【摘要】： 目的抗CD45RB抗體可以抑制T細(xì)胞的增殖,誘導(dǎo)移植免疫耐受,而樹突狀細(xì)胞通過與T細(xì)胞相互作用而發(fā)揮免疫調(diào)節(jié)作用。本研究通過觀察抗CD45RB單克隆抗體在體內(nèi)外對DCs成熟和功能的影響,以闡明DCs在抗CD45RB單克隆抗體誘導(dǎo)免疫耐受中的作用機(jī)制。方法無菌分離C57BL/6小鼠骨髓細(xì)胞,體外用rmGM-CSF和IL-4誘導(dǎo)骨髓細(xì)胞生成DCs,LPS刺激其為成熟DCs,同時加入不同劑量的抗CD45RB單克隆抗體,第6天用流式細(xì)胞儀檢測對成熟DCs表型、細(xì)胞周期和抗原吞噬能力的影響,用ELISA檢測培養(yǎng)上清中IL-12含量。通過多次過濾法提取成熟DCs外分泌體(exosomes, Dex),利用混合淋巴細(xì)胞實驗檢測DCs和Dex對T細(xì)胞增殖能力的影響。體內(nèi)實驗部分,將Balb/C小鼠心臟移植到C57BL/6小鼠,分別在移植0、1、3、5、7天經(jīng)腹腔注射抗CD45RB單克隆抗體0.1mg／次／天；在0、1、3、5、7天分別處死受體鼠提取骨髓細(xì)胞,在體外用rmGM-CSF、IL-4和LPS將其誘導(dǎo)為成熟DCs,觀察形態(tài)學(xué)改變,流式細(xì)胞術(shù)檢測DCs細(xì)胞表型和吞噬能力,ELISA檢測培養(yǎng)上清IL-12含量。 結(jié)果體外實驗表明隨著抗CD45RB單克隆抗體的增加,BMC來源DCs特異性標(biāo)記CDllc無明顯變化,而DCs成熟度標(biāo)記CD83、共刺激分子CD80、CD86和MHC-Ⅱ分子I-Ab的表達(dá)則隨著抗CD45RB單克隆抗體劑量增加而降低；抗CD45RB單克隆抗體使成熟DCs抗原吞噬能力增強(qiáng),IL-12的分泌量降低,細(xì)胞呈增殖狀態(tài),抗CD45RB單克隆抗體抑制DCs成熟具有劑量依賴型。混合淋巴細(xì)胞實驗表明經(jīng)抗CD45RB單克隆抗體處理的DCs和分泌的Dex,可抑制T細(xì)胞增殖,外分泌體Dex在DCs增強(qiáng)或抑制T細(xì)胞增殖作用中也具有明顯的劑量依賴性。體內(nèi)實驗表明,經(jīng)抗CD45RB單克隆抗體治療后,受體鼠骨髓細(xì)胞在體外誘導(dǎo)的mDCs,在形態(tài)學(xué)與對照組無明顯變化,細(xì)胞表型CDllc的表達(dá)也無明顯區(qū)別,CD83、CD80、CD86和I-Ab的表達(dá)則隨著時間的延長而降低,DCs抗原吞噬能力隨著時間的延長而增加,IL-12分泌能力逐漸下降,其結(jié)果與體外實驗相似。 結(jié)論體內(nèi)外實驗表明抗CD45RB單克隆抗體誘導(dǎo)免疫耐受的機(jī)制,主要是抑制DCs的成熟和功能,形成一種耐受性樹突狀細(xì)胞(tolerogenic dendritic cells,tDCs)以及外分泌體Dex可顯著抑制T細(xì)胞的增殖,發(fā)揮免疫抑制作用,誘導(dǎo)免疫耐受的產(chǎn)生。
[Abstract]:Aim Anti-CD45RB antibody can inhibit the proliferation of T cells and induce transplanted immune tolerance. Dendritic cells play an immunomodulatory role by interacting with T cells. In this study, we observed the effect of anti-CD45RB monoclonal antibody on the maturation and function of DCs in vitro and in vivo, in order to elucidate the mechanism of DCs in the induction of immune tolerance by anti-CD45RB monoclonal antibody. Methods Bone marrow cells of C57BL/6 mice were isolated aseptically. Bone marrow cells were induced to produce DCs,LPS by rmGM-CSF and IL-4 in vitro. The cells were stimulated into mature DCs, with different doses of anti-CD45RB monoclonal antibody. On the 6th day, flow cytometry was used to detect the effect on the phenotype, cell cycle and antigen phagocytosis of mature DCs, and the content of IL-12 in culture supernatant was detected by ELISA. Mature DCs exocrine (exosomes, Dex), was extracted by multiple filtration method. The effects of DCs and Dex on T cell proliferation were detected by mixed lymphocyte assay. In vivo, the heart of Balb/C mice was transplanted into C57BL/6 mice. Anti-CD45RB monoclonal antibody 0.1mg/ was injected intraperitoneally on day 0, 1, 3, 5, 7 days after transplantation, respectively, and the mice were injected intraperitoneally with anti-CD45RB monoclonal antibody 0.1mg/ once a day. At 0, 1, 3, 5, 7 days, the recipient mice were killed to extract bone marrow cells and induced them into mature DCs, in vitro by rmGM-CSF,IL-4 and LPS to observe morphological changes. Flow cytometry was used to detect the phenotype and phagocytosis of DCs cells. The content of IL-12 in culture supernatant was detected by ELISA. Results the results showed that with the increase of anti-CD45RB monoclonal antibody, the specific CDllc labeled by BMC-derived DCs did not change significantly, but the CD83, costimulatory molecule CD80, was labeled with DCs maturity. The expression of CD86 and MHC- 鈪，

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