香烟烟雾提取物对肺泡Ⅱ型上皮细胞热休克蛋白27及γ-谷氨酰半胱氨酸合成酶表达的调控作用

Regulatory effect of cigarette smoke extract on HSP27 and γ-GCS expression of alveolar type Ⅱ cells

目的探讨香烟烟雾提取物（cigarette smoke extract,CSE）对肺泡Ⅱ型上皮细胞热休克蛋白2γ（heat shock protein 27, HSP2γ）及γ-谷氨酰半胱氨酸合成酶（gamma glutamyl cysteine syntase, γ- GCS）表达的影响。方法体外培养肺泡Ⅱ细胞（A549），加入5％浓度的CSE作用于细胞0、6、12、24、48h以及不同浓度的CSE（0％，2．5％，5％，10％）作用细胞24h，运用逆转录聚合酶链反应（reverse transcription-polymerase chain reaction, RT-PCR）检测γ-GCSmRNA含量的变化；不同浓度的CSE（0％，2．5％，5％，10％）作用细胞12、24h以及5％的CSE作用细胞0、6、12、24、48h，采用免疫蛋白印迹（western bloting，WB）检测γ-GCS、HSP2γ及其磷酸化HSP2γ（p-HSP2γ）在蛋白表达水平的变化。结果①5％CSE的作用下，随着干预时间的延长，γ-GCSmRNA含量呈明显增加趋势（P〈0．05）；②不同浓度的CSE作用细胞24h，随着浓度的增加，γGCSmRNA含量呈明显增加趋势（Pd0．05）；③不同浓度的CSE（0％，2．5％，5％，10％）作用细胞12、24h以及5％的CSE作用细胞0、6、12、24、48h，HSP2γ及p-HSP2γ的蛋白水平有明显上调趋势（P〈0．05），γ-GCS蛋白含量也呈浓度和时间依赖上升。结论CSE能够引起肺泡上皮细胞应激反应，通过上调HSP2γ磷酸化及表达γGCS对肺泡上皮细胞具有保护作用机制。CSE可通过上调HSP27的磷酸化γ-CCS的表达启动肺泡上皮细胞的应激反应，以保护细胞。

Objective To investigate the effects of cigarette smoke extract （CSE） on heat shock protein 27 （HSP27） and γ-GCS expression in the cultured A549s that are rat alveolar type Ⅱ cells lines. Methods RT-PCR were used to analyze the changes of γ-GCS mRNA in different time （0,6,12,24,48 h） exposure 5% concentration of CSE,and different concentrations of CSE （0%,2.5%,5%,10%） in 24 h. Western bloting was performed to detect the level of 7-GCS, HSP27 and the phosphorylation of HSP27 protein expression exposure to different concentrations of CSE in 12,24 h and 50/00 concentration of CSE effect on the cells in different time （0, 6, 12, 24, 48 h）. Results ① With the elongation of drug interference, the expression of ）, GCS mRNA elevated on 5% concentration of CSE （ P 〈0.05）. The expression of 7-GCS mRNA was significantly increased with the increase of concentration in 24 h （ P〈0.05）. ②Different concentrations of CSE in 12,24 h and 5% concentration of CSE effect on the cells in different time was significantly up-regulated the expression of HSP27 and its phosphorylation, the expression of y-GCS protein also increased accompaning by the the elongation of concentration and time. Conclusions CSE can start stress responses of alveolar type Ⅱ cells protect by up regulated phosphorylation of HSP27 and γ-GCS expression,which futher to protect the cells.