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

【摘要】：研究目的：支氣管哮喘是由多種因素導(dǎo)致的慢性氣道炎癥,雖然現(xiàn)有的藥物能夠在相當(dāng)程度上控制哮喘的癥狀,但卻不能有效抑制哮喘的病情進(jìn)展。氣道上皮細(xì)胞(AEC)被認(rèn)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(zhǎng)因子(VEGF)是參與哮喘發(fā)病的十分重要的細(xì)胞因子,支氣管上皮細(xì)胞(NHBE)是VEGF的主要來(lái)源之一,不同的刺激因素作用下,NHBE對(duì)VEGF表達(dá)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(xì)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(xì)胞有很強(qiáng)的分泌膠原和胞外基質(zhì)的能力,其因特征性表達(dá)a-平滑肌肌動(dòng)蛋白(a-SMA)從而具有對(duì)環(huán)境刺激產(chǎn)生收縮反應(yīng)的能力,但其細(xì)胞來(lái)源尚不清楚。屋塵螨(HDM)是誘發(fā)和加重哮喘一個(gè)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(duì)象,與原代培養(yǎng)的人的氣道平滑肌細(xì)胞共培養(yǎng),探討HDM對(duì)NHBE分泌VEGF的影響及對(duì)氣道上皮細(xì)胞單層通透性以及上皮下平滑肌細(xì)胞遷移的影響,試圖尋找治療哮喘的新的藥物靶點(diǎn)。研究?jī)?nèi)容和方法：1. 人的支氣管上皮細(xì)胞的原代培養(yǎng)；2. 人的氣道平滑肌細(xì)胞的原代培養(yǎng)；3. 通過(guò)ELISA、熒光定量PCR檢測(cè)屋塵螨刺激人的支氣管上皮細(xì)胞后對(duì)VEGF表達(dá)的影響,通過(guò)檢測(cè)細(xì)胞的跨膜電阻了解屋塵螨對(duì)人的支氣管上皮細(xì)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(duì)ASMC的趨化作用,酶聯(lián)免疫吸附法檢測(cè)培養(yǎng)上清中的細(xì)胞因子的水平。5. 統(tǒng)計(jì)學(xué)方法：采用SPSS13.0統(tǒng)計(jì)軟件分析,計(jì)量資料用均數(shù)±標(biāo)準(zhǔn)差(X±S)表示,方差齊時(shí),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(shí)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(yàn)。以P0.05具有統(tǒng)計(jì)學(xué)意義。結(jié)果：1. 成功培養(yǎng)了可傳代達(dá)5代的人支氣管上皮細(xì)胞。2. 成功培養(yǎng)了可傳代達(dá)8代的人氣道平滑肌細(xì)胞。3. 不同屋塵螨濃度對(duì)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(duì) NHBE活力均沒(méi)有顯著影響(P0.05),濃度為1000 U/ml時(shí)顯著降低細(xì)胞活力(P0.001)。4. 屋塵螨可以誘導(dǎo)人的支氣管上皮細(xì)胞VEGF的表達(dá),且呈明顯的時(shí)間依賴(lài)性和濃度依賴(lài)性。在濃度為200U/ml刺激24小時(shí)作用最明顯,同對(duì)照組相比具有顯著的統(tǒng)計(jì)學(xué)意義(P0.05)。同對(duì)照組相比,200U/ml塵螨刺激24小時(shí)后可明顯的增加支氣管上皮細(xì)胞的通透性,具有顯著的統(tǒng)計(jì)學(xué)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(xì)胞24小時(shí)后增加了對(duì)氣道平滑肌細(xì)胞遷移作用,同對(duì)照組相比具有顯著的統(tǒng)計(jì)學(xué)意義(P0.05),貝伐單抗可明顯阻斷這一效應(yīng)(P0.05)。同步檢測(cè)transwell下室支氣管上皮細(xì)胞上清中VEGF濃度,屋塵螨刺激24小時(shí)VEGF明顯升高,同對(duì)照組相比具有顯著的統(tǒng)計(jì)學(xué)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(xì)胞上清中檢測(cè)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(jiǎn)便、易行、可重復(fù)的人原代支氣管上皮細(xì)胞的培養(yǎng)方法。2.組織貼塊法是一種簡(jiǎn)便、易行的人原代氣道平滑肌細(xì)胞的培養(yǎng)方法。3. HD M通過(guò)上調(diào)VEGF可以直接增加NHBE的通透性。4. HDM通過(guò)上調(diào)NHBE表達(dá)VEGF增加了對(duì)氣道平滑肌細(xì)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(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R562.25

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 方正旭 ,王伶,劉東;關(guān)于動(dòng)脈平滑肌細(xì)胞培養(yǎng)的幾個(gè)問(wèn)題[J];江西醫(yī)學(xué)院學(xué)報(bào);2002年05期

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本文編號(hào)：2090727