本文選題：支氣管哮喘 + 氣道重塑�。� 參考：《南方醫(yī)科大學》2012年博士論文

【摘要】：研究目的：支氣管哮喘是由多種因素導致的慢性氣道炎癥,雖然現(xiàn)有的藥物能夠在相當程度上控制哮喘的癥狀,但卻不能有效抑制哮喘的病情進展。氣道上皮細胞(AEC)被認為在哮喘的病理生理中處于中心地位,氣道上皮損傷、脫落及上皮下纖維化是哮喘氣道重塑的重要病理生理特征。而血管內(nèi)皮生長因子(VEGF)是參與哮喘發(fā)病的十分重要的細胞因子,支氣管上皮細胞(NHBE)是VEGF的主要來源之一,不同的刺激因素作用下,NHBE對VEGF表達調(diào)控狀況尚不甚清楚。哮喘患者氣道壁網(wǎng)狀基底膜中肌纖維母細胞(myofibroblast)數(shù)量是增多的,其數(shù)量與基底膜的增厚相關(guān)。肌纖維母細胞有很強的分泌膠原和胞外基質(zhì)的能力,其因特征性表達a-平滑肌肌動蛋白(a-SMA)從而具有對環(huán)境刺激產(chǎn)生收縮反應(yīng)的能力,但其細胞來源尚不清楚。屋塵螨(HDM)是誘發(fā)和加重哮喘一個重要的致敏因素。本課題利用HDM刺激原代培養(yǎng)的NHBE作為研究對象,與原代培養(yǎng)的人的氣道平滑肌細胞共培養(yǎng),探討HDM對NHBE分泌VEGF的影響及對氣道上皮細胞單層通透性以及上皮下平滑肌細胞遷移的影響,試圖尋找治療哮喘的新的藥物靶點。研究內(nèi)容和方法：1. 人的支氣管上皮細胞的原代培養(yǎng)；2. 人的氣道平滑肌細胞的原代培養(yǎng)；3. 通過ELISA、熒光定量PCR檢測屋塵螨刺激人的支氣管上皮細胞后對VEGF表達的影響,通過檢測細胞的跨膜電阻了解屋塵螨對人的支氣管上皮細胞通透性的影響。4. 采納共培養(yǎng)裝置,熒光顯微鏡下觀察屋塵螨刺激NHBE 對ASMC的趨化作用,酶聯(lián)免疫吸附法檢測培養(yǎng)上清中的細胞因子的水平。5. 統(tǒng)計學方法：采用SPSS13.0統(tǒng)計軟件分析,計量資料用均數(shù)±標準差(X±S)表示,方差齊時,總體均數(shù)的比較采用單向方差分析(one-way ANOVA),各組間多重比較采用LSD法,方差不齊時總體均數(shù)的比較采用Welkch法,各組間多重比較采用Tamhane's法檢驗。以P0.05具有統(tǒng)計學意義。結(jié)果：1. 成功培養(yǎng)了可傳代達5代的人支氣管上皮細胞。2. 成功培養(yǎng)了可傳代達8代的人氣道平滑肌細胞。3. 不同屋塵螨濃度對NHBE活力的影響：屋塵螨濃度為100 U/ml、200 U/ml、400 U/ml、800 U/ml對 NHBE活力均沒有顯著影響(P0.05),濃度為1000 U/ml時顯著降低細胞活力(P0.001)。4. 屋塵螨可以誘導人的支氣管上皮細胞VEGF的表達,且呈明顯的時間依賴性和濃度依賴性。在濃度為200U/ml刺激24小時作用最明顯,同對照組相比具有顯著的統(tǒng)計學意義(P0.05)。同對照組相比,200U/ml塵螨刺激24小時后可明顯的增加支氣管上皮細胞的通透性,具有顯著的統(tǒng)計學意義(P0.05),VEGF的單克隆抗體(貝伐單抗)可以明顯阻斷這一效應(yīng)(P0.05)5. 屋塵螨刺激支氣管上皮細胞24小時后增加了對氣道平滑肌細胞遷移作用,同對照組相比具有顯著的統(tǒng)計學意義(P0.05),貝伐單抗可明顯阻斷這一效應(yīng)(P0.05)。同步檢測transwell下室支氣管上皮細胞上清中VEGF濃度,屋塵螨刺激24小時VEGF明顯升高,同對照組相比具有顯著的統(tǒng)計學意義(P0.05),加用貝伐單抗后明顯下降(P0.05)。損傷的支氣管上皮細胞上清中檢測不到TNF-α、HMGB1相應(yīng)的增高。結(jié)論：1.纖支鏡直視下支氣管粘膜活檢+組織貼塊法是一種簡便、易行、可重復的人原代支氣管上皮細胞的培養(yǎng)方法。2.組織貼塊法是一種簡便、易行的人原代氣道平滑肌細胞的培養(yǎng)方法。3. HD M通過上調(diào)VEGF可以直接增加NHBE的通透性。4. HDM通過上調(diào)NHBE表達VEGF增加了對氣道平滑肌細胞的遷移作用。
[Abstract]:Objective : To study the effect of HDM on the expression of VEGF in asthmatic patients , and to explore the effect of HDM on the expression of VEGF in asthmatic patients . Primary culture of human bronchial epithelial cells ;
2 . Primary culture of human airway smooth muscle cells ;
3 . The influence of house dust mites on the expression of VEGF was detected by ELISA and fluorescence quantitative PCR . The effect of house dust mites on the permeability of human bronchial epithelial cells was investigated by detecting the transmembrane resistance of cells . A co - culture device was adopted to observe the chemoattractant action of NHBE on ASMC under fluorescence microscope , and the level of cytokines in culture supernatant was determined by enzyme linked immunosorbent assay . Statistical methods : SPSS 13.0 was used to analyze the statistical software . The mean per mean difference ( X 鹵 S ) of the measurement data was expressed by mean 鹵 standard deviation ( X 鹵 S ) . One - way ANOVA was used for the comparison of the total mean number between the groups . The comparison between the groups was performed by using the method of LSD . The comparison between the groups was examined by the Tamhane ' s method . The results were as follows : 1 . Human bronchial epithelial cells were successfully cultured for up to 5 generations . Eight generations of human airway smooth muscle cells were successfully cultured . The concentration of house dust mites was 100 U / ml , 200 U / ml , 400 U / ml and 800 U / ml had no significant effect on the activity of NHBE ( P0.05 ) . In comparison with the control group , the effect of 200 U / ml dust mites on the permeability of bronchial epithelial cells was significantly increased ( P0.05 ) . Conclusion : 1 . The tissue labeling method is a simple , easy and reproducible method for culturing human primary bronchial epithelial cells . Conclusion : 1 . The tissue labeling method is a simple , easy and feasible method for culturing human primary bronchial epithelial cells . HD M increases the permeability of NHBE directly by up - regulation of VEGF . HDM increased the migration of vascular smooth muscle cells by up - regulating the expression of VEGF in NHBE .
【學位授予單位】：南方醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R562.25

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相關(guān)期刊論文 前1條

1 方正旭 ,王伶,劉東;關(guān)于動脈平滑肌細胞培養(yǎng)的幾個問題[J];江西醫(yī)學院學報;2002年05期

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