辛伐他汀對(duì)COPD大鼠肺組織細(xì)胞凋亡的影響
發(fā)布時(shí)間：2019-06-02 00:34
【摘要】：[目的]通過觀察辛伐他汀對(duì)慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)模型大鼠肺組織凋亡細(xì)胞及凋亡相關(guān)因子內(nèi)皮型一氧化氮合酶(eNOS)、誘導(dǎo)型一氧化氮合酶(iNOS)及半胱氨酸蛋白酶-3(Caspase-3)蛋白和mRNA表達(dá)的影響,探討辛伐他汀在COPD中的作用機(jī)制,為其在COPD臨床治療中的應(yīng)用提供理論依據(jù)。 [方法] 1、將30只健康雄性Wistar大鼠隨機(jī)分為：正常組(n=10)、對(duì)照組(n=10)和治療組(n=10)。 2、采用熏香煙和氣管內(nèi)滴入脂多糖(lipopolysaccharides,LPS)的方法建立COPD大鼠模型,造模開始兩周后,治療組同時(shí)給予辛伐他汀(2.5mg/kg)灌胃治療,治療6周。 3、HE染色觀察各組大鼠肺組織病理改變；采用脫氧核糖核酸末端轉(zhuǎn)移酶介導(dǎo)的dUTP缺口末端標(biāo)記技術(shù)(TUNEL)觀察各組大鼠肺組織中凋亡的細(xì)胞,并計(jì)算凋亡指數(shù)(AI)；應(yīng)用免疫組織化學(xué)技術(shù)及RT-PCR檢測(cè)各組大鼠肺組織中eNOS、iNOS和Caspase-3蛋白及mRNA的表達(dá)。 [結(jié)果] 1、HE染色肺組織病理學(xué) 正常組大鼠肺組織中氣管黏膜假?gòu)?fù)層纖毛柱狀上皮完整,氣道周圍無炎癥細(xì)胞浸潤(rùn),毛細(xì)血管無擴(kuò)張充血,肺泡大小及結(jié)構(gòu)正常。對(duì)照組大鼠氣管和支氣管黏膜纖毛柱狀上皮層增厚,纖毛倒伏、粘連、壞死、脫落,粘膜下腺體肥大,粘膜下層和肌層大量炎癥細(xì)胞浸潤(rùn),血管周圍大量炎性細(xì)胞浸潤(rùn),毛細(xì)血管擴(kuò)張充血,肺泡結(jié)構(gòu)破壞,肺泡壁變薄斷裂融合。治療組大鼠氣道纖毛柱狀上皮細(xì)胞脫落、杯狀細(xì)胞增生、血管周圍炎性細(xì)胞浸潤(rùn)較對(duì)照組呈不同程度減輕,肺泡破壞斷裂融合較對(duì)照組少。 2、病理形態(tài)學(xué)定量 對(duì)照組和治療組大鼠肺組織中肺平均內(nèi)襯間隔(Mean linear intercept,MLI)高于正常組(P0.05),治療組MLI與對(duì)照組比較降低(P0.05)；對(duì)照組和治療組大鼠肺組織中平均肺泡數(shù)(Mean alveolar numbers,MAN)少于正常組(P0.05),治療組MAN與對(duì)照組比較增多(P0.05)。 3、TUNEL法檢測(cè)肺組織細(xì)胞凋亡 大鼠肺組織中凋亡細(xì)胞的細(xì)胞核可被染為淡棕色至深棕色不等,主要為肺泡上皮細(xì)胞、氣道上皮細(xì)胞,亦可見血管內(nèi)皮細(xì)胞、血管、支氣管平滑肌細(xì)胞及少量炎癥細(xì)胞。對(duì)照組及治療組與正常組比較,肺泡上皮細(xì)胞和氣道上皮細(xì)胞的凋亡增多(P0.05)；與對(duì)照組比較,治療組肺泡上皮細(xì)胞和氣道上皮細(xì)胞凋亡減少(P0.05)。 4、SP法檢測(cè)肺組織蛋白表達(dá) eNOS表達(dá)于氣道上皮細(xì)胞、肺泡上皮細(xì)胞和血管內(nèi)皮細(xì)胞,對(duì)照組及治療組與正常組比較,大鼠肺組織中eNOS表達(dá)減少(P0.05),與對(duì)照組比較,治療組eNOS表達(dá)增多(P0.05)。iNOS主要表達(dá)于肺泡上皮細(xì)胞、血管內(nèi)皮細(xì)胞和炎癥細(xì)胞,對(duì)照組及治療組與正常組比較,大鼠肺組織中iNOS表達(dá)增多P0.05),與對(duì)照組比較,治療組iNOS表達(dá)減少(P0.05)。Caspase-3的表達(dá)分布于肺泡上皮細(xì)胞、氣道上皮細(xì)胞、血管內(nèi)皮細(xì)胞、氣道和血管平滑肌細(xì)胞,對(duì)照組和治療組與正常組比較,大鼠肺組織中Caspase-3表達(dá)增加(P0.05),與對(duì)照組比較,治療組Caspase-3表達(dá)減少(P0.05)。 5、RT-PCR檢測(cè)基因的表達(dá) 對(duì)照組及治療組與正常組比較,大鼠肺組織中eNOS mRNA表達(dá)減少(P0.05),與對(duì)照組比較,治療組eNOS mRNA表達(dá)增多(P0.05)。對(duì)照組和治療組與正常組比較,iNOS和Caspase-3mRNA表達(dá)增多(P0.05),與對(duì)照組比較,治療組iNOS和Caspase-3mRNA表達(dá)減少(P0.05)。 6、大鼠肺組織Caspase-3與eNOS及iNOS蛋白表達(dá)之間的相關(guān)性分析 正常組大鼠肺組織中Caspase-3與eNOS蛋白表達(dá)之間呈負(fù)相關(guān)(r=0.86,P0.05),與iNOS呈正相關(guān)(r=0.80,P0.05)；對(duì)照組Caspase-3與eNOS蛋白表達(dá)之間呈負(fù)相關(guān)(r=-0.67,P0.05),與iNOS呈正相關(guān)(r=0.77,P0.05)；治療組Caspase-3與eNOS蛋白表達(dá)之間呈負(fù)相關(guān)(r=-0.85,P0.05),與iNOS呈正相關(guān)(r=0.88,P0.05)。 7、各組大鼠肺組織中Caspase-3與eNOS及iNOS mRNA表達(dá)之間的相關(guān)性分析 正常組大鼠肺組織Caspase-3與eNOS mRNA表達(dá)之間呈負(fù)相關(guān)(r=-0.86,P0.05),與iNOS mRNA表達(dá)呈正相關(guān)(r=0.94,P0.05)；對(duì)照組Caspase-3與eNOS mRNA表達(dá)之間呈負(fù)相關(guān)(r=-0.65,P0.05),與iNOS mRNA表達(dá)呈正相關(guān)(r=0.89,P0.05)；治療組Caspase-3與eNOS mRNA表達(dá)之間呈負(fù)相關(guān)(r=-0.92,P0.05),與iNOS mRNA的表達(dá)呈正相關(guān)(r=O.90,PO.05)。 [結(jié)論] 1、辛伐他汀可以減輕COPD模型大鼠支氣管及肺血管周圍炎癥。 2、辛伐他汀可以減輕COPD模型大鼠支氣管及肺組織細(xì)胞凋亡。 3、辛伐他汀通過增加肺組織eNOS的表達(dá),降低iNOS的表達(dá),進(jìn)而減少Caspase-3的表達(dá),從而減輕COPD肺組織細(xì)胞凋亡,在COPD中發(fā)揮保護(hù)性作用,為其在COPD I臨床治療中的應(yīng)用提供理論依據(jù)。
[Abstract]:[Objective] To observe the effect of simvastatin on the apoptosis and apoptosis-related factor endothelial nitric oxide synthase (eNOS) in the lung of rats with chronic obstructive pulmonary disease (COPD). The effect of simvastatin on the expression of inducible nitric oxide synthase (iNOS) and caspase-3 (Caspase-3) protein and mRNA was discussed. [Party Method] 1. Thirty healthy male Wistar rats were randomly divided into normal group (n = 10), control group (n = 10) and treatment group (n = 10). 10).2. The model of chronic obstructive pulmonary disease (COPD) was established by the method of dropping the lipopolysaccharides (LPS) into the cigarette and the trachea, and the treatment group was given simvastatin (2.5 mg/ kg) in the treatment group at the same time after the start of the model for two weeks. 