【作者】 洪渊智；

【导师】 李娜萍；

【作者基本信息】 华中科技大学 ， 病理和病理生理学， 2006， 硕士

【摘要】 急性肺损伤(acute lung injury ,ALI)是伴有严重呼吸障碍的一个复杂多变的病理过程,损伤导致肺内广泛的炎症反应,严重危及气道上皮细胞的完整性和功能的正常。β-连环素(β-catenin,β-cat)是一种具有双重功能的蛋白,它以两种形式存在,一种为结合型,即与跨膜的上皮钙粘附素(E-cadherin,E-cad)结合形成E-cad/β-cat复合体,是维持上皮极性和完整性的重要的细胞粘附结构;另一种为游离型,参与Wnt信号传导途径,调控细胞增生和组织修复等过程。本研究通过观察β-cat在细胞膜以及细胞质内的表达变化,同时检测导致β-cat在胞质内聚积的相关激酶的表达变化,旨在阐明由脂多糖(lipopolysaccharide,LPS)诱发的小鼠ALI时,β-cat在气道上皮损伤修复过程中可能发挥的作用及有关的调节机制。方法在建立LPS气管注射致小鼠ALI模型基础上,采用免疫组织化学及免疫荧光染色技术,应用光镜、共聚焦显微镜检测气道上皮细胞膜上β-cat及细胞质内糖原合成酶激酶3-β(GSK3-β)在的表达及共定位关系;应用Western Blotting技术检测肺组织内蛋白激酶C(PKC)、GSK3-β、磷酸化GSK3-β(P-GSK3-β)以及β-cat的蛋白含量。结果免疫组化与免疫荧光方法显示与正常组小鼠相比,LPS致ALI的小鼠气道上皮中,胞膜上β-cat及胞质内GSK3-β的表达均明显下降,胞质内β-cat表达增强(P < 0.05); Western Blotting结果显示实验组肺组织内PKC、P-GSK3-β及β-cat蛋白含量均明显升高(P < 0.05), GSK3-β蛋白含量显著下降(P < 0.05)。结论1.由LPS所致的小鼠ALI时可引起气道上皮细胞膜上行使连接功能的β-cat表达下降以及胞质内游离β-cat表达增高;2.由LPS所致的小鼠ALI时伴有PKC表达增高,并促进GSK3-β磷酸化;3.胞质内游离型β-cat增多与GSK3-β磷酸化失活调节有关。更多还原

【Abstract】 Acute lung injury (ALI) is a complicated pathologic process with serious respiratory disturbance, and it can lead to extensive inflammatory reaction in the lung and loss of the integrity and function of the airway epithelial cell.β-catenin (β-cat) is a kind of protein with double functions, existing by two forms: One is to combine with E-cadherin, and the complex they formed is the important cell adherent structure for maintaining the polarity and integrity of epithelium; Another is free in the cytoplasm, taking part in Wnt signal pathway that regulates the key factors of cell proliferation and tissue repair. The present study was undertaken to observe the alteration ofβ-cat both on the cell membrane and in the cytoplasm, meanwhile, to detect the change of the kinases that causedβ-cat accumulation in the cytoplasm. So the objective of this article was to illustrate the function ofβ-cat and it’s correlated regulatory mechanism during the injury and repair process of the airway epithelial cell in the ALI mice model induced by lipopolysaccharide(LPS).Methods Establishing the mice model of acute lung injury (ALI) after treated with LPS, both S-P immunohistochemical method and immunofluorescence were used to examine the expression ofβ-cat on the membrane as well as GSK3-βin the cytoplasm of the airway epithelial cell by light microscope and confocal microscopy; Western Blotting was used to examine the expression of proteins of PKC、GSK3-β、P-GSK3-βandβ-cat in the cytoplasm. Results The results of S-P immunohistochemical method and immunofluorescence showed that the expression ofβ-cat on the membrane and GSK3-βin the cytoplasm were remarkably decreased, butβ-cat in the cytoplasm was increased after ALI compared with control group (both P < 0.05). The results of Western Blotting showed that the expression of PKC、P-GSK3-βandβ-cat in the cytoplasm of the experimental mice’s airway epithelial cell were all significantly increased in contrast to control group (all P < 0.05); meanwhile, the expression of GSK3-βwas obviously lower than that in control group (P < 0.05).Conclusions 1. The level ofβ-cat on the membrane of the airway epithelial cell for cell adhesion was decreased after ALI, on the contrary, the freeβ-cat in the cytoplasm was increased;2. The expression of PKC was increased after ALI induced by LPS, and thus promoted the the GSK3-βto be phosphorylated;3. The increase of freeβ-cat in the cytoplasm was related with the phosphorylation of GSK3-β.更多还原