慢性阻塞性肺疾病大鼠中磷酸化蛋白激酶B与γ-谷氨酰半胱氨酸合成酶的表达研究

Expression of Phosphorylated Protein Kinases B and γ-glutamylcysteine Synthetase in Rats with Chronic Obstructive Pulmonary Disease

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 investigate the expression of γ-glutamylcysteine synthetase （γ-GCS） and phosphorylated protein kinases B （p-PKB） in lungs of rats with chronic obstructive pulmonary disease. Methods Twenty-eight male Wistar rats were randomly divided into chronic obstructive pulmonary disease （COPD） group and the control groups. The rat COPD model was established by intratracheal instillation with lipopolysaccharide （LPS 200 μg/200 μL） twice and then exposed to cigarette smoke daily. Activity of γ-GCS in the lung tissue was measured. The level of γ-GCS mRNA expression in lung tissue were measured by in situ hybridization （ISH）. The protein expression of γ-GCS and p-PKB were determined by immunohistochemistry and Western blot. Results The activity of γ-GCS was higher in COPD group （3.242±0.529） U than the control group （2.267 U±0.430 U, P〈0.05）. The levels of expression of γ-GCS mRNA in COPD group （0.445±0.059） were significantly higher than the control group （0.318±0.052, P〈0.05）. Positive immunoreactivity of γ-GCS and p-PKB could be detected in the cytoplasm of pulmonary alveolar wall, intrapulmonary vascular and bronchial wall in COPD group. The level of protein expression of γ-GCS and p-PKB was significantly higher in the COPD group （0.361±0.050, 0.317±0.042） than the control group （0.248±0.041 and 0.221±0.038, respectively, all P〈0.05）. Western blot analysis demonstrated that the protein expression of γ-GCS and p-PKB in COPD group （ 0.22 ±0.038, 0.204 ±0.033 ） were significantly higher than the control group （ 0.129±0.027,0.074±0.014, respectively, all P〈0.05）. By correlation analysis, there was a positive correlation between the levels of p-PKB and the γ-GCS activity, and between the level of γ-GCS mRNA and the proteint of γ-GCS. Conclusion The high level of expression of γ-GCS and p-PKB in the COPD group indicated that γ-GCS might play an important role in the development of COPD, and p-PKB might also be involved in the signal transduction of γ-GCS.