小窝蛋白-1在大鼠肺微血管内皮细胞炎性损伤中的作用初探

发布时间：2018-02-13 01:42

本文关键词： 肺微血管内皮细胞小窝蛋白-1 原位杂交脂多糖蛋白激酶C　出处：《安徽医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的研究脂多糖（lipopolysaccharide, LPS）对大鼠肺微血管内皮细胞（ratpulmonary microvascular endothelial cell, RPMVEC）中小窝蛋白-1（caveolin-1,Cav-1）mRNA表达的影响，观察蛋白激酶C（protein kinase C, PKC）抑制剂对LPS诱导的Cav-1mRNA表达的干预作用，为进一步研究Cav-1在急性肺损伤/急性呼吸窘迫综合征（acute lung injury/acute respiratory distress syndrome, ALI/ARDS）发病机制中的作用及地位奠定实验基础。方法体外分离培养RPMVEC；分别采用逆转录聚合酶链反应（reverse transcriptpolymerase chain reaction, RT-PCR）及免疫荧光检测RPMVEC中Cav-1mRNA及蛋白的表达；根据LPS刺激的浓度和时间差异，将RPMVEC随机分为LPS刺激量效组和时效组：量效组分别以0.1μg/ml、1μg/ml、10μg/ml LPS与RPMVEC孵育6h，时效组以10μg/ml LPS与RPMVEC分别孵育1h、2h、3h、6h，以上均设置相应对照组； RPMVEC与PKC抑制剂双吲哚基顺丁烯二酰亚胺（bis-indolylmaleimide, BIM）预孵育1h，再与10μg/ml的LPS继续孵育6h，以上设置相应对照组；原位杂交（In situ hybridization, ISH）与JD801形态学图像分析系统检测不同条件下RPMVEC中Cav-1mRNA的表达变化。结果1.体外成功进行RPMVEC的分离、培养，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. RT-PCR和免疫荧光检测出RPMVEC中表达Cav-1基因和蛋白。3. LPS以浓度依赖方式诱导Cav-1mRNA表达增加：0.1μg/ml、1μg/ml、10μg/ml LPS分别孵育RPMVEC6h后，Cav-1mRNA表达量随其浓度增加逐渐升高，与正常组比较差异均显著（P 0.05）；10μg/ml LPS刺激RPMVEC不同时间（1h、2h、3h、6h）后，RPMVEC中Cav-1mRNA表达水平呈动态变化：1h开始上升，2h达峰值，之后逐渐下降，，但至6h仍高于正常水平（P 0.05）。4.10μmol/L BIM预孵育RPMVEC1h后，可显著下调10μg/ml LPS对Cav-1mRNA表达的诱导效应（P 0.05）。结论1.成功进行RPMVEC的分离、培养和鉴定。2. RPMVEC表达Cav-1。3. LPS以浓度及时间依赖方式上调RPMVEC中Cav-1mRNA的表达。4. PKC抑制剂BIM能够下调LPS对RPMVEC中Cav-1mRNA的诱导效应。
[Abstract]:Objective to study the effect of lipopolysaccharide (LPS) on the expression of fossa protein (-1) caveolin-1 (Cav-1) mRNA in rat pulmonary microvascular endothelial cells (RPMVECs), and to observe the effect of protein kinase kinase (kinase) inhibitor on Cav-1mRNA expression induced by LPS. To further study the role and role of Cav-1 in the pathogenesis of acute lung injury/acute respiratory distress syndrome (Ali / ARDS) in patients with acute lung injury / acute respiratory distress syndrome (ARDS). Methods RPMVECs were isolated and cultured in vitro. Reverse transcriptpolymerase chain reactionation (RT-PCRR) was used to detect the expression of Cav-1mRNA and protein in RPMVEC by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence. RPMVEC was randomly divided into two groups: 1 渭 g / ml 1 渭 g / ml LPS and 10 渭 g / ml LPS were incubated with RPMVEC for 6 h respectively, and 10 渭 g / ml LPS and RPMVEC were incubated with 10 渭 g / ml LPS for 6 h, respectively. All of them were set up as control group; RPMVEC was incubated with PKC inhibitor diindole maleic maleic acid for 6 h. Bis-indolylmaleimide (bimm) was preincubated for 1 hour and then incubated with 10 渭 g / ml LPS for 6 h. In situ hybridization of in situ (ISH) and JD801 morphological image analysis system were used to detect the expression of Cav-1mRNA in RPMVEC under different conditions. Results 1. RPMVEC was isolated and cultured successfully in vitro. Morphological and isothiocyanate-labeled plant agglutinin binding assay confirmed that the expression of Cav-1 gene and protein in RPMVEC was detected by RT-PCR and immunofluorescence. LPS induced Cav-1mRNA expression to increase 1 渭 g / ml LPS 10 渭 g / ml LPS in a concentration-dependent manner after RPMVEC6h was incubated with 1 渭 g / ml LPS, respectively. The expression of Cav-1 mRNA increased with the increase of its concentration. Compared with the control group, the difference was significant (P 0.05g / ml LPS stimulated RPMVEC at different time (1h / h) / 3h / h / 6h). The expression of Cav-1mRNA in RPMVEC began to rise to its peak at 1: 1 h, and then decreased gradually, but it was still higher than the normal level (P 0.05 .4.10 渭 mol/L BIM) after preincubation of RPMVEC1h at 6 h, and the expression of Cav-1mRNA in RPMVEC was significantly higher than that in the control group (P 0.05. 4. 10 渭 mol/L BIM preincubated at 6 h). The effect of 10 渭 g / ml LPS on Cav-1mRNA expression was significantly down-regulated (P 0.05). The expression of Cav-1.3. LPS up-regulated the expression of Cav-1mRNA in RPMVEC in a dose-and time-dependent manner. PKC inhibitor BIM could down-regulate the inductive effect of LPS on Cav-1mRNA in RPMVEC. 2.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R563.8

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