N-乙酰半胱氨酸对高氧致新生大鼠肺损伤中ROS、p-JNK及细胞凋亡的影响

发布时间：2018-07-03 20:04

本文选题：高氧 + 肺损伤　；参考：《南昌大学》2015年硕士论文

【摘要】：目的:1.参照Ozdemir[1]所建立的大鼠高氧肺损伤模型,观察高氧暴露后新生大鼠的肺组织病理变化情况。2.研究高氧致新生大鼠肺损伤肺组织中ROS、p-JNK蛋白的表达、细胞凋亡的情况及相互关系,探讨三者在高氧致新生大鼠肺损伤中的作用3.研究N-乙酰半胱氨酸(NAC)对高氧致新生大鼠肺损伤肺组织中ROS、p-JNK及细胞凋亡的影响,并初步探讨其可能的作用机制,为NAC成为早期防治新生儿高氧肺损伤的药物提供实验依据。方法:1.实验分组:将72只生后第3d的SD大鼠随机分为以下3组(每组n=24):空气+生理盐水组(空气组对照组)、高氧+生理盐水组(高氧暴露组)和高氧+N-乙酰半胱氨酸组(NAC干预组)。2.模型制作及药物干预:自制高氧氧箱,氧箱内持续通入100%氧气,将高氧暴露组和NAC干预组的新生大鼠置于高氧氧箱中,空气对照组置于同一室内的常压空气中。NAC组于实验第1d开始腹腔注射NAC 150mg/kg,连续14d,空气对照组和高氧暴露组于相同时间点腹腔注射等容积的生理盐水。3.标本的采集与检测指标:分别在实验第3d,7d,14d从各组中取8只新生鼠断头处死,开胸取肺组织。将右侧肺组织置于少量的PBS中,然后置于液氮中速冻,用Elisa法检测肺组织中ROS浓度;将左侧肺组织置于中性甲醛溶液中固定后石蜡包埋,用HE染色观察肺组织病理学变化,用免疫组化法检测p-JNK蛋白表达并计算累积光密度值(IOD),用TUNEL法检测肺组织细胞凋亡并计算凋亡指数(AI)。结果:1.肺组织病理学改变:在光镜下观察,空气对照组肺组织结构正常。高氧暴露组3d可见肺泡间隔充血水肿,少量炎症细胞渗出。高氧暴露组7d可见肺泡间隔增宽,充血水肿加重,大量炎症细胞浸润,部分肺泡腔内可见渗出液,肺组织结构紊乱。高氧暴露组14d可见肺泡间隔充血水肿减轻,炎症细胞减少,肺组织结构紊乱。NAC干预组各时间点较高氧暴露组肺泡间隔充血水肿减轻,炎症细胞减少,肺泡间隔厚度变薄,肺组织结构未见明显紊乱。2.肺组织中ROS浓度变化:高氧暴露组、NAC干预组各时间点的ROS浓度均高于空气对照组(P0.01);NAC干预组各时间点ROS浓度均低于高氧暴露组(P0.01)。空气对照组中各时间点之间的ROS浓度比较无统计学意义(P0.05);高氧暴露组中3d的ROS浓度开始升高,7d达到高峰,14d ROS浓度下降,各时间点两两比较差异均有显著统计学意义(P0.01);NAC干预组中3d ROS浓度开始升高,7d达到高峰,14d ROS浓度下降,各时间点两两比较差异有显著统计学意义(P0.01)。3.肺组织中p-JNK蛋白变化及分布:高氧暴露组、NAC干预组各时间点的p-JNK蛋白表达均高于空气对照组(P0.01),肺泡上皮细胞、血管内皮细胞、巨噬细胞及炎症细胞中均可见大量阳性细胞的表达;NAC干预组各时间点的p-JNK蛋白表达低于高氧暴露组(P0.01)。空气对照组中各时间点之间的p-JNK蛋白表达比较无统计学意义(P0.05);高氧暴露组中3d的p-JNK蛋白表达开始升高,7d达到高峰,14d p-JNK蛋白表达下降,各时间点两两比较差异有显著统计学意义(P0.01);NAC干预组中3d的p-JNK蛋白表达开始升高,7d达到高峰,14d p-JNK蛋白表达下降,各时间点两两比较差异有显著统计学意义(P0.01)。4.肺组织细胞凋亡的变化及分布:高氧暴露组、NAC干预组的凋亡细胞明显增加,肺泡上皮细胞、血管内皮细胞,巨噬细胞、炎症细胞中均可见大量凋亡细胞,各时间点凋亡指数与空气对照组相比显著升高(P0.01);NAC干预组各时间点凋亡指数均低于高氧暴露组(P0.01)。空气对照组中各时间点之间的凋亡指数比较无统计学意义(P0.05);高氧暴露组中3d的凋亡指数开始升高,7d达到高峰,14d凋亡指数下降,各时间点两两比较差异有显著统计学意义(P0.01);NAC干预组中3d的凋亡指数开始升高,7d达到高峰,14d凋亡指数下降,各时间点两两比较差异有显著统计学意义(P0.01)。5.ROS、p-JNK蛋白、AI的之间相关分析:高氧暴露组新生大鼠肺组织中ROS、p-JNK、AI两两均呈正相关(P0.01)。结论:1.我们已成功建立新生大鼠高氧肺损伤模型,高氧暴露后新生大鼠肺组织病理可见肺泡间膈增宽,充血水肿,炎症反应,肺组织结构紊乱等。2.高氧暴露后新生大鼠肺组织中ROS、p-JNK蛋白表达及细胞凋亡增加,提示ROS-JNK-细胞凋亡通路可能是高氧致新生大鼠肺损伤原因之一。3.高氧暴露组新生大鼠肺组织中ROS、p-JNK及凋亡指数两两相关性分析均成正相关,提示ROS、p-JNK和细胞凋亡可能在高氧致新生大鼠肺损伤中起协同作用。4.NAC可能通过抑制ROS的产生,减少p-JNK蛋白的表达,最终减轻细胞凋亡,从而发挥对高氧致新生大鼠肺损伤的保护作用。
[Abstract]:Objective: 1. to observe the rat model of hyperoxic lung injury established by Ozdemir[1] and observe the pathological changes of lung tissue in neonatal rats after hyperoxia exposure..2. studies the expression of ROS, p-JNK protein, apoptosis and their relationship in lung tissue of neonatal rats with hyperoxia induced lung injury, and explore the role of three in the lung injury induced by hyperoxia induced neonatal rats 3 The effects of N- acetyl cysteine (NAC) on ROS, p-JNK and apoptosis in lung tissue of neonatal rat lung injury induced by hyperoxia were investigated, and its possible mechanism was preliminarily explored to provide experimental basis for the early prevention and treatment of neonatal hyperoxic lung injury by NAC. Methods: 1. experimental groups were divided into 3 3D SD rats randomly divided into the following 3. Group (group n=24): air + saline group (air group control group), hyperoxia + physiological saline group (high oxygen exposure group) and hyperoxic +N- acetylcysteine group (NAC intervention group).2. model making