【作者】 石亮；

【导师】 胡丽华；

【作者基本信息】 华中科技大学 ， 临床免疫学， 2010， 博士

【摘要】 第一部分自身免疫调节因子与大分子自噬目的自身免疫调节因子（AIRE）具有诱导中枢免疫耐受的潜能,但在外周免疫耐受中,其功能未明。考虑大分子自噬可参与免疫耐受的诱导,我们初步探讨了自身免疫调节因子在外周中的表达及其对外周单核细胞系THP-1大分子自噬的影响。方法真核表达载体pEGFP-AIRE经双限制性酶切、PCR扩增及DNA测序分析鉴定；依据密度梯度离心与贴壁培养法进行外周血单个核细胞及单核细胞的分离；THP-1细胞由佛波醇酯诱导分化后通过瑞氏-姬姆萨染色进行形态学鉴定；细胞转染按脂质体法进行；AIRE表达抑制采用siRNA的方法；荧光显微镜观察GFP-AIRE融合蛋白的表达和亚细胞定位；转染效率经RT-PCR与Western Blot进行验证；自噬小体在胞内的形成和定位通过间接免疫荧光染色指示；大分子自噬的抑制采用PI3K抑制剂（渥曼青霉素）进行诱导；LC3B-Ⅱ与p62/SQSTM1的蛋白表达变化经Western Blot进行检测。结果质粒DNA经双酶切、PCR扩增和琼脂糖凝胶电泳后可见特异性条带；DNA测序结果显示测定序列与AIRE （NM₀00383.2）基因序列符合率99%（BLAST评分1698）。外周血单个核细胞、单核细胞、U937和THP-1细胞存在AIRE mRNA表达；单核细胞和THP-1细胞仅表达低水平AIRE蛋白。诱导后,THP-1细胞呈贴壁生长,胞体增大,形态多样性,核不规则,胞浆内空泡,吞噬细胞碎片等典型成熟分化形态。质粒转染48小时后,荧光显微镜显示GFP-AIRE融合蛋白以斑点状定位于细胞核内；在mRNA和蛋白水平,AIRE均呈明显高表达；而AIRE siRNA则明显抑制GFP-AIRE融合蛋白及AIRE mRNA与蛋白的表达。过表达AIRE可上调LC3B-Ⅱ的表达和自噬小体的形成；而大分子自噬抑制剂与AIRE siRNA可抑制AIRE介导的上调效应。过表达AIRE或大分子自噬抑制剂与AIRE siRNA对p62/SQSTM1蛋白表达无影响。结论真核表达载体pEGFP-AIRE构建正确,可应用于体外转染研究。AIRE可表达于外周单核细胞并通过上调LC3B-Ⅱ的表达和自噬小体的形成促进单核细胞大分子自噬。AIRE介导单核细胞大分子自噬的信号转导通路可能与p62/SQSTM1蛋白无关。通过促进单核细胞大分子自噬,AIRE可能在外周免疫耐受中具有功能。第二部分自身免疫调节因子与凋亡细胞的吞噬清除目的凋亡细胞是自身抗原成分的主要来源之一,适时清除凋亡细胞对维持免疫自稳具有重要意义。作为细胞死亡的两种不同形式——大分子自噬与凋亡共享许多调节因子,基于本文第一部分研究结果,为继续探讨自身免疫调节因子（AIRE）在外周免疫耐受中的功能,我们在非“职业”吞噬细胞——上皮细胞系16HBE中探讨了AIRE对吞噬凋亡细胞的影响。方法16HBE细胞的转染使用来源于第一部分研究的真核表达载体pEGFP-AIRE通过FuGENEHD转染法进行；转染效率经荧光显微镜、RT-PCR与Western Blot进行验证；HL-60细胞的凋亡由喜树碱诱导后通过Annexin V-FITC/碘化丙啶双染色进行流式细胞仪检测：吞噬凋亡细胞的检测通过髓过氧化物酶（MPO）染色进行显示；吞噬相关基因（Rac1）的表达变化经RT-PCR与Western Blot进行检测。结果pEGFP-AIRE转染48小时后,荧光显微镜显示GFP-AIRE融合蛋白以斑点状定位于16HBE细胞核内；通过RT-PCR与Western Blot检测,AIRE mRNA和蛋白均呈明显高表达。对比未经凋亡诱导的阴性对照组,流式细胞仪检测显示,喜树碱诱导的HL-60细胞凋亡率上升约3倍[（4.08±0.26）%Vs（13.46±1.71）%]。在与凋亡细胞共孵育1小时后,可见转AIRE基因16HBE细胞吞噬率为（25.5±3.67）%,而转染空载体16HBE细胞吞噬率为（6.25±1.58）%,两者间具有显著性差异（P<0.05）；但过表达AIRE对16HBE细胞吞噬未凋亡细胞无明显影响[（1.0±0.67）%]。同时,过表达AIRE可上调16HBE细胞Rac 1 mRNA和蛋白表达。结论16HBE细胞对凋亡细胞具有基础吞噬率,可作为体外研究吞噬凋亡细胞的细胞模型。MPO染色法对检测吞噬来源于MPO阳性靶细胞的吞噬效应具有较好对比度,可作为体外显示吞噬效应的检测方法。AIRE可促进上皮细胞对凋亡细胞的吞噬,通过上调Rac1的表达促进对凋亡细胞吞噬清除防止“自我”抗原“暴露”,AIRE可能在外周免疫耐受中具有功能。更多还原

