【摘要】：目的 研究脂多糖(lipopolysaccharide, LPS)致肺損傷大鼠肺微血管內(nèi)皮細(xì)胞(pulmonary microvascular endothelial cell, PMVEC)及肺組織內(nèi)小窩蛋白-1(caveolin-1, Cav-1)表達(dá)的變化,觀察LPS對PMVEC通透性的影響,探討蛋白激酶A (protein kinase A, PKA)抑制劑在LPS誘導(dǎo)PMVEC通透性變化及Cav-1表達(dá)中的干預(yù)作用,為進(jìn)一步研究Cav-1在大鼠LPS性肺損傷發(fā)病機(jī)制中的作用提供實驗依據(jù)。 方法 體外分離培養(yǎng)RPMVEC;通過檢測跨內(nèi)皮細(xì)胞電阻以及伊文思藍(lán)-白蛋白法觀察LPS對RPMVEC單層通透性的影響；采用免疫印跡技術(shù)(western-blotting technique)檢測RPMVEC中Cav-1蛋白的表達(dá)；構(gòu)建LPS致大鼠ALI模型,通過蘇木素-伊紅染色(hematoxylin-eosin staining, HE)觀察肺組織病理學(xué)變化；采用逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(reverse transcript polymerase chain reaction, RT-PCR)、 Western-blotting及免疫組織化學(xué)法技術(shù)分別檢測肺組織內(nèi)Cav-1蛋白及mRNA表達(dá)。 結(jié)果 1.體外成功分離、培養(yǎng)出RPMVEC,并經(jīng)形態(tài)學(xué)及異硫氰酸標(biāo)記的植物凝集素結(jié)合實驗鑒定證實。 2.10μg/ml LPS刺激RPMVEC不同時間(0h、1h、3h、6h、12h、24h)后發(fā)現(xiàn),LPS時間依賴性增加RPMVEC單層通透性：Pd于刺激后1h即升高,3h后達(dá)高峰、顯著增加的Pd持續(xù)到刺激后24h；同Pd變化,LPS刺激RPMVEC單層時,TER呈時間依賴性下降。10μmol/L PKI預(yù)孵育,能進(jìn)一步加重LPS誘導(dǎo)增加的RPMVEC單層通透性。 3.10μg/ml LPS刺激RPMVEC, Cav-1蛋白表達(dá)于1h開始上升,3h達(dá)峰值,之后漸下降,但至24h仍高于孵育0h組,各組間差異有統(tǒng)計學(xué)意義(P0.01)； 4.LPS刺激RPMVEC10min后,Cav-1氨基端的第14位酪氨酸被磷酸化,30min達(dá)高峰,然后開始下降,120min后恢復(fù)到基礎(chǔ)水平。而PKI預(yù)孵育后,LPS誘導(dǎo)增加的Cav-1及其磷酸化表達(dá)進(jìn)一步增加。 5.5μg/ml LPS成功復(fù)制大鼠肺損傷模型。 6.LPS以時間依賴方式下調(diào)大鼠肺組織Cav-1mRNA及蛋白表達(dá)。 7.與LPS刺激6h組相比,甲潑尼龍組肺組織病理損傷程度減輕,可部分抑制LPS對肺組織Cav-1mRNA及蛋白表達(dá)的下調(diào)作用。 結(jié)論 1.LPS呈時間依賴性增加RPMVEC單層通透性。 2.LPS以時間依賴的方式上調(diào)RPMVEC中Cav-1蛋白及其磷酸化蛋白的表達(dá)。 3.PKA抑制劑單獨作用可引起RPMVEC單層通透性增加,且對LPS的致通透性損傷作用有協(xié)同作用,其機(jī)制可能與其上調(diào)LPS對RPMVEC中Cav-1及磷酸化Cav-1的誘導(dǎo)效應(yīng)有關(guān)。 4.LPS致大鼠肺損傷模型中肺組織Cav-1表達(dá)下調(diào)。 5.甲潑尼龍可部分抑制LPS對大鼠肺組織Cav-1表達(dá)的下調(diào)作用,有效減輕肺損傷程度。
[Abstract]:Objective to study the changes of (pulmonary microvascular endothelial cell, PMVEC) and caveolin-1, Cav-1 expression in lung microvascular endothelial cells induced by lipopolysaccharide (lipopolysaccharide, LPS) in rats with lung injury, and to observe the effect of LPS on the permeability of PMVEC. To investigate the effect of protein kinase A (protein kinase A, PKA) inhibitor on the changes of PMVEC permeability induced by LPS and the expression of Cav-1, and to provide experimental evidence for the further study of the role of Cav-1 in the pathogenesis of LPS induced lung injury in rats. Methods the effect of LPS on the permeability of RPMVEC monolayer was observed by detecting the resistance of transendothelial cells and the effect of LPS on the permeability of RPMVEC monolayers by detection of the resistance of transendothelial cells and the expression of Cav-1 protein in RPMVEC by Western blotting (western-blotting technique). The rat model of ALI induced by LPS was established and the histopathological