| 题名 | 细胞连接分子和粘蛋白在胃肠上皮的表达及 幽门螺杆菌感染对其的影响 |
| 作者 | 张虹雨 |
| 学位类别 | 博士 |
| 答辩日期 | 2012-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 曹毅 |
| 关键词 | 细胞连接 粘蛋白 胃肠上皮 肿瘤 幽门螺杆菌 |
| 其他题名 | Expressions of Adhesion Molecules and Mucins in Gastrointestinal Epithelium, and Their Alterations Induced by Helicobacter pylori Infection |
| 学位专业 | 细胞生物学 |
| 中文摘要 | 胃肠粘膜屏障保护着消化道免于各种酶类、细菌和毒素的损伤，维护胃肠上皮内环境的稳定。胃肠上皮的细胞连接和上皮细胞分泌的粘蛋白构成了胃肠粘膜屏障的主要成分。上皮细胞间连接主要有紧密连接（tight junctions）、附着连接（adherens junctions）和桥粒（desmosomes）。细胞连接分子的异常与包括肿瘤在内的多种疾病的发生密切相关。粘蛋白的异常表达是多种癌症和炎性疾病的显著特性之一。在本研究中，我们系统检测了细胞连接分子和粘蛋白在正常人和恶性胃肠上皮组织，以及猕猴胃肠上皮的表达。在正常人胃肠上皮，zonula occludens-1 (ZO-1)、 α-catenin、β-catenin、γ-catenin 和 desmoglein-2 (DSG2)定位于细胞膜，而symplekin定位于细胞核。ZO-1、三种连接蛋白和DSG2在胃肠肿瘤组织显示表达降低或者异位表达，symplekin在部分胃肠肿瘤样本表达缺失。ZO-1和symplekin的免疫组织化学染色结果与培养细胞的免疫细胞化学染色、蛋白质免疫印迹分析结果相一致。未经高碘酸盐氧化作用，MUC1在正常人胃肠上皮细胞呈阴性反应，而在胃肠肿瘤细胞显示强阳性染色。MUC2在正常胃肠上皮细胞阳性染色，在胃肠肿瘤细胞显示过度表达。这些结果表明上皮细胞连接分子和粘蛋白的表达改变与胃肠细胞的恶性转化相关。猕猴胃肠上皮细胞表达ZO-1、symplekin、α-catenin、β-catenin、γ-catenin、DSG2和MUC2。经高碘酸盐氧化作用，MUC1在人与猕猴胃肠上皮细胞显示阳性表达。其结果表明猕猴胃肠上皮细胞与人类相似，也表达上述连接分子和粘蛋白。这提示对于细胞连接分子和粘蛋白的研究而言，猕猴是一种合适的实验动物模型。 胃粘膜屏障的破坏可以诱发胃炎、胃溃疡和胃癌等疾病，而这些疾病与幽门螺杆菌Helicobacter pylori (H. pylori)感染密切相关，H. pylori感染是削弱胃粘膜屏障的最重要因素之一。H. pylori 定植于胃上皮细胞顶面，可能经多种途径影响上皮细胞的极性、通透性与完整性，从而损伤粘膜屏障，继发炎症反应，甚至诱导肿瘤的发生。在本研究中，我们系统检测了H. pylori感染对细胞连接分子和粘蛋白MUC1表达的影响。在H. pylori感染的细胞，ZO-1在细胞膜的染色明显减弱，伴有紧密连接的破坏，免疫细胞化学染色结果与免疫组织化学染色、蛋白质免疫印迹分析和RT-Q-PCR的结果相一致。Symplekin、E-cadherin、β-catenin和γ-catenin在H. pylori感染细胞的胞膜染色明显减弱，伴有细胞连接的破坏，免疫细胞化学染色结果与蛋白质免疫印迹分析的结果相一致。α-catenin和DSG2在H. pylori感染细胞的定位和定量表达，及其mRNA水平无明显改变。免疫细胞化学染色结果显示MUC1在H. pylori感染细胞的表达明显减弱。这些结果表明H. pylori感染能够影响紧密连接和附着连接分子，以及粘蛋白MUC1的表达，从而破坏胃肠粘膜屏障。 紧密连接是维持上皮细胞间通透性，防止细菌和毒素等通过细胞间隙侵入组织器官的主要屏障。在本研究中，我们应用Boyden-chamber细胞通透性实验检测了H. pylori感染对胃肠上皮细胞紧密连接功能的影响，结果显示H. pylori感染引起细胞通透性明显升高。其结果表明H. pylori感染可以破坏胃肠上皮紧密连接的功能。这为进一步认识H. pylori感染的临床意义，以及研究H. pylori感染与上皮细胞连接的关系提供了有价值的基础资料。 关键词：细胞连接，粘蛋白，胃肠上皮，肿瘤，幽门螺杆菌 |
| 英文摘要 | Epithelial junctions and mucins play key roles in the gastrointestinal mucosal barrier, and their alterations are associated with numerous diseases, including carcinomas. Epithelial junctions are mainly composed of tight junctions, adherens junctions and desmosomes. Mucins are classified into membrane-associated (MUC1) and membrane-secreted (MUC2) types. The systematic expression of adhesion molecules and mucins in normal and malignant human gastrointestinal cells was investigated in this study. In normal human gastrointestinal cells, zonula occludens-1 (ZO-1), α-catenin, β-catenin, γ-catenin and desmoglein-2 (DSG2) were located in the cytoplasmic membranes, whereas symplekin stained in the nuclei. ZO-1, the three catenins, and DSG2 were observed in the gastric and colorectal carcinomas with reduced and heterogeneous expression and with abnormal distribution. Symplekin was detected in the nuclei of tumor cells in most tumors but not observed in some others. The