慢性阻塞性肺疾病大鼠磷脂酰肌醇-3激酶、蛋白激酶B及γ-谷氨酰半胱氨酸合成酶表达研究

The study of Phosphoinositide-3 kinases, protein kinase B and γ-glutamylcysteine synthetase in rats with chronic obstructive pulmonary disease

目的通过观察COPD大鼠中磷脂酰肌醇-3激酶（phosphoinositide-3 kinases，P13K）、蛋白激酶B（protein kinaseB，PKB）与γ-谷氨酰半胱氨酸合成酶（gamma glutamylcysteine synthetase，γ-GCS）的表达，研究P13K、PKB通路和rGCS的关系及其在COPD中可能的参与机制。方法30只健康雄性Wistar大鼠随机分COPD组和对照组。采用每日熏香烟和两次气管内注入脂多糖法制作COPD大鼠模型。检测大鼠肺组织中7-GCS活性，原位杂交检测肺组织中rGCSmRNA的表达，免疫组织化学分析肺组织中P13K、PKB与γ-GCS蛋白水平。结果COPD组大鼠肺组织中γ-GCS差异（P〈0．01）。P13K、PKB与γ-GCS免疫组织化学在COPD组可见肺泡、支气管活性明显高于对照组，组间差异有统计学意义（P〈0．01）。原位杂交示COPD组大鼠支气管，肺泡上皮细胞与小动脉平滑肌细胞7-GCSmRNA广泛表达，与对照组有显著性壁细胞及小血管平滑肌细胞胞浆中皆有蛋白阳性信号表达；图象定量分析示大鼠COPD组P13K、PKB与γ-GCS蛋白表达显著高于对照组（P〈0．01）。直线相关分析得出P13K、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7-GCS蛋白和mRNA表达增高，P13K、p-PKB蛋白也有相应高表达，提示P13K、PKB和γ-GCS可能在COPD的发病机制中发挥作用，且可能参与了γ-GCS的信号转导通路。

Objective To investigate the expression and relationship levels of PI3K, p-PKB and γ-glutamylcysteine synthetase （γ-GCS） in lung of rats with chronic obstructive pulmonary disease. Methods Thirty male Wistar rats were randomly divided into COPD model and control groups. The rat COPD model was established by intratracheal instillation of lipopolysaccharide （LPS 200 μG/200 μl） twice and exposed to cigarette smoke daily. The level of the activity of γ-GCS in rat lung tissue was measured, the levels of γ-GCS mRNA expression in rat lung tissue was measured by in site hybridization （ISH）. The protein expression of PI3K, p-PKB and γ-GCS were observed by immunohistochemistry. Results The levels of the activity of γ-GCS were higher in COPD group （21. 608±3. 112） U than that in control group [-（13. 256±2. 351） U, P 〈0.01]. The ISH showed that the levels of γ-GCS mRNA expression in COPD group （0. 369 ± 0. 035） were significantly higher than that in the control group （0. 231± 0. 026, P 〈 0.01）. In immunohistochemical （IH）, the granules of γ-GCS, PI3K and p-PKB, positively stained, were observed in the cytoplasm of pulmonary alveolar wall, intrapulmonary vascular and bronchial wall in COPD group. The protein expression of γ-GCS, PI3K and p-PKB was significantly higher in the COPD group （0. 351 ＋0. 043,0. 356 ± 0. 039,0. 342± 0. 043） than the control group （0. 237±0. 035,0. 231 ± 0.038, 0. 256 ± 0. 034） respectively, all （ P 〈 0.01）. By correlation analysis, we found there were a positive correlation between the protein levels of PI3K, p-PKB and the γ-GCS activity. Conclusions The high expression of PI3K,p-PKB and γ-GCS in the COPD group suggested that γ-CICS play a important role in the mechanism of COPD formation,and PI3K, PKB were involved in the signal transduction of γ-GCS.