changes of lung were observed by hematoxylin-eosin staining (hematoxylin-eosin staining, HE). Reverse transcriptase polymerase chain reaction (reverse transcript polymerase chain reaction, RT-PCR), Western-blotting and immunohistochemistry were used to detect the expression of Cav-1 protein and mRNA in lung tissue respectively. Result 1. RPMVEC, was isolated in vitro and confirmed by morphological and phytoagglutinin binding assay. 2. 10 渭 g/ml LPS stimulated RPMVEC at different time (0 h, 1 h, 3 h, 6 h and 12 h / h) showed that LPS increased monolayer permeability in a time-dependent manner: Pd increased at 1 h after stimulation and reached its peak at 3 h, and the significantly increased Pd continued until 24 h after stimulation. When LPS stimulated RPMVEC monolayer, TER decreased in a time dependent manner, and 10 渭 mol/L PKI preincubation increased the RPMVEC monolayer permeability induced by LPS. The expression of RPMVEC, Cav-1 protein increased at 1 h, reached the peak at 3 h, then decreased gradually, but was still higher than that in 0 h incubation group at 24 h, the difference was statistically significant (P0.01). After stimulation of RPMVEC10min by 4.LPS, tyrosine at the amino terminal of Cav-1 was phosphorylated, 30min peaked, then began to decrease, and 120min returned to basic level. However, after preincubation with PKI, the expression of Cav-1 and its phosphorylation induced by LPS was further increased. Rat lung injury model was successfully established at 5.5 渭 g/ml LPS. 6.LPS down-regulated the expression of Cav-1mRNA and protein in rat lung tissues in a time dependent manner. 7. Compared with LPS stimulation group for 6 h, methylprednisolone group had less pathological injury and partly inhibited the down-regulation of Cav-1mRNA and protein expression in lung tissue by LPS. Conclusion 1.LPS increases monolayer permeability of RPMVEC in a time dependent manner. 2.LPS up-regulated the expression of Cav-1 protein and phosphorylated protein in RPMVEC in a time dependent manner. 3.PKA inhibitor alone can increase the permeability of RPMVEC monolayer and has synergistic effect on the permeability injury of LPS. The mechanism may be related to the up-regulation of LPS induced Cav-1 and phosphorylated Cav-1 in RPMVEC. The expression of Cav-1 was down-regulated in 4.LPS induced lung injury in rats. 5. Methylprednisolone partly inhibited the down-regulation of Cav-1 expression in rat lung tissue by LPS, and effectively alleviated the severity of lung injury.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R563

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 徐智,吳國明,錢桂生,王興勝,陳維中,李淑平,薛橋,王士雯;油酸-內(nèi)毒素序貫致傷致老年鼠MODS模型的建立[J];第三軍醫(yī)大學(xué)學(xué)報;2004年10期

，

本文編號：2408887