埃兹蛋白在TNF-α致大鼠肺微血管内皮细胞炎性损伤中表达变化及Rac1的影响

发布时间：2018-02-16 12:08

本文关键词： 埃兹蛋白肿瘤坏死因子肺微血管内皮细胞 Rac1 急性呼吸窘迫综合征　出处：《安徽医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：背景与目的埃兹蛋白(Ezrin)是细胞骨架桥接蛋白ERM(Ezrin-Radixin-Moesin)家族成员之一,主要分布于细胞皮层。Ezrin作为膜蛋白和肌动蛋白连接蛋白,通过ERM家族特征性FERM(four-point-one protein,ezrin,redixin,moesin)功能域将胞膜蛋白与肌动蛋白连接,参与多种信号传导,在调控细胞的形态、生长、生存、黏附、增殖和迁徙等生物学功能中发挥重要作用。研究发现,用特异性Ezrin小干扰RNA作用于肿瘤坏死因子α(tumor necrosis factor,TNF-α)刺激的人肺微血管内皮细胞(Human pulmonary microvascular endothelial cell,HPMVEC),磷酸化的Ezrin减少,可显著抑制内皮细胞通透性的增加。但在肺炎症损伤过程中,Ezrin具体通过哪些信号传导通路参与其中,尚未见报道。本研究通过TNF-α建立大鼠肺微血管内皮细胞(rat pulmonary microvascular endothelial cell,RPMVEC)损伤模型,探讨RPMVEC炎性损伤与Ezrin表达量及磷酸化之间的关系,以及Ezrin在急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)发病机制中的作用和小G蛋白家族成员Rac1对Ezrin在TNF-α致RPMVEC炎性损伤中的影响。方法1)体外肺组织块贴壁法分离培养原代RPMVECs;2)蛋白质免疫印迹技术(Western-blotting technique)检测TNF-α刺激RPMVECs后各时间点Ezrin及其磷酸化的表达水平。3)蛋白质免疫印迹技术测定NSC23766刺激RPMVECs后不同时间点Ezrin及p-Ezrin的表达;结果1)体外成功分离培养出原代RPMVECs,经光镜下的细胞形态学及植物凝集素结合实验鉴定。2)RPMVECs中仅低量表达Ezrin,且TNF-α刺激对Ezrin无明显影响。10μg/L TNF-α刺激RPMVECs不同时间(0h、0.25h、0.5h、1h、3h、6h、12h、24h)后发现:Ezrin表达无变化。而TNF-α可呈时间依赖性增加Ezrin磷酸化,相对表达量分别为(0.21±0.33)、(0.53±0.19)、(0.95±0.24)、(1.07±0.30)、(1.68±0.30)、(1.32±0.37)、(0.93±0.20)、(0.87±0.18),0.25 h(0.53±0.19)时开始高于0 h(0.21±0.33),3h(1.68±0.30)达峰值且显著高于0 h、0.25 h、0.5 h、1 h、6 h、12 h、24 h(均P0.001),24 h(0.87±0.18)仍高于0 h。各组间比较差异有统计学意义(F=62.2,P0.001)。3)与空白对照组(0.68±0.16)比较,单独使用10μg/L TNF-α(0.92±0.12)或200μmol/L NSC 23766(1.33±0.24)刺激均能诱导p-Ezrin表达量增加(t=-2.864、P=0.020,t=-5.429、P=0.000);将NSC 23766与TNF-α共同刺激(2.14±0.18)能明显提高p-Ezrin表达量,与TNF-α(0.92±0.12)单独刺激组比较有统计学差异(t=-14.670,P0.001)。结论1)RPMVECs仅低量表达Ezrin,TNF-α刺激对Ezrin无明显影响。2)TNF-α以时间依赖的方式上调PMVEC中磷酸化Ezrin的表达。3)抑制Rac1信号通路可通过上调Ezrin磷酸化表达参与TNF-α对RPMVECs的致伤效应。
[Abstract]:Background & objective Ezrinin is a member of the Ezrin-Radixin-Moesin family of cytoskeleton bridging proteins. Ezrin mainly distributes in the cell cortex as membrane protein and actin junction protein. Cellular membrane proteins are connected with actin through the functional domain of ERM family FERM(four-point-one protein FERM(four-point-one protein redix in moesin, which play an important role in regulating cell morphology, growth, survival, adhesion, proliferation, migration and other biological functions, and play an important role in the regulation of cell morphology, growth, survival, adhesion, proliferation and migration. Human pulmonary microvascular endothelial cells (HPMVECs) stimulated by tumor necrosis factor- 伪 (TNF- 伪) were treated with specific Ezrin small interfering RNA, and phosphorylated Ezrin was decreased. But in the process of lung inflammation injury, which signal transduction pathways does Ezrin take part in? In this study, we established rat pulmonary microvascular endothelial cell model by TNF- 伪 to investigate the relationship between RPMVEC inflammatory injury and Ezrin expression and phosphorylation. The role of Ezrin in the pathogenesis of acute respiratory distress syndrome (ARDS) and the effect of small G protein family member Rac1 on Ezrin in RPMVEC inflammatory injury induced by TNF- 伪. Methods 1) isolation and culture of primary RPMVECs2 eggs by adherent lung tissue mass in vitro). Western blotting technique was used to detect the expression level of Ezrin and its phosphorylation at different time points after TNF- 伪 stimulated RPMVECs. The expression of Ezrin and p-Ezrin at different time points after RPMVECs stimulated by NSC23766 were detected by Western blotting technique. Results 1) Primary RPMVECs were isolated and cultured successfully in vitro. The results of cell morphology and phytoagglutinin binding experiments under light microscope showed that the expression of Ezrinin in the RPMVECs was only low, and TNF- 伪 stimulation had no significant effect on Ezrin. 10 渭 g / L TNF- 伪 stimulated RPMVECs for 0 h 0.25 h, 0.5 h 1 h, 1 h 3 hU 6 h ~ 12 h ~ 24 h). TNF- 伪 could increase the phosphorylation of Ezrin in a time dependent manner. The relative expression levels were 0.21 卤0.33, 0.53 卤0.19, 0.95 卤0.24, 1.07 卤0.30, 1.32 卤0.371.32 卤0.371.32 卤0.371.32 卤0.370.87 卤0.18h0.53 卤0.19, respectively, and reached a peak value of 0.21 卤0.33, 1.68 卤0.30) and significantly higher than 0 h, 0.25 h, 0.5 h, 1 h, 6 h, 12 h, 24 h (all P 0.001h, 0.87 卤0.18). The difference between the two groups was statistically significant compared with that of the control group (0.68 卤0.16). 10 渭 g / L TNF- 伪 0.92 卤0.12) or 200 渭 mol/L NSC 237661.33 卤0.24) increased the expression of p-Ezrin, increased the expression of p-Ezrin, increased the expression of p-Ezrin, increased the expression of p-Ezrin by co-stimulation of NSC 23766 and TNF- 伪. Compared with TNF- 伪 0.92 卤0.12) alone, there was a significant difference between TNF- 14.670 and P0.0010.Conclusion only the low expression of Ezrinine TNF- 伪 in RPMVECs has no effect on Ezrin. 2TNF- 伪 upregulated the expression of phosphorylated Ezrin in PMVEC in a time-dependent manner. 3) inhibited Rac1 signaling pathway by up-regulating Ezrin phosphorylation. The expression of TNF- 伪 was involved in the injury effect of TNF- 伪 on RPMVECs.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R563.8

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