CTNNAL1对人支气管上皮细胞粘附功能的影响

发布时间：2018-04-11 00:32

  本文选题：CTNNAL1 + 人支气管上皮细胞　； 参考：《中南大学》2012年硕士论文

【摘要】：国内外及本实验室多项研究证实,粘附分子在气道炎症及损伤修复中发挥重要作用。支气管上皮细胞表达的粘附分子参与气道上皮对应激刺激的应答,表现为：通过与基质的结构性粘附维持上皮的结构完整,并参与细胞的抗氧化、增殖迁移和损伤修复功能；通过与白细胞的炎症性粘附激活传递炎症或应激信号。 本课题组前期研究中发现连环素(catenin)家族中的CTNNAL1(catenin alpha-like1)基因在哮喘病人的外周血和支气管粘膜中表达下调,提示其与哮喘相关,但是在在急性应激的动物模型及培养的人支气管上皮细胞(human bronchial epithelial cells, HBECs)损伤边缘高表达,提示CTNNAL1可能参与气道应激反应,参与维持气道上皮的完整,其表达下调可能导致上皮功能缺陷及完整性破坏。因此,为探讨CTNNAL1在气道上皮应激反应中的功能意义,本课题拟研究CTNNAL1对HBECs粘附功能的影响。 目的： 观察不同表达水平的CTNNAL1对HBECs增殖和粘附功能以及部分粘附分子表达的影响,初步探讨CTNNAL1在气道上皮应激反应中的功能意义。 方法： (1)构建CTNNAL1不同表达水平的重组质粒,用G418筛选稳定转染重组质粒的HBECs,建立稳定细胞系。用实时荧光定量PCR和流式细胞术检测稳定细胞系中CTNNAL1的表达。 (2)将CTNNAL1不同表达水平的HBECs系用细胞核荧光染料碘化丙啶(PI)进行染色,采用流式细胞术检测细胞周期。 (3)MTT法检测CTNNAL1不同表达水平的HBECs系与鼠尾胶原的基质性粘附能力。 (4)用FITC标记人WBC的CD45分子,与CTNNAL1不同表达水平的HBECs系粘附,用流式细胞术检测粘附的WBC的数量,反映各种HBECs系的炎症性粘附能力。 (5)采用RT-PCR观察CTNNAL1不同表达水平的HBECs系中上皮钙粘附素(E-cadherin)和细胞间粘附分子1(ICAM-1)的表达变化。 (6)ELISA法检测各组HBECs系中IL-8的分泌量。结果： (1)成功构建pcDNA3.1(-)-cyc-his/CTNNAL1高表达质粒和pGCsilencerU6/Neo/RFP/CTNNAL1-RNAi沉默质粒,并以此分别转染HBECs,成功构建稳定转染细胞系,与空载体相比,pcDNA3.1(-)-cyc-his/CTNNAL1重组质粒的HBECs系中CTNNAL1mRNA水平及蛋白水平均显著升高；与阴性对照质粒的HBECs系相比,CTNNAL1-RNAi沉默质粒的HBECs系中CTNNAL1mRNA水平及蛋白水平均显著降低。 (2) CTNNAL1高表达HBECs系增殖活跃,表现为(G2+S)/G1细胞百分比增加199.84%。 (3) CTNNAL1高表达增强了HBECs对基质的粘附能力,CTNNAL1沉默后降低了HBECs的基质粘附率,且在30min,120min有统计学差异。 (4) CTNNAL1高表达使HBECs与人总WBC的炎性粘附细胞百分比降低了35.3%。 (5) CTNNAL1高表达增加了HBECs中粘附分子E-cadherin mRNA水平的表达(56.5%),降低了ICAM-1的表达(90.4%)；CTNNAL1沉默后降低了E-cadherin mRNA水平的表达(59.9%) (6) CTNNAL1高表达使HBECs分泌的IL-8减少了53.87%。 结论： CTNNAL1可显著促进HBECs增殖,通过促进E-cadherin的表达提高HBECs对基质的粘附能力,通过降低ICAM-1的表达和减少IL-8的分泌减弱HBECs与人WBC的炎症性粘附能力。
[Abstract]:Study at home and abroad and the number of laboratory confirmed that adhesion molecules play an important role in airway inflammation and injury repair. The expression of adhesion molecules in bronchial epithelial cells in airway epithelium on the stress response, as by the structural adhesion with matrix to maintain the structural integrity of the epithelium, and involved in cell proliferation and migration of antioxidant, damage repair function; through inflammatory adhesion and leukocyte activation transfer inflammation or stress signals.
Found in the previous study of - Catenin (catenin) family CTNNAL1 (catenin alpha-like1) by gene expression in peripheral blood of patients with asthma and bronchial mucosa, suggesting that its associated with asthma, but in the animal model of acute stress and cultured human bronchial epithelial cells (human bronchial epithelial cells. HBECs) high expression of injury margin, suggesting that CTNNAL1 may be involved in stress response of the airway, involved in the maintenance of airway epithelium integrity, its expression may cause epithelial function defects and damage the integrity. Therefore, the functional significance of CTNNAL1 in airway epithelium in response to stress, this paper intends to study the effect of CTNNAL1 on HBECs adhesion function.
Objective:
Objective To observe the effects of different expression levels of CTNNAL1 on the proliferation and adhesion function of HBECs and the expression of some adhesion molecules, and to preliminarily explore the functional significance of CTNNAL1 in airway epithelial stress response.
Method锛，

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