【作者】 唐庆娟；

【导师】 耿美玉；

【作者基本信息】 中国海洋大学 ， 药物化学， 2007， 博士

【副题名】pIgR介导潘氏细胞参与获得性免疫与pIgR介导MDCK细胞恶性转化

【摘要】 多聚免疫球蛋白受体(pIgR)属于I型跨膜糖蛋白,可以与多聚免疫球蛋白A和多聚免疫球蛋白M特异性结合,通过穿胞转运,将它们从上皮细胞基底侧膜转运到顶膜,并最终分泌到外分泌液中去。pIgR通过介导细胞内多聚免疫球蛋白的转运,可以在粘膜的腔面阻止病原体粘附,在上皮细胞内中和病毒,也可以将固有层内的抗原分泌出去。因此,pIgR的有效分泌是多聚免疫球蛋白发挥粘膜防御功能的必要条件,在粘膜免疫中起着举足轻重的作用。最初对人小肠粘膜的免疫组织化学研究发现:人小肠pIgR分布于柱状吸收细胞,尤其是那些分布在肠隐窝内的吸收细胞,而潘氏细胞、杯状细胞、内分泌细胞均未发现有pIgR的存在。几十年来人们普遍认为在小肠,只有柱状吸收细胞可以合成pIgR、介导IgA转运并分泌sIgA。然而,有研究表明:在大鼠小肠组织中,pIgR的免疫组织化学阳性染色特异性地位于潘氏细胞内,提示潘氏细胞与pIgR有一定的关联。潘氏细胞是存在于肠隐窝底部的锥形细胞,已经证实潘氏细胞有许多功能,如分泌磷脂酶A2、溶菌酶、防御素(defensins)、抗菌肽等,这些物质是肠道先天性免疫的重要基础。近年来的研究表明,潘氏细胞在炎症性肠道疾病(IBD)的发生、发展中也起重要作用。潘氏细胞是否合成pIgR并参与IgA的转运,对于IBD的发生机制研究及临床治疗手段的改进而言,具有重要的科学意义。本论文首先采用分子生物学的方法,转录合成了特异性针对大鼠与人pIgR的分子探针;然后采用荧光双标记原位杂交和荧光双标记免疫组织化学的方法,结合潘氏细胞的特异性蛋白标志物-溶菌酶,检测了大鼠和人小肠组织中pIgR的mRNA和蛋白的表达情况。实验结果显示:大鼠与人潘氏细胞内均可以检测到pIgR的mRNA和蛋白质,提示潘氏细胞可以合成pIgR。本论文又采用荧光双标记免疫组织化学和免疫胶体金电镜的方法,检测了潘氏细胞与小肠IgA分布之间的关系。结果发现:潘氏细胞的分泌颗粒和细胞基底膜上均含有大量的IgA,提示潘氏细胞所合成的pIgR可能参与IgA的转运。在大鼠小肠中,只有潘氏细胞内能够合成pIgR,而在人小肠中潘氏细胞和肠腺吸收细胞均可以合成pIgR,这表明大鼠与人小肠IgA的转运途径可能不一致。虽然多聚免疫球蛋白受体(pIgR)在粘膜免疫中起着至关重要的作用,但有报道指出该蛋白在体内的功能具有多面性。研究表明某些上皮肿瘤组织和细胞中pIgR的表达量高于正常组织或细胞,提示pIgR过表达可能与上皮来源肿瘤的发生、发展有一定的相关性。但是迄今为止,pIgR能否成为肿瘤的Marker或者成为肿瘤治疗的分子靶点目前尚未见报道。发现及阐明pIgR在肿瘤发生及发展中的重要作用,将对肿瘤这一人类尚未克服的疑难病症的诊断及治疗具有深远的意义。本论文首先利用真核转染技术,成功构建了人pIgR稳定高表达的MDCK细胞株,然后对细胞的特性进行观察与比较。结果发现:与对照组相比,pIgR过表达的MDCK细胞生长速度明显加快,细胞形态发生显著改变,细胞容易被胰酶消化提示粘附力降低。本论文利用SRB法、PI染色法、western blotting方法以及平皿克隆生长实验方法对有关细胞增殖的特征性指标进行了检测。结果显示:人pIgR过表达可以使MDCK细胞的增殖能力显著增强,细胞周期进程加快,细胞增殖指数明显升高,PCNA表达量增高,ERK1/2磷酸化显著增强,而且单个细胞生长能力增强。在上皮细胞形态变化和细胞与基质的粘附力下降的发生机制中,上皮细胞-间充质转化(EMT)往往起着至关重要的作用。EMT的主要特征有细胞粘附分子(如E-钙粘素)表达的减少、细胞骨架发生变化、形态上具有间充质细胞的特征等。这种转换允许上皮细胞摆脱细胞-细胞间连接,而表现得更具侵袭性。本论文又利用荧光免疫细胞化学染色法、transwell小室法对EMT相关的指标进行了检测。结果显示:人pIgR过表达的MDCK细胞形态上具有间充质细胞的特征,细胞骨架发生改变,具有粘附力下降、迁移和侵袭能力增强等EMT细胞行为学特征,并且有E钙粘素减少这一典型EMT分子特征。软琼脂克隆形成实验进一步表明:人pIgR过表达的MDCK细胞具备了一定的体外成瘤能力。上述结果表明,糖蛋白pIgR高表达具有明显的促MDCK细胞恶性转化的作用,pIgR在细胞恶性增殖和EMT中均发挥一定作用。综上所述,本论文首次证明潘氏细胞不仅参与先天性免疫,而且可能参与IgA介导的获得性免疫。本研究给潘氏细胞参与炎症性肠道疾病(IBD)提供了更多的证据,也给IBD的临床治疗研究提供了新思路。本研究还发现糖蛋白pIgR高表达具有明显的促MDCK细胞恶性转化的作用,为今后深入研究其中的作用机制、进而为肿瘤药物开发提供潜在靶点奠定了理论基础。更多还原