immunohistochemical results for ZO-1 and symplekin on the tissues were consistent with the data for the cultured cells obtained by immunocytochemical staining and Western blot analysis. MUC1 was not stained in the normal gastrointestinal cells without periodate oxidation, but it was strongly labeled in the malignant gastrointestinal cells. MUC2 was detected in the normal and malignant gastrointestinal cells without the periodate treatment. These findings indicate that alterations in the expression of the epithelial junctions and mucins are associated with the malignant transformation of gastrointestinal cells. In addition, the gastrointestinal epithelial cells of rhesus macaques expressed these adhesion molecules and mucins, as did the human cells, suggesting that the rhesus monkey is a suitable experimental animal model for research on adhesion molecules and mucins. The injury of gastric mucosal barrier induces gastritis, peptic ulcer and gastric cancer, which have close association with Helicobacter pylori (H. pylori). H. pylori infection belongs to the most important factors weaken the gastric mucosal barrier. Expressions of adhesion molecules and MUC1 in gastrointestinal epithelial cells infected with H. pylori were detected in the present study. Membranous distribution of ZO-1 was decreased in H. pylori infected cells, along with the destruction of tight junctions, as shown in immunofluorescence. The immunohistochemical staining of ZO-1 weakened in gastric epithelial tissue with H. pylori infection. And the reduction of ZO-1 expression in infected cells was confirmed by Western blot analysis and RT-Q-PCR. The decreased membranous staining of symplekin, E-cadherin, β-catenin and γ-catenin were detected in H. pylori infected cells, along with the destruction of cell-cell junctions. The immunocytochemistry results were consistent with those of Western blot analysis. Expressions of α-catenin and DSG2 in H. pylori infected cells resembled the control. MUC1 showed weak immunocytochemical staining in H. pylori infected cells. These data indicate that H. pylori infection could induce the alteration of adhesion molecules and MUC1. Tight junctions maintain epithelial intercellular permeability. Cell monolayer integrity assessment was applied to detect the dysfunction of tight junctions in this study. The permeability of NCI-N87, Caco-2 and HT-29 cells increased significantly after H. pylori infection for 6 h. The result suggests that H. pylori infection could destroy the function of gastrointestinal epithelial tight junctions. Keywords: adhesion molecule, mucin, gastrointestinal epithelium, carcinoma, Helicobacter pylori |
| 语种 | 中文 |
| 公开日期 | 2012-07-03 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6993]  |
| 专题 | 昆明动物研究所_分子病理学 |
| 推荐引用方式 GB/T 7714 | 张虹雨. 细胞连接分子和粘蛋白在胃肠上皮的表达及 幽门螺杆菌感染对其的影响[D]. 北京. 中国科学院研究生院. 2012. |
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