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APC蛋白、GSK3#beta#在吸烟小鼠气道上皮损伤修复中的动态变化

刘明阁, 李娜萍, 吴人亮, 马燕, 洪渊智, 田丹, 朱敏

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

摘要

为了探讨结肠腺瘤性息肉病(adenomatous polyposis coli,APC)蛋白、糖原合成酶激酶3#beta#(glycogen synthase kinase 3#beta#,GSK3#beta#)在吸烟致气道上皮细胞(airway epithelial cell,AEC)损伤修复中的作用,该实验建立了吸烟导致AEC损伤修复的小鼠模型,采用HE染色、免疫组织化学染色、免疫荧光共聚焦成像和Western blot方法,观察损伤修复过程中APC蛋白、GSK3#beta#在AEC中表达及分布的动态变化。结果显示：(1)随着吸烟时间延长,AEC形态学上呈现损伤(1、4周)、修复(8周)、再损伤(12周)的变化。(2)免疫组化染色显示：AEC中APC蛋白表达在吸烟1周时较对照组明显增强,4周时较对照组明显减弱,8、12周时均较4周时增强但与对照组无差异；对照组GSK3#beta#表达较强,吸烟组表达较对照组均减弱。Western blot检测小鼠肺组织中APC蛋白、GSK3#beta#的表达变化与免疫组化结果一致,磷酸化GSK3#beta#(p--GSK3#beta#)在吸烟组的表达较对照组均有不同程度增高,尤以吸烟1周时明显。(3)荧光共聚焦成像显示：对照组APC蛋白在AEC胞质内均匀分布，吸烟1、8周时定位发生改变，呈簇状聚集于AEC腔面和侧面质膜下；GSK3#beta#在对照组和吸烟组AEC胞质内均匀分布，无定位改变。由上述结果可见，吸烟致小鼠AEC损伤修复过程中APC蛋白表达量及在胞质内分布呈动态变化，同时伴有GSK3#beta#表达量下调和磷酸化水平增高，提示二者可能通过参与修复中细胞迁移、分裂增殖等活动，在气道上皮损伤修复过程中发挥重要作用。

关键词： 结肠腺瘤性息肉病蛋白; 糖原合成酶激酶3#beta#; 吸烟; 气道上皮; 损伤修复

Dynamic changes of adenomatous polyposis coli protein and glycogen synthase kinase 3#beta# in the repair of the injured airway epithelial cells in smoking mice

Liu Mingge, Li Naping, Wu Renliang, Ma Yan, Hong Yuanzhi, Tian Dan, Zhu Min

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

Abstract

To investigate the roles of adenomatous polyposis coli (APC) protein and glycogen synthase kinase3#beta# (GSK3#beta#) of smoking murine model in the repair of the injured airway epithelial cells (AECs) in different stages, 30 male Kun-Ming mice were randomly divided into two groups, the control group and the smoking group. There were 24 mice in smoking group, 6 animals were separately killed at the end of the 1st, 4th, 8th and 12th week after smoking. Then the following tests were undertaken: (1) HE staining of lung section to observe the morphological changes of the bronchi in the smoking mice. (2) Immunohistochemical staining of APC protein and GSK3#beta# in the AEC. (3) Western blotting was used to detect the levels of APC protein, GSK3#beta# and phosphoric GSK3#beta# (p-GSK3#beta#) in pulmonary tissue. (4) Observing the localizations of APC protein and GSK3#beta# in the AEC by immunofluorescence technique. The results showed: (1) Airway epithelial cells showed predominant injury (1-, 4-week), repair (8-week) and reinjury (12-week). The experimental results indicated that the model of smoking mice was duplicated successfully. (2) Immunohistochemical results showed that the expression of APC protein in the AEC increased after 1-week smoking (0.458±0.062 vs 0.399±0.060, P<0.05 vs control), but was significantly decreased at the end of 4th week (0.339±0.056, P<0.01 vs control) and increased at the end of the 8th and 12th week (0.387±0.041, 0.378±0.037, P<0.05 vs 4-week). The expressions of GSK3#beta# in the AEC of smoking mice obviously decreased (P<0.01 or P<0.05 vs control). (3) Western blotting showed that the expressions of APC protein and GSK3#beta# in lung tissue were consistent with the results of immunohistochemistry; the levels of P-GSK3#beta# in all smoking model were higher than that in control. (4) The results of immunofluorescence showed that APC protein was localized mainly near the regions of epithelial cell membrane at the end of 1st and 8th week after smoking, which are dissimilar with the localization in control, and this change was not seen in the locations of GSK3#beta#. Taken together, these results demonstrate that the expressions and localizations of APC protein, GSK3#beta# and the activity of GSK3#beta# are dynamically changed in the AEC with experimental smoking injury at different phases, suggesting that APC protein and GSK3#beta# may be involved in the regulation of migration and proliferation of airway epithelial cell, and play an important role in the process of repair of airway epithelium injury.

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

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引用本文：

刘明阁, 李娜萍, 吴人亮, 马燕, 洪渊智, 田丹, 朱敏. APC蛋白、GSK3#beta#在吸烟小鼠气道上皮损伤修复中的动态变化[J]. 生理学报 2006; 58 (3): .

Liu Mingge, Li Naping, Wu Renliang, Ma Yan, Hong Yuanzhi, Tian Dan, Zhu Min. Dynamic changes of adenomatous polyposis coli protein and glycogen synthase kinase 3#beta# in the repair of the injured airway epithelial cells in smoking mice. Acta Physiol Sin 2006; 58 (3): (in Chinese with English abstract).