HSP27磷酸化对CSE诱导肺氧化应激的保护作用及与γ-GCS表达的相关研究

发布时间：2018-02-08 19:58

本文关键词： 香烟烟雾提取物热休克蛋白27 γ-谷氨酰半胱氨酸合酶慢性阻塞性肺疾病　出处：《南华大学》2012年硕士论文　论文类型：学位论文

【摘要】：【目的】检测被动吸烟大鼠肺组织中HSP27表达水平及磷酸化水平。研究香烟烟雾提取物（CSE）处理的大鼠II型肺泡上皮细胞（AEC II）中HSP27表达水平及磷酸化水平，以及与γ-GCS表达的相关性。【方法】本研究分为动物实验和细胞实验两部分。（1）动物实验部分：40只雄性Wistar大鼠随机分为四组，，分别为对照组、吸烟2个月组、吸烟4个月组和戒烟组（吸烟4个月然后戒烟1个月），复制被动吸烟大鼠模型，收集对照组和实验组大鼠肺组织标本，Western-blot法检测各组肺组织中的HSP27表达水平及磷酸化状态。（2）细胞实验部分：酶消化法分离Wistar大鼠AEC II并传代培养，取第3代对数生长期细胞用于实验。用0%、2.5%、5%、10%的CSE分别处理大鼠AECII6h、12h，采用Western-blot检测各组细胞中的HSP27、p-HSP27和γ-GCS的蛋白表达水平，RT-PCR法检测γ-GCSmRNA的表达水平。再用5%CSE分别对细胞处理0h、6h、12h、24h、48h，采用Western-blot法检测各组细胞中的HSP27、p-HSP27和γ-GCS的蛋白表达水平，RT-PCR法检测γ-GCSmRNA的表达水平。【结果】（1）动物实验部分：对照组、吸烟2个月组、吸烟4个月组、戒烟组大鼠肺组织HSP27表达水平分别为0.73±0.01、0.77±0.01、0.78±0.03和0.77±0.01，4组之间无显著性差异（p0.05）。吸烟2个月组大鼠肺组织p-HSP27水平显著高于对照组（0.37±0.02VS0.14±0.01，p 0.05），吸烟4个月组较吸烟2个月组进一步升高（0.48±0.02VS0.37±0.02，p 0.05），戒烟组（0.41±0.01）较吸烟4个月组降低（p0.05），但仍然高于吸烟2个月组（p 0.05）。各组大鼠肺组织HSP27磷酸化比例变化趋势与p-HSP27水平变化趋势一致。（2）细胞实验部分：大鼠AECII分别经0%(对照组)、2.5%、5%和10%的CSE处理6h及12h后，总HSP27及p-HSP27蛋白质水平随CSE浓度升高而升高，各浓度12h处理组的HSP27及p-HSP27蛋白质水平均高于6h处理组。大鼠AECII经5%CSE分别处理0h（对照组）、6h、12h、24h、48h， HSP27水平分别为0.12±0.01、0.16±0.02、0.23±0.03、0.29±0.01、0.36±0.01，p-HSP27水平分别为0.02±0.00、0.06±0.01、0.13±0.02、0.23±0.03、0.23±0.03，HSP磷酸化比例分别为0.17±0.04、0.39±0.07、0.57±0.09、0.80±0.06、0.64±0.09（其中6h组和12h显著高于对照组，24h组高于6h组和12h，48h组低于24h但高于6h组和12h，p 0.05）。AECII经0、2.5%、5%、10%的CSE处理24h，γ-GCS mRNA表达水平分别为0.21、0.32、0.44、0.54；经5%的CSE处理0h、6h、12h、24h、48h，γ-GCS mRNA的表达水平分别为0.27、0.43、0.50、0.72和0.61。AECII经5%的CSE处理0h、6h、12h、24、48h，γ-GCS蛋白表达水平随处理时间进行性升高，于24小时达到高峰。直线相关分析表明AECII γ-GCS mRNA和蛋白表达水平与HSP27、p-HSP27表达水平以及HSP27磷酸化比例成正相关。【结论】 1． HSP27磷酸化参与了被动吸烟大鼠肺内抗氧化应激机制。 2. HSP27对CSE处理的大鼠AECII可能具有抗氧化保护作用。 3. HSP27磷酸化可能通过上调γ-GCS的表达水平对CSE诱导的大鼠AECII氧化应激产生保护作用。
[Abstract]:[objective] to detect the expression and phosphorylation of HSP27 in the lung tissue of passive smoking rats. To study the expression and phosphorylation of HSP27 in type II alveolar epithelial cells treated with cigarette smoke extract and its correlation with 纬 -GCS expression. [methods] A total of 40 male Wistar rats were randomly divided into four groups: control group and smoking group for 2 months. Four months smoking group and smoking cessation group (smoking for 4 months and then quitting for 1 month) were used to make passive smoking rat model. The expression level of HSP27 and phosphorylation of AEC II were detected by Western-blot in lung tissue of control group and experimental group. The experiment part: isolation and passage culture of AEC II from Wistar rats by enzyme digestion. In the third generation of logarithmic growth phase cells, the cells were treated with 10% CSE for 12 h, and the protein expression levels of HSP27, p-HSP27 and 纬 -GCS were detected by Western-blot. The expression of 纬 -GCS mRNA was detected by RT-PCR, and the cells were treated with 5CSE for 0 h, 6 h, 12 h, 24 h and 48 h, respectively. The protein expression level of HSP27p-HSP27 and 纬 -GCS was detected by Western-blot assay and the expression level