【Abstract】 Part 1 Autoimmune Regulator and MacroautophayPurposeAutoimmune regulator （AIRE） has the potential to induce central immune tolerance. However its function in peripheral immune tolerance has so far remained unknown. Assuming that macroautophagy may have a role in the induction of immune tolerance, we intended to explore the expression of AIRE in periphery and its possible effect upon the macroautophagy of THP-1 human monocytes.MethodspEGFP-AIRE, the eukaryotic expression vector, was identified by double restriction enzyme digestion, then amplified by PCR and verified identified by DNA sequencing analysis. Using density gradient centrifugation and adhesive culture, peripheral blood mononuclear cells （PBMC） and monocytes were separated from peripheral blood. The phorbol ester （PMA） was adopted to induce the differentiation of THP-1 cells, which then was morphologically indentified by Wright-Giemsa staining and transfected by the Llipofectamine 2000. AIRE expression was inhibited by siRNA. Under the fluorescence microscope, the expression of GFP-AIRE fusion protein was observed and the subcellular location of proteins was indicated. The transfection efficiency was confirmed by RT-PCR and Western Blot. The intracellular formation and localization of autophagosome was indicated by indirect immunofluorescence staining. Macroautophagy was inhibited by PI3K inhibitor （wortmannin）; the protein expression of LC3B-Ⅱand p62/SQSTM1 were detected by Western Blot.Results Specific bands appeared after plasmid DNA was treated by restriction enzyme digestion, PCR amplification and agarose gel electrophoresis. According to the result of DNA sequencing, the compliance rate is as high as 99% with that of AIRE （NM₀00383.2） （BLAST scores 1698）. AIRE mRNA expression existed in PBMC, monocytes, U937 and THP-1 cells; AIRE protein expression is low in monocytes and THP-1 cells. Followed by induction, THP-1 cells present a series of typical manifestations of mature differentiation: an increased adherence to tissue culture plastic, expanded cell body, morphological diversity, irregular nucleus, phagocytic vacuoles and cellular debris in cytoplasm.48 hours after plasmid transfection, the dot-like GFP-AIRE fusion protein was spotted in the nucleus under fluorescence microscopy; in mRNA and protein level, AIRE showed significantly high expression while AIRE siRNA markedly inhibited the GFP-AIRE fusion protein expression as well as AIRE mRNA and protein expression. The over-expression of AIRE upregulated the LC3B-Ⅱexpression and promoted the formation of autophagosome. But this effect could be counteracted by macroautophagy inhibitors and AIRE siRNA. Neither over-expression of AIRE nor macroautophagy inhibitors and AIRE siRNA had any effect on the expression of p62/SQSTM1.ConclusionThe correct construction of pEGFP-AIRE, the eukaryotic expression vector, can be applied to the in vitro research of transfection. AIRE can be expressed in peripheral monocytes and by upregulating both the expression of LC3B-Ⅱand the formation of autophagosome, it is able to promote monocytic macroautophagy. The signal transduction pathway through which AIRE mediated monocytic macroautophagy may bear no relation to p62/SQSTM1 protein. It is probably by promoting monocytic macroautophagy that AIRE functions in peripheral immune tolerance. Part 2 Autoimmune Regulator and Phagocytosis of Apoptotic CellsPurposeAn opportune clearance of apoptotic cells, which are a major source of self-antigenic components, is of great significance for maintaining immunological homeostasis. As two different forms of programmed cell death, autophagic cell death and apoptosis share many common regulators. Based on the findings of the first part in this study, we continue to explore the function of autoimmune regulator （AIRE） in peripheral immune tolerance by focusing on the effect AIRE has upon phagocytosis of apoptotic cells by non-professional phagocytes-epithelial 16HBE cells.Methods16HBE cells were transfected by pEGFP-AIRE, the same plasmid in Part 1, using FuGENE HD transfection reagent. The transfection efficiency was verified by fluorescence microscopy, RT-PCR and Western Blot. The apoptosis of HL-60 cells was first induced by the CPT （Camptothecin）, then double stained by Annexin V-FITC/propidium iodide （PI） and finally detected by flow cytometry. Phagocytosis of apoptotic cells was displayed by myeloperoxidase （MPO） staining. Both RT-PCR and Western Blot were adopted to detect the expression of the phagocytosis-related gene （Rac 1）.Results48 hours after the pEGFP-AIRE transfection, we observed the predominant location of the GFP-AIRE fusion proteins in the nuclear dots by using fluorescence microscope; through RT-PCR and Western Blot assays, AIRE mRNA and protein expression were both significantly high. Compared to the negative control group with no apoptosis induction, the data from flow cytometry indicated that the percentage of apoptosis was approximately increased threefold [（4.08±0.26）% Vs （13.46±1.71）%] in HL-60 cells induced by camptothecin. Being incubated with apoptotic cells for 1 hour, the AIRE-transfected 16HBE cells enjoyed a phagocytosis rate of （25.5±3.67）%, while those transfected by empty vector had a phagocytosis rate of （6.25±1.58）%, indicating a significant differences （P<0.05）. However, over-expression of AIRE had little effect on 16HBE phagocytosis of non-apoptotic cells [（1.0±0.67）%] but upregulated the Rac 1 mRNA and protein expression.Conclusion16HBE cells possess a basic phagocytic rate in removing apoptotic cells and thus can be used as an alternative model in the study of phagocytosis of apoptotic cells in vitro. MPO staining is an optional testing method since it provides a sharp contrast in detecting engulfment of MPO-positive target cells in vitro. AIRE can promote the phagocytosis of apoptotic cells by epithelial cells. The upregulation of the expression of Rac 1 by AIRE can strengthen the phagocytic clearance of apoptotic cells and thus prevent the exposure of self-antigens, suggesting that AIRE might play a role in peripheral immune tolerance.更多还原