肺气肿大鼠全病程中膈肌功能的研究
摘要
目的
本研究应用被动吸烟复制大鼠肺气肿模型,采用酶组织化学、免疫印记技术等不同的检测方法,检测肺气肿大鼠全病程中的膈肌纤维类型和mTor蛋白的变化,探讨肺气肿全病程中膈肌功能异常的分子生物学机制。
实验材料及方法
一、实验动物和分组
选取7周龄健康雄性Wistar大鼠120只,体重250—280g,由中国医科大学实验动物中心提供,清洁度Ⅰ级,常规饲料自由饮水,温度22±1℃,湿度50%左右,明／暗周期为12h。随机被分为3组,分别为对照组(C)、肺气肿吸烟组(E)、肺气肿戒烟组(N),分别在肺气肿形成0天、3月、6月、12月取材,每组10只。
二、被动吸烟复制大鼠肺气肿模型
将肺气肿大鼠每天放入自制的烟熏舱(体积为0.25 m3),暴露于10支黄果树牌香烟,每次被动吸烟1小时5天／周,持续75天。
三、主要实验方法
(一)肺组织病理：HE染色
(二)膈肌纤维检测：采用琥珀酸脱氢酶方法,将肌纤维分为氧化型和酵解型,其中染色深的是氧化型纤维,染色浅的酵解型纤维。
(三)mTOR蛋白测定：Western Blotting
四、统计学分析
所有数据用均数±标准差(x±s)表示,采用完全随机设计资料单因素方差分析进行显著性检验,若P<0.05,再进行q检验,比较任意两组间的差异有无统计学意义。采用单因素直线回归分析,比较干预因素与测量因素有无相关性。所有分析用SPSS13.0软件进行。
结果
1、在肺气肿形成0天和3个月时,E组、N组的氧化型纤维比例高于对照组(P<0.05)。在肺气肿形成6个月时,N组的氧化型纤维比例高于对照组(P<0.05),E组的氧化型纤维比例与对照组比较无显著差异(P>0.05)。在肺气肿形成12个月时,E组、N组的氧化型纤维比例低于对照组(P<0.05)。
2、年龄与氧化型纤维比例的相关分析结果显示正相关(P<0.05)。
3、在肺气肿形成0天和3个月时,E组、N组的mTOR蛋白高于对照组(P<0.05)。在肺气肿形成6个月时,N组的mTOR蛋白高于对照组(P<0.05),E组的mTOR蛋白与对照组比较无显著差异(P>0.05)。在肺气肿形成12个月时,E组、N组的mTOR低于对照组(P<0.05)。
结论
1、肺气肿大鼠膈肌氧化型纤维比例随疾病进展先增加后下降,说明膈肌出现代偿和失代偿过程。
2、随着年龄的增加,膈肌氧化型纤维比例逐渐升高。
3、肺气肿早期调节骨骼肌增生的信号蛋白mTOR蛋白表达高于对照组,肺气肿晚期mTOR蛋白表达低于对照组。
Purpose
The passive smoking was used to duplicate rat emphysema model. To detect the diaphragm fiber types in the whole course of emphysema with enzyme histochemistry. The mTor protein was detected by Western blotting techniques, which in order to explore the mechanism of diaphragmatic dysfunction in the molecular biological level.
Experimental materials and methods
One hundred and twenty healthy seven-week-old male Wistar rats were selected for study, weighting 250-280g, which came from the Experimental Animal Center of China Medical University. They were randomly divided into 3 groups:control group (C), emphysema and smoking group (E) and emphysema and smoking cessation group (N). On the very day of emphysema formation, and after 3 months,6 months and 12 months, we will randomly choose 10 rats for experiment. The rats were put into the home-made smoking capsule (volume 0.25 m3), exposure to 10 Huangguoshu cigarettes,1 hour each day,5 days/week, continued to 75 days. The main experimental methods including lung pathology with HE staining, detection of diaphragm muscle fiber with succinic acid dehydrogenase method and mTOR protein detection with Western Blotting. SPSS13.0 software was used for all data analysis. If P<0.05, statistics is of significance.
Results
1.In 3 months of pulmonary emphysema, and on the day of emphysema, oxidized fibers in E group and N group were significantly higher than that in C group(P<0.05). In 6 months of emphysema, oxidized fibers of N group were significantly higher than that of C group (P<0.05), the proportion of oxidized fibers in E group compared with the control group, there is no significant difference (P> 0.05). In emphysema of 12 months, E group, N group the proportion of oxidized fibers were higher than the control group (P<0.05).
2. The age and the proportion of oxidized fiber correlation analysis showed a positive correlation (P<0.05).
3. In 3 months of pulmonary emphysema, and on the day of emphysema, mTOR protein in E group and N group were significantly higher than that in C group(P<0.05). In 6 months of emphysema, mTOR protein of N group were significantly higher than that of C group (P<0.05), mTOR protein in E group compared with the control group, there is no significant difference (P> 0.05). In emphysema of 12 months, mTOR protein of E group and N group were higher than the control group (P<0.05).
Conclusions
1. The ratio of oxidized fibers in the emphysema rat diaphragm with disease progression first increased and then decreased, indicating compensation and decompensation occurred.
2. Increasing with age, the proportion of oxidized fibers gradually increased.
3. At early stage of emphysema, signaling protein mTOR protein in emphysema group were higher than that in control group.
