【作者】 刘金鹏；

【导师】 李富荣；

【作者基本信息】 暨南大学 ， 病理学与病理生理学， 2010， 硕士

【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。更多还原

【Abstract】 Objective Anti-CD45RB monoclonal antibody has been reported to inhibit proliferation of T cells and immunologic tolerance.The dendritic cells (DCs) play a immune regulation role by interacting with T cells. This experiment has explored anti-CD45RB monoclonal antibody on maturation and functions of DCs during the differentiation of monocytes into mature DCs(mDCs) in vivo and vitro, to clarify the mechanisms of anti-CD45RB monoclonal antibody inducing immune tolerance.Methods The mature dendritic cells(mDCs) from C57BL/6 mice were cultured with rmGM-CSF、IL-4 and LPS for six days.The bone marrow cells treated with varying doses of anti-CD45RB monoclonal antibody in the experiment group.The phenotype, cell cycle and the phagocytic capability of mDCs were detected by flow cytometry. IL-12 level was determined with an ELISA kit.The exosomes (Dex) from DCs were extracted via repeated filter method, the mDCs and mDex after anti-CD45RB monoclonal antibody treatment and those without treatment (Ab-mDCs and Ab-mDex, respectively) were used for the following experiment of mixed lymphocyte reaction.In vivo, the heart from donor Balb/C mouse were transplanted into C57BL/6 mouse received anti-CD45RB monoclonal antibody at a dose of 0.1 mg/times/day in 0,1,3,5,7 days after transplanting, then BM cells obtained from reciptient mice in 0,1,3,5,7 days, and induced to mDCs with rmGM-CSF、IL-4 and LPS in vitro, and to observe the phenotypes and functioning of mDCs and IL-12 level in supernatant were detected by ELISA.Results In vitro,the result indicated that the expression of CD83、CD80、CD86 and I-Abwere inhibited by anti-CD45RB monoclonal antibody in a dose dependent manner, but the expression of CD11c was no differently between experiment and control groups. The antigen phagocytosis of mDCs was higher and the IL-12 level was lower than that of control group. anti-CD45RB monoclonal antibody could inhibit DCs maturation in a dose dependent manner and the effects of Dex in DCs-induced proliferation of T cells were also in a dose dependent manner. In vivo, mDCs form the receptor mice were anti-CD45RB monoclonal antibody treated, the shape and expression of CD1 lc was no differently compared with control group, but the expression of CD83、CD80、CD86 and I-Ab was decreased in a dose dependent manner, the phagocytic activity was increased and IL-12 level was decreased with time, this result was similar with the result of experiment in vitro.Conclusions These findings suggested in the anti-CD45RB monoclonal antibody could profoundly inhibit the maturation and functioning of DCs in vitro and vivo and generate tolerogenic dendritic cells (tDCs) as well as Dex, thus resulting in obvious inhibition on T cell proliferation, immunosuppression and immune tolerance subsequently.更多还原