胞是通過激活T細(xì)胞分泌IL-10激活Th2亞群而抑制Th1亞群來誘發(fā)局部免疫抑制。 2.自體角朊細(xì)胞通過B7-1、B7-2以外的其他分子提供共刺激信號(hào),而且其負(fù)向調(diào)節(jié)作用與CTLA-4無(wú)關(guān)。 3.自體角朊細(xì)胞是通過B7-H1提供共刺激信號(hào),進(jìn)而激活分泌IL-10的Th2細(xì)胞,最終遏制Th1功能誘導(dǎo)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- 緯 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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392.4

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