本文選題：CTNNAL1 + 人支氣管上皮細(xì)胞�。� 參考：《中南大學(xué)》2012年碩士論文

【摘要】：國(guó)內(nèi)外及本實(shí)驗(yàn)室多項(xiàng)研究證實(shí),粘附分子在氣道炎癥及損傷修復(fù)中發(fā)揮重要作用。支氣管上皮細(xì)胞表達(dá)的粘附分子參與氣道上皮對(duì)應(yīng)激刺激的應(yīng)答,表現(xiàn)為：通過(guò)與基質(zhì)的結(jié)構(gòu)性粘附維持上皮的結(jié)構(gòu)完整,并參與細(xì)胞的抗氧化、增殖遷移和損傷修復(fù)功能；通過(guò)與白細(xì)胞的炎癥性粘附激活傳遞炎癥或應(yīng)激信號(hào)。 本課題組前期研究中發(fā)現(xiàn)連環(huán)素(catenin)家族中的CTNNAL1(catenin alpha-like1)基因在哮喘病人的外周血和支氣管粘膜中表達(dá)下調(diào),提示其與哮喘相關(guān),但是在在急性應(yīng)激的動(dòng)物模型及培養(yǎng)的人支氣管上皮細(xì)胞(human bronchial epithelial cells, HBECs)損傷邊緣高表達(dá),提示CTNNAL1可能參與氣道應(yīng)激反應(yīng),參與維持氣道上皮的完整,其表達(dá)下調(diào)可能導(dǎo)致上皮功能缺陷及完整性破壞。因此,為探討CTNNAL1在氣道上皮應(yīng)激反應(yīng)中的功能意義,本課題擬研究CTNNAL1對(duì)HBECs粘附功能的影響。 目的： 觀察不同表達(dá)水平的CTNNAL1對(duì)HBECs增殖和粘附功能以及部分粘附分子表達(dá)的影響,初步探討CTNNAL1在氣道上皮應(yīng)激反應(yīng)中的功能意義。 方法： (1)構(gòu)建CTNNAL1不同表達(dá)水平的重組質(zhì)粒,用G418篩選穩(wěn)定轉(zhuǎn)染重組質(zhì)粒的HBECs,建立穩(wěn)定細(xì)胞系。用實(shí)時(shí)熒光定量PCR和流式細(xì)胞術(shù)檢測(cè)穩(wěn)定細(xì)胞系中CTNNAL1的表達(dá)。 (2)將CTNNAL1不同表達(dá)水平的HBECs系用細(xì)胞核熒光染料碘化丙啶(PI)進(jìn)行染色,采用流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期。 (3)MTT法檢測(cè)CTNNAL1不同表達(dá)水平的HBECs系與鼠尾膠原的基質(zhì)性粘附能力。 (4)用FITC標(biāo)記人WBC的CD45分子,與CTNNAL1不同表達(dá)水平的HBECs系粘附,用流式細(xì)胞術(shù)檢測(cè)粘附的WBC的數(shù)量,反映各種HBECs系的炎癥性粘附能力。 (5)采用RT-PCR觀察CTNNAL1不同表達(dá)水平的HBECs系中上皮鈣粘附素(E-cadherin)和細(xì)胞間粘附分子1(ICAM-1)的表達(dá)變化。 (6)ELISA法檢測(cè)各組HBECs系中IL-8的分泌量。結(jié)果： (1)成功構(gòu)建pcDNA3.1(-)-cyc-his/CTNNAL1高表達(dá)質(zhì)粒和pGCsilencerU6/Neo/RFP/CTNNAL1-RNAi沉默質(zhì)粒,并以此分別轉(zhuǎn)染HBECs,成功構(gòu)建穩(wěn)定轉(zhuǎn)染細(xì)胞系,與空載體相比,pcDNA3.1(-)-cyc-his/CTNNAL1重組質(zhì)粒的HBECs系中CTNNAL1mRNA水平及蛋白水平均顯著升高；與陰性對(duì)照質(zhì)粒的HBECs系相比,CTNNAL1-RNAi沉默質(zhì)粒的HBECs系中CTNNAL1mRNA水平及蛋白水平均顯著降低。 (2) CTNNAL1高表達(dá)HBECs系增殖活躍,表現(xiàn)為(G2+S)/G1細(xì)胞百分比增加199.84%。 (3) CTNNAL1高表達(dá)增強(qiáng)了HBECs對(duì)基質(zhì)的粘附能力,CTNNAL1沉默后降低了HBECs的基質(zhì)粘附率,且在30min,120min有統(tǒng)計(jì)學(xué)差異。 (4) CTNNAL1高表達(dá)使HBECs與人總WBC的炎性粘附細(xì)胞百分比降低了35.3%。 (5) CTNNAL1高表達(dá)增加了HBECs中粘附分子E-cadherin mRNA水平的表達(dá)(56.5%),降低了ICAM-1的表達(dá)(90.4%)；CTNNAL1沉默后降低了E-cadherin mRNA水平的表達(dá)(59.9%) (6) CTNNAL1高表達(dá)使HBECs分泌的IL-8減少了53.87%。 結(jié)論： CTNNAL1可顯著促進(jìn)HBECs增殖,通過(guò)促進(jìn)E-cadherin的表達(dá)提高HBECs對(duì)基質(zhì)的粘附能力,通過(guò)降低ICAM-1的表達(dá)和減少IL-8的分泌減弱HBECs與人WBC的炎癥性粘附能力。
[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錛，

本文編號(hào)：1733657