本文關(guān)鍵詞： 肺微血管內(nèi)皮細(xì)胞 通透性 小窩蛋白-1RNAi　出處：《安徽醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的構(gòu)建caveolin-1基因RNA干擾慢病毒，觀察抑制caveolin-1基因表達(dá)后對大鼠肺微血管內(nèi)皮細(xì)胞通透性的影響。為進(jìn)一步研究caveolin-1在ALI/ARDS發(fā)病機制中的作用奠定實驗基礎(chǔ)。 方法體外分離培養(yǎng)RPMVEC；采用RT-PCR及Western blot檢測RPMVEC中caveolin-1的表達(dá)；選定大鼠caveolin-1基因RNA干擾的靶序列，，合成靶序列DNA，退火成雙鏈后與經(jīng)XhoⅠ和HpaⅠ雙酶切后的慢病毒載體pLentilox3.7連接；重組pLL3.7-caveolin-1慢病毒載體轉(zhuǎn)化大腸桿菌DH5α，提取質(zhì)粒后，經(jīng)限制性內(nèi)切酶XbaⅠ和XhoⅠ酶切鑒定后進(jìn)行測序鑒定；慢病毒載體質(zhì)粒pLL3.7和包裝質(zhì)粒VSV-G,RSV-REV,pMDL在脂質(zhì)體2000的介導(dǎo)下共轉(zhuǎn)染包裝細(xì)胞293T細(xì)胞，包裝慢病毒，病毒上清經(jīng)濃縮后，按一定比例稀釋后感染293T細(xì)胞，熒光顯微鏡下觀察GFP蛋白的表達(dá)，測定病毒滴度；通過Western blot檢測Caveolin-1shRNA慢病毒感染RPMVEC后對caveolin-1基因表達(dá)的抑制；通過檢測跨內(nèi)皮細(xì)胞電阻觀察caveolin-1基因表達(dá)抑制對TNF-α所致RPMVEC單層通透性增高的影響。 結(jié)果1.體外成功分離、培養(yǎng)RPMVEC。2.通過RT-PCR和Western blot證實RPMVEC表達(dá)caveolin-1。3.經(jīng)酶切和測序證實，成功構(gòu)建慢病毒表達(dá)載體pLL3.7-caveolin-1shRNA。4.包裝的慢病毒經(jīng)濃縮后的病毒懸液滴度為2×108TU/ml。5.Western blot證實慢病毒感染RPMVEC后，caveolin-1的表達(dá)明顯下調(diào)，72h后caveolin-1表達(dá)被抑制85%。6.Caveolin-1表達(dá)抑制能顯著改善TNF-α所致RPMVEC單層TER的降低，與正常RPMVEC刺激組相比，抑制組30min時即出現(xiàn)差異，6h時差異最大，24h后差異不明顯。（P＜0.05）。 結(jié)論1.RPMVEC表達(dá)caveolin-1。2.成功構(gòu)建大鼠caveolin-1基因RNAi慢病毒。3.慢病毒感染RPMVEC后顯著抑制caveolin-1的表達(dá)。4.抑制RPMVEC中caveolin-1的表達(dá)可顯著改善TNF-α致RPMVEC單層通透性的增高。
[Abstract]:Objective to construct caveolin-1 gene RNA interfering lentivirus and observe the effect of inhibiting the expression of caveolin-1 gene on the permeability of rat pulmonary microvascular endothelial cells in order to further study the role of caveolin-1 in the pathogenesis of ALI/ARDS. Methods RPMVECs were isolated and cultured in vitro, the expression of caveolin-1 in RPMVEC was detected by RT-PCR and Western blot, the target sequence of rat caveolin-1 gene RNA interference was selected, the target DNA was synthesized and annealed into double strand, and then connected with the Lentivirus vector pLentilox3.7 which was digested by Xho 鈪

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