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目的通过观察慢性阻塞性肺疾病(COPD)大鼠模型中γ-谷氨酰半胱氨酸合成酶(γ-GCS)与磷酸化蛋白激酶B(p-PKB)的表达,研究p-PKB和γ-GCS在COPD中可能的参与机制。方法28只健康雄性Wistar大鼠随机分为COPD组和对照组。采用每日熏香烟和两次气管内注入脂多糖(LPS)法制作COPD大鼠模型。检测肺组织中γ-GCS活性,原位杂交检测肺组织中γ-GCSmRNA的表达,免疫组化和Western印迹分析肺组织中γ-GCS与p-PKB蛋白水平。结果γ-GCS活性在COPD组大鼠中明显高于对照组,组间差异有统计学意义(P<0.05)。原位杂交显示COPD组大鼠支气管、肺泡上皮细胞与小动脉平滑肌细胞γ-GCSmRNA广泛表达,与对照组比较差异有统计学意义(P<0.05)。COPD组大鼠γ-GCS与p-PKB免疫组化可见肺泡、支气管壁细胞及小血管平滑肌细胞胞浆有较强蛋白阳性信号;图像定量分析显示COPD组γ-GCS与p-PKB蛋白表达高于对照组,两组比较差异有统计学意义(P<0.05)。Westernblot显示,γ-GCS与p-PKB在COPD组蛋白表达高于对照组,且差异有统计学意义(P<0.05)。SPSS10.0直线相关分析得出p-PKB蛋白表达与γ-GCS活性、mRNA及蛋白表达呈正相关。结论COPD大鼠肺组织γ-GCS蛋白和mRNA表达增高,p-PKB蛋白也有相应高表达,提示p-PKB和γ-GCS可能在COPD的发病机制中起作用,且PKB可能参与了γ-GCS的信号转导。
Objective To observe the expression of γ-glutamylcysteine synthase (γ-GCS) and phosphorylated protein kinase B (p-PKB) in the rat model of chronic obstructive pulmonary disease (COPD) Possible Mechanism of γ-GCS Participation in COPD. Methods Twenty-eight healthy male Wistar rats were randomly divided into COPD group and control group. COPD rat model was made by daily cigarette smoking and twice intratracheal injection of lipopolysaccharide (LPS). The activity of γ-GCS in lung tissue was detected. The expression of γ-GCS mRNA in lung tissue was detected by in situ hybridization. The levels of γ-GCS and p-PKB in lung tissue were analyzed by immunohistochemistry and Western blot. Results γ-GCS activity in COPD group was significantly higher than that in control group, the difference was statistically significant (P <0.05). In situ hybridization showed that the expression of γ-GCS mRNA in bronchial, alveolar epithelial cells and arteriolar smooth muscle cells of COPD rats was significantly higher than that of control rats (P <0.05). The expression of γ-GCS and p-PKB protein in COPD group was significantly higher than that in COPD group (P <0.05). There was a strong positive signal in the cytoplasm of alveolar, bronchial wall and small smooth muscle cells In the control group, the difference between the two groups was statistically significant (P <0.05). Western blot showed that the protein expression of γ-GCS and p-PKB in COPD group was higher than that in control group, and the difference was statistically significant (P <0.05). SPSS10.0 linear correlation analysis showed that p-PKB protein expression and γ-GCS activity, mRNA and protein expression was positively correlated. Conclusions The expression of γ-GCS protein and mRNA in lung tissue of COPD rats is increased, and the expression of p-PKB protein is also high in COPD rats, suggesting that p-PKB and γ-GCS may play a role in the pathogenesis of COPD and PKB may be involved in γ-GCS Of signal transduction.