【Abstract】 Polymeric immunoglobulin receptor (pIgR) belongs to type I trans-membrane glycoprotein. It could specifically bind to polymeric IgA or polymeric IgM, and transport them into the secretion via the process of transcytosis. On basal to apical transport across epithelial cells, the pIgR extracellular domain is cleaved, releasing secreotry component (SC) in association with pIgA. pIgR can mediate the transcytosis of polymeric IgA, and thus ensure the multiple functions of sIgA, such as luminal prevention of pathogen attachment, intracellular neutralization of viruses, and antigen secretion from lamina propria. Thus, efficient secretion of pIgR is a prerequisite of polymeric IgA-mediated mucoscal protection.pIgR is always expressed in all types of secretory epithelia, with the highest levels observed in the intestine. In human small intestine, pIgR is expressed predominantly on immature cells within crypts and at lower levels on the villi cells. The localization of pIgR and IgA suggest that absorptive columnar cells, but not other cells, are primarily responsible for sIgA secretion into the intestine. Accordingly, it is generally accepted that it is the absorptive columnar cells but not other cells, including Paneth, goblet, or endocrine cells, that account for the synthesis of the pIgR, transportation of IgA, and secretion of sIgA in the small intestine. However, it was showed that positive staining of pIgR is specifically localized in Paneth cells of the rat small intestine, which suggested the possible relationship of Paneth cells and pIgR. Paneth cells are pyramidal epithelial cells found at the base of the crypts of Lieberkühn in the small intestines of many mammalian species. Paneth cells have been commonly recognized as important effectors of intestinal innate immunity. Resent researches showed that Paneth cells were involved in intestinal inflammation. Whether Paneth cells can synthesize pIgR and participate in IgA transportation or not is an important issue for the research work of IBD. It will be helpful for the improvement of clinical therapy of IBD.Firstly, the techniques of molecular biology was use to synthesize the specific digoxigenin-labeled cRNA probe of rat and human pIgR. Then, with the specific marker of Paneth cells- lysozyme, double-labeled fluorescent immunohistochemistry and double-labeled fluorescent in situ hybridization were used to determine RNA and protein expression in rat and human small intestine. Our results showed that pIgR mRNA and protein could be detected in rat and human Paneth cells, suggested that Paneth cells could synthesize pIgR. Another noteworthy finding is that, in humans, pIgR is expressed both in absorptive cells and Paneth cells, but in rats, it is found only in Paneth cells.Although pIgR plays an essential role in mucosal immunity, it was still reported to have varied functions in the body. Previous studies indicated that pIgR was over-expressed in some epithelial tumor tissues or cells, which suggested that there might be some relationship between pIgR and epithelial tumor. But the relationship is still unclear now. Whether or not pIgR will be used as a specific marker of tumor, or will be treated as a molecular target of anti-tumor medicines hasn’t been reported. It will be useful to investigate and illustrate the important role of pIgR in tumorgenesis.Firstly, MDCK- hpIgR cells which over-express human pIgR was successfully constructed with the technique of transfection. Then, the common characters of MDCK-hpIgR cells and MDCK-mock cells were detected and compared. It was shown that MDCK-hpIgR cells proliferate more quickly than MDCK-mock cells. Moreover, the shape of these cells changed significantly and they were digested easily from the flat. The cell proliferation was detected using flow cytometry, western blotting and other techniques. The results showed that MDCK-hpIgR cells have the characters of high proliferation index, increasing expression of PCNA and increasing phosphorylation of ERK1/2. Flat clone experiments confirmed that over-expression of human pIgR made MDCK cells have malignant proliferation. In mechanism of the shape change and the decrease of adhesion, the epithelial mesenchymal transition always plays an important role. EMT is the process of polar epithelia transforming into the cells capable of free movement. The main characters of EMT include the decrease of E-cadherin, the different cell framework, and the shape of mesenchymal cells. This transition allows the tumor cells shake off the cell-cell connection and to be more invadable. The results showed that the cell shape and framework formed by F-actin of MDCK-hpIgR were similar as mesenchymal cells. Over-expression of human pIgR inhibited the adhesion of MDCK cells on fibronectin, and promoted migration and invasion of them. In addition, MDCK-hpIgR cells have the molecular character of EMT- decrease expression of E-cadherin. The degree of cell malignant was tested by the soft agar clone experiment. It was shown that, in vivo, over-expression of human pIgR could promote the cells gain the ability of tumor formation. These results indicated that over-expression of human pIgR made MDCK cells have the characters of malignant proliferation and EMT, and finally be the malignant cells.In sum, Paneth cells may play a critical role in IgA-mediated acquired immunity in the gastrointestinal tract in addition to their well-recognized role in innate immunity. This will add to the accumulating evidences that Paneth cells are involved in intestinal inflammation, and, therefore, support a potential role in mucosal inflammation-driven pathologic disorders in humans. Furthermore, the new finding that over-expression of human pIgR made MDCK cells become into malignant cells, will be helpful for the future researches that promote pIgR to be a new molecular target of anti-cancer medicines.更多还原