and drug intervention: self-made hyperoxia box, oxygen box continuously through 100% oxygen, hyperoxic exposure group and NAC intervention group of newborn rats in hyperoxic box, The air control group was placed in the same indoor atmospheric pressure air in group.NAC to start the intraperitoneal injection of NAC 150mg/kg, continuous 14d, air control group and hyperoxic exposure group at the same time point of equal volume of equal volume of normal saline.3. samples collection and detection indicators: respectively in the experimental 3D, 7d, 14d from each group of 8 newborn rats at the end of the head. The right lung tissue was placed in the lung tissue. The right lung tissue was placed in a small amount of PBS and then frozen in liquid nitrogen. The concentration of ROS in the lung tissue was detected by Elisa. The left lung tissue was embedded in the neutral Formaldehyde Solution and embedded in paraffin. The pathological changes of lung tissue were observed by HE staining, and the expression of p-JNK protein was detected by immunohistochemical method and the cumulative light was calculated. The density value (IOD), TUNEL method was used to detect the apoptosis of lung tissue and calculate the apoptosis index (AI). Results: 1. the pathological changes of lung tissue: the lung tissue structure of the air control group was normal under the light microscope. The hyperoxia exposed group 3D showed alveolar septal congestion edema and a small amount of inflammatory cells exudate. The 7d of the hyperoxic exposure group showed the widening of the alveolar septum, hyperemia and edema. Heavy, large number of inflammatory cells infiltration, part of the alveolar cavity visible exudative fluid, lung tissue structure disorder. Hyperoxia exposure group 14d visible alveolar septal congestion edema, inflammatory cells decreased, lung tissue disorder.NAC intervention group at each time point higher oxygen exposure group alveolar septal filling water swelling, inflammatory cells decreased, the thickness of alveolar septum thinner, The concentration of ROS in.2. lung tissue was not significantly altered in the lung tissue structure: the concentration of ROS in the NAC intervention group was higher than that in the air control group (P0.01) at all time points in the hyperoxic exposure group, and the concentration of ROS in the NAC intervention group was lower than that of the hyperoxia group (P0.01) at all time points. The concentration of ROS was not statistically significant (P0.05) in the air control group (P0.05); The ROS concentration of 3D in the exposed group began to rise, the 7d reached the peak, the concentration of 14d ROS decreased, and the difference of each time point 22 was statistically significant (P0.01). The ROS concentration in NAC intervention group began to rise, the 7d reached the peak, the 14d ROS concentration decreased, and the 22 ratio of each time point was significant statistically significant. White change and distribution: the expression of p-JNK protein at all time points in the NAC intervention group was higher than that in the air control group (P0.01). The expression of a large number of positive cells in the alveolar epithelial