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  本文關(guān)鍵詞： 香煙煙霧提取物 熱休克蛋白27 γ-谷氨酰半胱氨酸合酶 慢性阻塞性肺疾病　出處：《南華大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：【目的】檢測被動吸煙大鼠肺組織中HSP27表達(dá)水平及磷酸化水平。研究香煙煙霧提取物（CSE）處理的大鼠II型肺泡上皮細(xì)胞（AEC II）中HSP27表達(dá)水平及磷酸化水平，以及與γ-GCS表達(dá)的相關(guān)性。 【方法】本研究分為動物實驗和細(xì)胞實驗兩部分。（1）動物實驗部分：40只雄性Wistar大鼠隨機(jī)分為四組，，分別為對照組、吸煙2個月組、吸煙4個月組和戒煙組（吸煙4個月然后戒煙1個月），復(fù)制被動吸煙大鼠模型，收集對照組和實驗組大鼠肺組織標(biāo)本，Western-blot法檢測各組肺組織中的HSP27表達(dá)水平及磷酸化狀態(tài)。（2）細(xì)胞實驗部分：酶消化法分離Wistar大鼠AEC II并傳代培養(yǎng)，取第3代對數(shù)生長期細(xì)胞用于實驗。用0%、2.5%、5%、10%的CSE分別處理大鼠AECII6h、12h，采用Western-blot檢測各組細(xì)胞中的HSP27、p-HSP27和γ-GCS的蛋白表達(dá)水平，RT-PCR法檢測γ-GCSmRNA的表達(dá)水平。再用5%CSE分別對細(xì)胞處理0h、6h、12h、24h、48h，采用Western-blot法檢測各組細(xì)胞中的HSP27、p-HSP27和γ-GCS的蛋白表達(dá)水平，RT-PCR法檢測γ-GCSmRNA的表達(dá)水平。 【結(jié)果】（1）動物實驗部分：對照組、吸煙2個月組、吸煙4個月組、戒煙組大鼠肺組織HSP27表達(dá)水平分別為0.73±0.01、0.77±0.01、0.78±0.03和0.77±0.01，4組之間無顯著性差異（p0.05）。吸煙2個月組大鼠肺組織p-HSP27水平顯著高于對照組（0.37±0.02VS0.14±0.01，p 0.05），吸煙4個月組較吸煙2個月組進(jìn)一步升高（0.48±0.02VS0.37±0.02，p 0.05），戒煙組（0.41±0.01）較吸煙4個月組降低（p0.05），但仍然高于吸煙2個月組（p 0.05）。各組大鼠肺組織HSP27磷酸化比例變化趨勢與p-HSP27水平變化趨勢一致。 （2）細(xì)胞實驗部分：大鼠AECII分別經(jīng)0%(對照組)、2.5%、5%和10%的CSE處理6h及12h后，總HSP27及p-HSP27蛋白質(zhì)水平隨CSE濃度升高而升高，各濃度12h處理組的HSP27及p-HSP27蛋白質(zhì)水平均高于6h處理組。大鼠AECII經(jīng)5%CSE分別處理0h（對照組）、6h、12h、24h、48h， HSP27水平分別為0.12±0.01、0.16±0.02、0.23±0.03、0.29±0.01、0.36±0.01，p-HSP27水平分別為0.02±0.00、0.06±0.01、0.13±0.02、0.23±0.03、0.23±0.03，HSP磷酸化比例分別為0.17±0.04、0.39±0.07、0.57±0.09、0.80±0.06、0.64±0.09（其中6h組和12h顯著高于對照組，24h組高于6h組和12h，48h組低于24h但高于6h組和12h，p 0.05）。AECII經(jīng)0、2.5%、5%、10%的CSE處理24h，γ-GCS mRNA表達(dá)水平分別為0.21、0.32、0.44、0.54；經(jīng)5%的CSE處理0h、6h、12h、24h、48h，γ-GCS mRNA的表達(dá)水平分別為0.27、0.43、0.50、0.72和0.61。AECII經(jīng)5%的CSE處理0h、6h、12h、24、48h，γ-GCS蛋白表達(dá)水平隨處理時間進(jìn)行性升高，于24小時達(dá)到高峰。直線相關(guān)分析表明AECII γ-GCS mRNA和蛋白表達(dá)水平與HSP27、p-HSP27表達(dá)水平以及HSP27磷酸化比例成正相關(guān)。 【結(jié)論】 1． HSP27磷酸化參與了被動吸煙大鼠肺內(nèi)抗氧化應(yīng)激機(jī)制。 2. HSP27對CSE處理的大鼠AECII可能具有抗氧化保護(hù)作用。 3. HSP27磷酸化可能通過上調(diào)γ-GCS的表達(dá)水平對CSE誘導(dǎo)的大鼠AECII氧化應(yīng)激產(chǎn)生保護(hù)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 緯 -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 緯 -GCS were detected by Western-blot. The expression of 緯 -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 緯 -GCS was detected by Western-blot assay and the expression level of 緯 -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 鹵0.01VS0.77 鹵0.01VS0.78 鹵0.03 and 0.77 鹵0.01C respectively. The level of p-HSP27 in lung tissue of smoking group was significantly higher than that of control group (0.37 鹵0.02VS0.14 鹵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 鹵0.02VS0.37 鹵0.02p0.05P, 0.41 鹵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 鹵0.040.39 鹵0.040.39 鹵0.070.57 鹵0.060.80 鹵0.060.64 鹵0.064 鹵0.64 鹵0.64 鹵0.012 鹵0.01 鹵0.16 鹵0.030.29 鹵0.030.29 鹵0.031 鹵0.031 鹵0.36 鹵0.031 鹵0.36 鹵0.01 鹵0.23 鹵0.03 鹵0.23 鹵0.03h, respectively. The expression levels of 緯 -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 緯 -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 緯 -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 緯 -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 緯 -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 緯 -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 緯 -GCS.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

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