DCs在抗CD45RB单克隆抗体诱导免疫耐受中的作用机制
发布时间：2019-04-15 13:01
【摘要】： 目的抗CD45RB抗体可以抑制T细胞的增殖,诱导移植免疫耐受,而树突状细胞通过与T细胞相互作用而发挥免疫调节作用。本研究通过观察抗CD45RB单克隆抗体在体内外对DCs成熟和功能的影响,以阐明DCs在抗CD45RB单克隆抗体诱导免疫耐受中的作用机制。方法无菌分离C57BL/6小鼠骨髓细胞,体外用rmGM-CSF和IL-4诱导骨髓细胞生成DCs,LPS刺激其为成熟DCs,同时加入不同剂量的抗CD45RB单克隆抗体,第6天用流式细胞仪检测对成熟DCs表型、细胞周期和抗原吞噬能力的影响,用ELISA检测培养上清中IL-12含量。通过多次过滤法提取成熟DCs外分泌体(exosomes, Dex),利用混合淋巴细胞实验检测DCs和Dex对T细胞增殖能力的影响。体内实验部分,将Balb/C小鼠心脏移植到C57BL/6小鼠,分别在移植0、1、3、5、7天经腹腔注射抗CD45RB单克隆抗体0.1mg／次／天；在0、1、3、5、7天分别处死受体鼠提取骨髓细胞,在体外用rmGM-CSF、IL-4和LPS将其诱导为成熟DCs,观察形态学改变,流式细胞术检测DCs细胞表型和吞噬能力,ELISA检测培养上清IL-12含量。 结果体外实验表明随着抗CD45RB单克隆抗体的增加,BMC来源DCs特异性标记CDllc无明显变化,而DCs成熟度标记CD83、共刺激分子CD80、CD86和MHC-Ⅱ分子I-Ab的表达则随着抗CD45RB单克隆抗体剂量增加而降低；抗CD45RB单克隆抗体使成熟DCs抗原吞噬能力增强,IL-12的分泌量降低,细胞呈增殖状态,抗CD45RB单克隆抗体抑制DCs成熟具有剂量依赖型。混合淋巴细胞实验表明经抗CD45RB单克隆抗体处理的DCs和分泌的Dex,可抑制T细胞增殖,外分泌体Dex在DCs增强或抑制T细胞增殖作用中也具有明显的剂量依赖性。体内实验表明,经抗CD45RB单克隆抗体治疗后,受体鼠骨髓细胞在体外诱导的mDCs,在形态学与对照组无明显变化,细胞表型CDllc的表达也无明显区别,CD83、CD80、CD86和I-Ab的表达则随着时间的延长而降低,DCs抗原吞噬能力随着时间的延长而增加,IL-12分泌能力逐渐下降,其结果与体外实验相似。 结论体内外实验表明抗CD45RB单克隆抗体诱导免疫耐受的机制,主要是抑制DCs的成熟和功能,形成一种耐受性树突状细胞(tolerogenic dendritic cells,tDCs)以及外分泌体Dex可显著抑制T细胞的增殖,发挥免疫抑制作用,诱导免疫耐受的产生。
[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，
本文编号：2458175
[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，
本文编号：2458175
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