of 纬 -GCS mRNA was detected by RT-PCR. [results] Animal experiment part: control group, smoking 2 months group, smoking 4 months group, The expression level of HSP27 in lung tissue of smoking cessation group was 0.73 卤0.01VS0.77 卤0.01VS0.78 卤0.03 and 0.77 卤0.01C respectively. The level of p-HSP27 in lung tissue of smoking group was significantly higher than that of control group (0.37 卤0.02VS0.14 卤0.01p0.05P). The level of p-HSP27 in lung tissue of smoking group was significantly higher than that of smoking group for 2 months. The level of p-HSP27 in lung tissue of smoking group was higher than that of smoking group for 2 months. The level of p-HSP27 in lung tissue of smoking group was significantly higher than that of control group. 0.48 卤0.02VS0.37 卤0.02p0.05P, 0.41 卤0.01) were lower than those of 4-month smoking group, but still higher than that of smoking group for 2 months (p 0.05). The change trend of HSP27 phosphorylation in lung tissue of each group was the same as that of p-HSP27 level. (2) Cell experiment: the levels of total HSP27 and p-HSP27 protein increased with the increase of CSE concentration in rat AECII treated with 5% and 10%% CSE for 6 h and 12 h, respectively. The protein levels of HSP27 and p-HSP27 in 12h treatment group were higher than those in 6h treatment group. The levels of HSP27 and p-HSP27 protein in rats treated with 5CSE were 0.17 卤0.040.39 卤0.040.39 卤0.070.57 卤0.060.80 卤0.060.64 卤0.064 卤0.64 卤0.64 卤0.012 卤0.01 卤0.16 卤0.030.29 卤0.030.29 卤0.031 卤0.031 卤0.36 卤0.031 卤0.36 卤0.01 卤0.23 卤0.03 卤0.23 卤0.03h, respectively. The expression levels of 纬 -GCS mRNA in control group were lower than those in 6h group and 12h / 48h group but higher than 6h group and 12h group respectively, and the expression levels of 纬 -GCS mRNA were 0.21 0.322.40.54, 0.21t 0.322.440.54, 0.270.430.72 and 0.61.AECII treated with 5% CSE for 24 hours, respectively, and the expression levels of 纬 -GCS mRNA were 0.270.43 0.72 and 0.61.AECII treated with 5% CSE for 0 h 6 h, 12 h 24 h 24 h, 48 h respectively, the expression levels of 纬 -GCS mRNA were 0.270.43 and 0.61.AECII were treated with 5% CSE for 0 h 6 h, 12 h 24 h 24 h and 48 h for 48 h, respectively. The expression levels of 纬 -GCS mRNA in control group were 0.270.43 and 0.61.AECII were 0.270.43 and 0.61.AECII treated with 5% CSE, respectively. The level increased gradually with the treatment time. Linear correlation analysis showed that the expression level of AECII 纬 -GCS mRNA and protein was positively correlated with the expression level of HSP27 p-HSP27 and the ratio of HSP27 phosphorylation. [conclusion]. 1. HSP27 phosphorylation participates in the mechanism of antioxidant stress in the lung of passive smoking rats. 2. HSP27 may have antioxidant protective effect on rat AECII treated with CSE. 3. HSP27 phosphorylation may protect CSE induced oxidative stress of AECII in rats by up-regulating the expression of 纬 -GCS.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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