3, HE staining was used to observe the pathological changes of lung tissue in each group, and the apoptotic cells in the lung tissues of each group were observed by the end-end labeling (TUNEL) of dUTP nick end-transferase mediated dUTP nick end labeling (TUNEL). The expression of eNOS, iNOS and Caspase-3 in lung tissues of each group was detected by immunohistochemistry and RT-PCR. mRNA Expression.[Results] 1. The tracheal mucosa pseudostratified ciliated columnar epithelium in the lung tissue of the normal group of HE stained lung tissue was intact, there was no inflammatory cell infiltration around the airway, and the capillary was not dilated. In the control group, the cilia of the trachea and the bronchial mucosa of the control group were thickened, the cilia lodging, the adhesion, the necrosis, the shedding, the hypertrophy of the submucosal glands, the infiltration of the submucosal and muscular layers of the inflammatory cells, the infiltration of a large number of inflammatory cells around the blood vessels, the capillary, vasodilation, congestion, alveolar junction in that treatment group, the ciliated columnar epithelial cells of the group of the treatment group fall off, the goblet cell hyperplasia and the infiltration of the peripheral inflammatory cells of the blood vessel are reduced to a different extent than the control group, Alveolar destruction and fracture fusion In the control group, the mean pulmonary lining interval (MLI) in the lung tissue of the control group and the treatment group was higher than that in the normal group (P0.05), and the mean number of alveoli in the lung tissues of the control group and the treatment group (Me An alveolar numbers (MAN) was less than that of the normal group (P0.05), and the treatment group M The number of AN and the control group increased (P0.05). .3. TUNEL method in the detection of apoptosis in the lung tissue of the lung tissue of rats can be stained with light brown to dark brown, mainly for the alveolar epithelial cells, airway epithelial cells, and also to the vascular endothelial cells. In the control group and the treatment group, the apoptosis of the alveolar epithelial cells and the airway epithelial cells increased (P0.05); and compared with the control group, the treatment group's alveoli Reduction of apoptosis in skin cells and airway epithelial cells (P0.05). The expression of eNOS in the lung tissue was detected by SP method in airway epithelial cells, alveolar epithelial cells and vascular endothelial cells. The expression of eNOS in the tissue was decreased (P0.05). The expression of eNOS in the treatment group was increased in comparison with the control group (P0.05). iNOS was mainly expressed in the alveolar epithelial cells, the vascular endothelial cells and the inflammatory cells, and the control group and the treatment group were compared with the normal group. The expression of iNOS in the lung tissue was increased (P0.05). The expression of Caspase-3 was decreased in the treatment group compared with the control group (P0.05). The expression of Caspase-3 was distributed in the alveolar epithelial cells, the airway epithelial cells, the vascular endothelial cells, the airway and the vascular smooth muscle cells, the control group and the treatment group were compared with the normal group, and the calcium in the lung tissues of the rats The expression of spase-3 was increased (P0.05) and compared with the control group. Table of Caspase-3 in the Treatment Group The expression of eNOS mRNA in the lung of rats was decreased (P0.05). Compared with the control group, the expression of eNOS mRNA in the treatment group increased (P0.05). Compared with the control