探讨脂多糖致急性肺损伤时Tribbles同源蛋白3表达变化

发布时间：2018-03-07 18:33

本文选题：脂多糖　切入点：血管内皮细胞　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的探讨Tribbles同源蛋白3(TRB3)在脂多糖(LPS)致急性肺损伤时的变化及其与p38-MAPK信号通路的关系。方法体内实验:复制LPS致急性肺损伤(ALI)大鼠模型,分生理盐水对照组(5ml/kg)和5 mg/kg LPS刺激组,免疫组织化学法检测肺组织中TRB3蛋白表达,逆转录聚合酶联反应(RT-PCR)检测肺组织TRB3 m RNA表达。体外实验:体外培养大鼠肺微血管内皮细胞(PMVEC),随机分为LPS量效组(0、2、4、10μg/ml LPS分别孵育4 h)、LPS时效组(10μg/ml LPS分别孵育0、4、8、12 h)和p38抑制剂(SB203580)干预组:分为正常对照组,10μg/ml LPS组、10μmol/L SB203580组、10μmol/L SB203580+10μg/ml LPS组,免疫印迹法检测TRB3蛋白、p-p38和p38-MAPK表达。结果免疫组织化学法显示大鼠肺泡壁和腺上皮均表达TRB3;RT-PCR法检测大鼠肺组织和大鼠PMVEC均表达TRB3 m RNA;与生理盐水组比较,LPS致ALI大鼠肺组织TRB3 m RNA表达显著增加(3.675±0.423 vs 1.056±0.209,t=15.524,P0.01);与未刺激组比较,LPS刺激的大鼠PMVEC中TRB3 m RNA表达增加(2.098±0.317 vs0.612±0.314,t=7.549,P0.01);免疫印迹法发现PMVEC表达TRB3蛋白:表达量随LPS浓度(0、2、4、10μg/ml)增加逐渐升高,分别为0.169±0.089、0.198±0.071、0.338±0.089、0.494±0.118,组间比较,差异有统计学意义(F=12.619,P0.001);时效组TRB3蛋白表达量于4 h达高峰(0.443±0.087),之后下降, 8 h(0.303±0.107),仍高于正常对照组(0.159±0.073),组间比较,差异有统计学意义(F=11.273,P0.001)。干预组:与正常对照组比较,10μg/ml LPS诱导大鼠PMVEC的p-p38蛋白表达量增高(0.660±0.100 vs 0.227±0.085,t=49.121,P0.001)以及TRB3蛋白表达增高(0.461±0.097 vs 0.178±0.084,t=15.113,P0.001);10μmol/L SB203580+10μg/ml LPS联合刺激组与LPS单独刺激组比较,p-p38蛋白表达量下降(0.557±0.125 vs 0.660±0.100,t=7.040,P0.05,TRB3表达量也下降(0.306±0.077 vs 0.461±0.097,t=11.900,P0.001);SB203580单独作用对大鼠PMVEC表达p-p38及TRB3蛋白无影响(与正常对照组比较,P0.05)。结论LPS致急性肺损伤时TRB3表达增加,TRB3表达受p38-MAPK信号通路调控。
[Abstract]:Objective to investigate the changes of Tribbles homologue protein 3 (TRB3) in acute lung injury induced by lipopolysaccharide (LPS) and its relationship with p38-MAPK signaling pathway. Methods the rat model of acute lung injury induced by LPS was established in vivo and divided into normal saline control group (5 ml / kg) and 5 mg/kg LPS stimulation group. Immunohistochemical method was used to detect the expression of TRB3 protein in lung tissue. Reverse transcriptase polymerase chain reaction (RT PCR) was used to detect the expression of TRB3 m RNA in lung tissue. In vitro, rat pulmonary microvascular endothelial cells were incubated with 10 渭 g / ml LPS (10 渭 g / ml LPS) and p38 (10 渭 g / ml LPS, respectively) and p38. The control group was divided into 10 渭 g / ml LPS group and 10 渭 mol/L SB203580 10 渭 g / ml LPS group, 10 渭 mol/L SB203580 group, 10 渭 mol/L SB203580 10 渭 g / ml LPS group, 10 渭 mol/L SB203580 10 渭 g / ml LPS group, 10 渭 mol/L SB203580 10 渭 g / ml LPS group. The expression of p-p38 and p38-MAPK in rat alveolar wall and glandular epithelium was detected by immunoblotting assay. Results the expression of TRB3 mRNA in rat lung tissue and rat PMVEC was detected by RT-PCR, and compared with that in normal saline group, the expression of TRB3 mRNA in rat alveolar wall and glandular epithelium was detected by immunohistochemistry. The expression of TRB3 m RNA increased significantly in lung tissue of rats (3.675 卤0.423 vs 1.056 卤0.209), and the expression of TRB3 m RNA in PMVEC was increased by 2.098 卤0.317 vs0.612 卤0.314 vs0.612 卤7.549P0.01.The expression of TRB3 protein was increased with the increase of LPS concentration (10 渭 g / ml). The expression of TRB3 protein was 0.169 卤0.089 卤0.198 卤0.071, 0.338 卤0.089 and 0.494 卤0.118, respectively. The difference between the two groups was statistically significant, and the expression of TRB3 protein reached the peak at 4 h, reached the peak at 4 h, then decreased, and decreased to 0.303 卤0.107 at 8 h, which was still higher than that of the normal control group (0.159 卤0.073). In the intervention group, the expression of p-p38 protein in PMVEC induced by 10 渭 g / ml LPS was significantly higher than that in the control group (0.660 卤0.100 vs 0.227 卤0.085) and the expression of TRB3 protein was 0.461 卤0.097 vs 0.178 卤0.084 渭 mol/L SB203580 10 渭 g / ml LPS combined with LPS alone. The decrease of protein expression (0.557 卤0.125 vs 0.660 卤0.100) P0.05TRB3 also decreased 0.306 卤0.077 vs 0.461 卤0.097 T11.900P0.001P0.001TRB3. The expression of p-p38 and TRB3 protein in PMVEC was not affected by LPS alone. Conclusion the increased expression of TRB3 in acute lung injury induced by LPS is regulated by p38-MAPK signaling pathway.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.8

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