cells, vascular endothelial cells, macrophages and inflammatory cells, and the expression of p-JNK protein in the NAC intervention group at each time point was lower than that of the hyperoxia group (P0.01). The expression of p-JNK protein in each time point in the group was not statistically significant (P0.05); the expression of p-JNK protein in 3D in the hyperoxic exposure group began to rise, the 7d reached the peak, the expression of 14d p-JNK protein decreased, and the difference of the time point 22 was statistically significant (P0.01). The p-JNK protein expression of 3D in the NAC intervention group began to rise and the 7d reached a higher level. The expression of peak and 14d p-JNK protein decreased, and there was significant statistical significance (P0.01) on the changes and distribution of apoptotic cells in the lung tissue of.4. (P0.01): the apoptotic cells in the hyperoxic exposure group, the NAC intervention group increased obviously, and the apoptotic cells in the alveolar epithelial cells, the vascular endothelial cells, the macrophages and the inflammatory cells were all seen in a large number of apoptotic cells, at all time points. The apoptotic index was significantly higher than that in the air control group (P0.01), and the apoptotic index at all time points in the NAC intervention group was lower than that in the hyperoxia group (P0.01). The apoptosis index was not statistically significant (P0.05) in the air control group (P0.05), and the number of apoptotic index of 3D in the hyperoxic exposure group began to rise, the 7d reached the peak, and the 14d apoptosis index decreased at every time. There was significant statistical significance (P0.01). The apoptosis index of 3D in the NAC intervention group began to rise, the 7d reached the peak, the 14d apoptosis index decreased, and the difference of the time point 22 had significant statistical significance (P0.01).5.ROS, p-JNK protein and AI: ROS, p-JNK, AI 22 in the lung tissue of the neonatal rats with hyperoxia exposure. Positive correlation (P0.01). Conclusion: 1. we have successfully established the neonatal rat model of hyperoxic lung injury. After hyperoxia exposure, the lung tissue of newborn rats can see the widening of the phrenic diaphragm, the congestion and edema, the inflammatory reaction, the disorder of the lung tissue, and so on. The expression of ROS, the expression of p-JNK protein and the increase of apoptosis in the lung tissue of the newborn rats after.2. hyperoxia are increased, suggesting ROS- JNK- cell apoptosis pathway may be one of the causes of lung injury in neonatal rats induced by hyperoxia.3. hyperoxic exposure group, ROS, p-JNK and apoptotic index 22 correlation analysis are all positive correlation, suggesting that ROS, p-JNK and apoptosis may play a synergistic role in the lung injury induced by hyperoxia rats,.4.NAC may be induced by the inhibition of ROS production. It can reduce the expression of p-JNK protein and ultimately reduce cell apoptosis, thereby playing a protective role in lung injury induced by hyperoxia in neonatal rats.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R722.1

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