group, the expression of iNOS and Caspase-3 mRNA was increased (P0.05). The expression of iNOS and Caspase-3 mRNA was decreased (P0.05). The correlation between the expression of Caspase-3 and eNOS and iNOS protein was negatively correlated with the expression of the expression of Caspase-3 and eNOS in the normal group (r = 0.86, P0.05). There was a negative correlation between the expression of e-3 and eNOS (r =-0.67, P <0.05) and positive correlation with iNOS (r = 0.77, P0.05); and the expression of Caspase-3 and eNOS in the treatment group was negatively correlated (r =-0. (85, P0.05), positive correlation with iNOS (r = 0.88, P0.05). There was a negative correlation between the expression of Caspase-3 and eNOS and iNOS mRNA in the normal group (r =-0.86, P0.05) and positive correlation with the expression of iNOS mRNA (r = 0.94, P0.05). There was a negative correlation between the expression of ase-3 and eNOS mRNA in the treatment group (r =-0.89, P0.05), and the expression of Caspase-3 and eNOS mRNA in the treatment group was negatively correlated (r =-0.92, P <0.05). P0.0 5) The expression of iNOS mRNA was positively correlated with the expression of iNOS mRNA (r = O.90, PO. 05).[Conclusion] 1, Simvastatin can reduce the COPD model rats 2. Simvastatin can reduce the expression of iNOS and decrease the expression of Caspase-3 by increasing the expression of eNOS in the lung, so as to reduce the COP.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R563.9
本文編號(hào)：2490693
[Abstract]:[Objective] To observe the effect of simvastatin on the apoptosis and apoptosis-related factor endothelial nitric oxide synthase (eNOS) in the lung of rats with chronic obstructive pulmonary disease (COPD). The effect of simvastatin on the expression of inducible nitric oxide synthase (iNOS) and caspase-3 (Caspase-3) protein and mRNA was discussed. [Party Method] 1. Thirty healthy male Wistar rats were randomly divided into normal group (n = 10), control group (n = 10) and treatment group (n = 10). 10).2. The model of chronic obstructive pulmonary disease (COPD) was established by the method of dropping the lipopolysaccharides (LPS) into the cigarette and the trachea, and the treatment group was given simvastatin (2.5 mg/ kg) in the treatment group at the same time after the start of the model for two weeks. 3, HE staining was used to observe the pathological changes of lung tissue in each group, and the apoptotic cells in the lung tissues of each group were observed by the end-end labeling (TUNEL) of dUTP nick end-transferase mediated dUTP nick end labeling (TUNEL). The expression of eNOS, iNOS and Caspase-3 in lung tissues of each group was detected by immunohistochemistry and RT-PCR. mRNA Expression.[Results] 1. The tracheal mucosa pseudostratified ciliated columnar epithelium in the lung tissue of the normal group of HE stained lung tissue was intact, there was no inflammatory cell infiltration around the airway, and the capillary was not dilated. In the control group, the cilia of the trachea and the bronchial mucosa of the control group were thickened, the cilia lodging, the adhesion, the necrosis, the shedding, the hypertrophy of the submucosal glands, the infiltration of the submucosal and muscular layers of the inflammatory cells, the infiltration of a large number of inflammatory cells around the blood vessels, the capillary, vasodilation, congestion, alveolar junction in that treatment group, the ciliated columnar epithelial cells of the group of the treatment group fall off, the goblet cell hyperplasia and the infiltration of the peripheral inflammatory cells of the blood vessel are reduced to a different extent than the control group, Alveolar destruction and fracture fusion In the control group, the mean pulmonary lining