本研究应用被动吸烟复制大鼠肺气肿模型,采用酶组织化学、免疫印记技术等不同的检测方法,检测肺气肿大鼠全病程中的膈肌纤维类型和mTor蛋白的变化,探讨肺气肿全病程中膈肌功能异常的分子生物学机制。
实验材料及方法
一、实验动物和分组
选取7周龄健康雄性Wistar大鼠120只,体重250—280g,由中国医科大学实验动物中心提供,清洁度Ⅰ级,常规饲料自由饮水,温度22±1℃,湿度50%左右,明／暗周期为12h。随机被分为3组,分别为对照组(C)、肺气肿吸烟组(E)、肺气肿戒烟组(N),分别在肺气肿形成0天、3月、6月、12月取材,每组10只。
二、被动吸烟复制大鼠肺气肿模型
将肺气肿大鼠每天放入自制的烟熏舱(体积为0.25 m3),暴露于10支黄果树牌香烟,每次被动吸烟1小时5天／周,持续75天。
三、主要实验方法
(一)肺组织病理：HE染色
(二)膈肌纤维检测：采用琥珀酸脱氢酶方法,将肌纤维分为氧化型和酵解型,其中染色深的是氧化型纤维,染色浅的酵解型纤维。
(三)mTOR蛋白测定：Western Blotting
四、统计学分析
所有数据用均数±标准差(x±s)表示,采用完全随机设计资料单因素方差分析进行显著性检验,若P<0.05,再进行q检验,比较任意两组间的差异有无统计学意义。采用单因素直线回归分析,比较干预因素与测量因素有无相关性。所有分析用SPSS13.0软件进行。
结果
1、在肺气肿形成0天和3个月时,E组、N组的氧化型纤维比例高于对照组(P<0.05)。在肺气肿形成6个月时,N组的氧化型纤维比例高于对照组(P<0.05),E组的氧化型纤维比例与对照组比较无显著差异(P>0.05)。在肺气肿形成12个月时,E组、N组的氧化型纤维比例低于对照组(P<0.05)。
2、年龄与氧化型纤维比例的相关分析结果显示正相关(P<0.05)。
3、在肺气肿形成0天和3个月时,E组、N组的mTOR蛋白高于对照组(P<0.05)。在肺气肿形成6个月时,N组的mTOR蛋白高于对照组(P<0.05),E组的mTOR蛋白与对照组比较无显著差异(P>0.05)。在肺气肿形成12个月时,E组、N组的mTOR低于对照组(P<0.05)。
结论
1、肺气肿大鼠膈肌氧化型纤维比例随疾病进展先增加后下降,说明膈肌出现代偿和失代偿过程。
2、随着年龄的增加,膈肌氧化型纤维比例逐渐升高。
3、肺气肿早期调节骨骼肌增生的信号蛋白mTOR蛋白表达高于对照组,肺气肿晚期mTOR蛋白表达低于对照组。
Purpose
The passive smoking was used to duplicate rat emphysema model. To detect the diaphragm fiber types in the whole course of emphysema with enzyme histochemistry. The mTor protein was detected by Western blotting techniques, which in order to explore the mechanism of diaphragmatic dysfunction in the molecular biological level.
Experimental materials and methods
One hundred and twenty healthy seven-week-old male Wistar rats were selected for study, weighting 250-280g, which came from the Experimental Animal Center of China Medical University. They were randomly divided into 3 groups:control group (C), emphysema and smoking group (E) and emphysema and smoking cessation group (N). On the very day of emphysema formation, and after 3 months,6 months and 12 months, we will randomly choose 10 rats for experiment. The rats were put into the home-made smoking capsule (volume 0.25 m3), exposure to 10 Huangguoshu cigarettes,1 hour each day,5 days/week, continued to 75 days. The main experimental methods including lung pathology with HE staining, detection of diaphragm muscle fiber with succinic acid dehydrogenase method and mTOR protein detection with Western Blotting. SPSS13.0 software was used for all data analysis. If P<0.05, statistics is of significance.
Results
1.In 3 months of pulmonary emphysema, and on the day of emphysema, oxidized fibers in E group and N group were significantly higher than that in C group(P<0.05). In 6 months of emphysema, oxidized fibers of N group were significantly higher than that of C group (P<0.05), the proportion of oxidized fibers in E group compared with the control group, there is no significant difference (P> 0.05). In emphysema of 12 months, E group, N group the proportion of oxidized fibers were higher than the control group (P<0.05).
2. The age and the proportion of oxidized fiber correlation analysis showed a positive correlation (P<0.05).
3. In 3 months of pulmonary emphysema, and on the day of emphysema, mTOR protein in E group and N group were significantly higher than that in C group(P<0.05). In 6 months of emphysema, mTOR protein of N group were significantly higher than that of C group (P<0.05), mTOR protein in E group compared with the control group, there is no significant difference (P> 0.05). In emphysema of 12 months, mTOR protein of E group and N group were higher than the control group (P<0.05).
Conclusions
1. The ratio of oxidized fibers in the emphysema rat diaphragm with disease progression first increased and then decreased, indicating compensation and decompensation occurred.
2. Increasing with age, the proportion of oxidized fibers gradually increased.
3. At early stage of emphysema, signaling protein mTOR protein in emphysema group were higher than that in control group.
引文
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2 Begin P, Grassino A. Inspiratory muscle dysfunction and chronic hypercapnia in chronic obstructive pulmonary disease. Am Rev Respir Dis.1991; 143:905-912.
3 Celli B. The diaphragm and respiratory muscles. Chest Surg Clin N Am.1998; 8:207-224.
4 Similowiski T, Yan S, Gauthier P, et al. Contractile properties of the humnan diaphragm during chronic hyperinflation. N Engl J Med.1991; 325:917-923.
5 Ottenheijm CA, Heunks LMA, Hafmans T, et al. Titin and diaphragm dysfunction in chronic obstructive pulmonary disease. Am J Respir Crit Care Med.2006; 173:527-534.
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