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糖原合酶激酶3#beta#和腺瘤性结肠息肉病蛋白在气道上皮细胞机械损伤修复过程的时空分布

朱敏, 李建莎, 田丹, 马燕, 李娜萍, 吴人亮

华中科技大学同济医学院病理学系,卫生部呼吸系统疾病重点实验室.湖北,武汉 430030

摘要

为了探讨糖原合酶激酶3#beta#(glycogen synthase kinase 3#beta#,GSK3#beta#)和腺瘤性结肠息肉病(adenomatous polyposis coli, APC)蛋白在气道上皮细胞(airway epithelial cells,AECs)损伤和修复中的作用,作者采用机械划线损伤的方法建立体外气道上皮损伤修复模型,采用Western blot、免疫荧光双标共聚焦成像和免疫沉淀的方法观察损伤修复过程中APC蛋白和GSK3#beta#在AECs中表达及分布的动态变化。结果显示:(1)用Western blot方法观察到划线损伤0.5 h后即有GSK3#beta#磷酸化增强({sl P}<0.05),6 h达到高峰({sl P}<0.05),持续到12 h({sl P}<0.05),24 h开始下降,而GSK3#beta#总量大致保持一致。(2)在免疫荧光双标共聚焦成像实验中,划线损伤0 h组APC蛋白主要表达于胞浆,而划线损伤6 h后APC蛋白主要聚集于损伤前沿区的迁移活跃细胞。(3)免疫共沉淀的实验结果显示,划线损伤0 h时GSK3#beta#和APC蛋白能共同沉淀,但在划线损伤6 h之后,两者发生了分离。以上结果表明:划线损伤后AECs立即启动修复过程,此时GSK3#beta#的活性被抑制，促使APC游离出来；游离出来的APC则与微管正极结合，增加了微管的稳定性，从而调节细胞骨架运动，促进气道上皮的损伤修复过程。

关键词： 糖原合酶激酶3#beta#; 腺瘤性结肠息肉病蛋白; 气道上皮细胞; 损伤修复

Spatial--temporal distribution of glycogen synthase kinase 3#beta# and adenomatous polyposis coli protein are involved in the injury and repair of airway epithelial cells induced by scratching

Zhu Min, Li Jiansha, Tian Dan, Ma Yan, Li Naping, Wu Renliang

Department of Pathology,Tongji Medical College,Huazhong University of Science and Technology,and Key Laboratory of Pulmonary Disease of Ministry of Health of China.Wuhan 430030,Hubei

Abstract

To investigate the roles of glycogen synthase kinase 3#beta#(GSK3#beta#) and adenomatous polyposis coli (APC) protein in wound repair of airway epithelial cells (AECs), we established a wound model of airway epithelium in vitro. Then the following tests were undertaken: (1) Western blot was used to detect the levels of total GSK3#beta# and phosphorylated GSK3#beta# in human bronchial epithelial (16HBE) cells; (2) The localizations of APC protein was observed by using immunofluorescence technique; (3) Immunoprecipitation was used to investigate the relationship between APC protein and GSK3#beta# during the repair of 16HBE cells. The results showed: (1) The level of phosphorylated GSK3#beta# increased 0.5 h after scratching (P<0.05), reached a maximum at 6 h (P<0.05), and maintained until 12 h, while the total levels of GSK3#beta# remained constant; (2) Results of immunofluorescence study showed that the APC protein clustered with tubulin in the region of the migrating leading cells 6 h after scratching, which was dissimilar with the localization of the protein in the control group; (3) GSK3#beta# and APC protein were immunoprecipitated and analysed on SDS-PAGE. We found that GSK3#beta# and APC protein were precipitated, indicating that the two proteins existed in a complex. After scratching, significant dissociation occurred between the proteins. Taken together, we conclude that scratching caused a decrease in phosphorylation of GSK3#beta#, and that reduced phosphorylation of GSK3#beta# promoted the APC protein to bind to the plus ends of microtubules and stabilize the growing ends. These observations suggest that APC protein and GSK3#beta# may synergistically play an important role in the repair of airway epithelium.

Key words: glycogen synthase kinase 3#beta#;adenomatous polyposis coli protein;airway epithelial cells;injury and repair

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引用本文：

朱敏, 李建莎, 田丹, 马燕, 李娜萍, 吴人亮. 糖原合酶激酶3#beta#和腺瘤性结肠息肉病蛋白在气道上皮细胞机械损伤修复过程的时空分布[J]. 生理学报 2007; 59 (2): 197-203.

Zhu Min, Li Jiansha, Tian Dan, Ma Yan, Li Naping, Wu Renliang. Spatial--temporal distribution of glycogen synthase kinase 3#beta# and adenomatous polyposis coli protein are involved in the injury and repair of airway epithelial cells induced by scratching. Acta Physiol Sin 2007; 59 (2): 197-203 (in Chinese with English abstract).