interval (MLI) in the lung tissue of the control group and the treatment group was higher than that in the normal group (P0.05), and the mean number of alveoli in the lung tissues of the control group and the treatment group (Me An alveolar numbers (MAN) was less than that of the normal group (P0.05), and the treatment group M The number of AN and the control group increased (P0.05). .3. TUNEL method in the detection of apoptosis in the lung tissue of the lung tissue of rats can be stained with light brown to dark brown, mainly for the alveolar epithelial cells, airway epithelial cells, and also to the vascular endothelial cells. In the control group and the treatment group, the apoptosis of the alveolar epithelial cells and the airway epithelial cells increased (P0.05); and compared with the control group, the treatment group's alveoli Reduction of apoptosis in skin cells and airway epithelial cells (P0.05). The expression of eNOS in the lung tissue was detected by SP method in airway epithelial cells, alveolar epithelial cells and vascular endothelial cells. The expression of eNOS in the tissue was decreased (P0.05). The expression of eNOS in the treatment group was increased in comparison with the control group (P0.05). iNOS was mainly expressed in the alveolar epithelial cells, the vascular endothelial cells and the inflammatory cells, and the control group and the treatment group were compared with the normal group. The expression of iNOS in the lung tissue was increased (P0.05). The expression of Caspase-3 was decreased in the treatment group compared with the control group (P0.05). The expression of Caspase-3 was distributed in the alveolar epithelial cells, the airway epithelial cells, the vascular endothelial cells, the airway and the vascular smooth muscle cells, the control group and the treatment group were compared with the normal group, and the calcium in the lung tissues of the rats The expression of spase-3 was increased (P0.05) and compared with the control group. Table of Caspase-3 in the Treatment Group The expression of eNOS mRNA in the lung of rats was decreased (P0.05). Compared with the control group, the expression of eNOS mRNA in the treatment group increased (P0.05). Compared with the control group, the expression of iNOS and Caspase-3 mRNA was increased (P0.05). The expression of iNOS and Caspase-3 mRNA was decreased (P0.05). The correlation between the expression of Caspase-3 and eNOS and iNOS protein was negatively correlated with the expression of the expression of Caspase-3 and eNOS in the normal group (r = 0.86, P0.05). There was a negative correlation between the expression of e-3 and eNOS (r =-0.67, P <0.05) and positive correlation with iNOS (r = 0.77, P0.05); and the expression of Caspase-3 and eNOS in the treatment group was negatively correlated (r =-0. (85, P0.05), positive correlation with iNOS (r = 0.88, P0.05). There was a negative correlation between the expression of Caspase-3 and eNOS and iNOS mRNA in the normal group (r =-0.86, P0.05) and positive correlation with the expression of iNOS mRNA (r = 0.94, P0.05). There was a negative correlation between the expression of ase-3 and eNOS mRNA in the treatment group (r =-0.89, P0.05), and the expression of Caspase-3 and eNOS mRNA in the treatment group was negatively correlated (r =-0.92, P <0.05). P0.0 5) The expression of iNOS mRNA was positively correlated with the expression of iNOS mRNA (r = O.90, PO. 05).[Conclusion] 1, Simvastatin can reduce the COPD model rats 2. Simvastatin can reduce the expression of iNOS and decrease the expression of Caspase-3 by increasing the expression of eNOS in the lung, so as to reduce the COP.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R563.9
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