小窝蛋白-1在大鼠脂多糖性肺损伤中的作用机制
发布时间：2019-01-14 16:43
【摘要】：目的 研究脂多糖(lipopolysaccharide, LPS)致肺损伤大鼠肺微血管内皮细胞(pulmonary microvascular endothelial cell, PMVEC)及肺组织内小窝蛋白-1(caveolin-1, Cav-1)表达的变化,观察LPS对PMVEC通透性的影响,探讨蛋白激酶A (protein kinase A, PKA)抑制剂在LPS诱导PMVEC通透性变化及Cav-1表达中的干预作用,为进一步研究Cav-1在大鼠LPS性肺损伤发病机制中的作用提供实验依据。 方法 体外分离培养RPMVEC;通过检测跨内皮细胞电阻以及伊文思蓝-白蛋白法观察LPS对RPMVEC单层通透性的影响；采用免疫印迹技术(western-blotting technique)检测RPMVEC中Cav-1蛋白的表达；构建LPS致大鼠ALI模型,通过苏木素-伊红染色(hematoxylin-eosin staining, HE)观察肺组织病理学变化；采用逆转录聚合酶链反应(reverse transcript polymerase chain reaction, RT-PCR)、 Western-blotting及免疫组织化学法技术分别检测肺组织内Cav-1蛋白及mRNA表达。 结果 1.体外成功分离、培养出RPMVEC,并经形态学及异硫氰酸标记的植物凝集素结合实验鉴定证实。 2.10μg/ml LPS刺激RPMVEC不同时间(0h、1h、3h、6h、12h、24h)后发现,LPS时间依赖性增加RPMVEC单层通透性：Pd于刺激后1h即升高,3h后达高峰、显著增加的Pd持续到刺激后24h；同Pd变化,LPS刺激RPMVEC单层时,TER呈时间依赖性下降。10μmol/L PKI预孵育,能进一步加重LPS诱导增加的RPMVEC单层通透性。 3.10μg/ml LPS刺激RPMVEC, Cav-1蛋白表达于1h开始上升,3h达峰值,之后渐下降,但至24h仍高于孵育0h组,各组间差异有统计学意义(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达高峰,然后开始下降,120min后恢复到基础水平。而PKI预孵育后,LPS诱导增加的Cav-1及其磷酸化表达进一步增加。 5.5μg/ml LPS成功复制大鼠肺损伤模型。 6.LPS以时间依赖方式下调大鼠肺组织Cav-1mRNA及蛋白表达。 7.与LPS刺激6h组相比,甲泼尼龙组肺组织病理损伤程度减轻,可部分抑制LPS对肺组织Cav-1mRNA及蛋白表达的下调作用。 结论 1.LPS呈时间依赖性增加RPMVEC单层通透性。 2.LPS以时间依赖的方式上调RPMVEC中Cav-1蛋白及其磷酸化蛋白的表达。 3.PKA抑制剂单独作用可引起RPMVEC单层通透性增加,且对LPS的致通透性损伤作用有协同作用,其机制可能与其上调LPS对RPMVEC中Cav-1及磷酸化Cav-1的诱导效应有关。 4.LPS致大鼠肺损伤模型中肺组织Cav-1表达下调。 5.甲泼尼龙可部分抑制LPS对大鼠肺组织Cav-1表达的下调作用,有效减轻肺损伤程度。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563
本文编号：2408887
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563
【参考文献】
相关期刊论文 前1条
1 徐智,吴国明,钱桂生,王兴胜,陈维中,李淑平,薛桥,王士雯;油酸-内毒素序贯致伤致老年鼠MODS模型的建立[J];第三军医大学学报;2004年10期
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