本文選題：高氧 + 肺損傷��； 參考：《南昌大學(xué)》2015年碩士論文

【摘要】：目的:1.參照Ozdemir[1]所建立的大鼠高氧肺損傷模型,觀察高氧暴露后新生大鼠的肺組織病理變化情況。2.研究高氧致新生大鼠肺損傷肺組織中ROS、p-JNK蛋白的表達(dá)、細(xì)胞凋亡的情況及相互關(guān)系,探討三者在高氧致新生大鼠肺損傷中的作用3.研究N-乙酰半胱氨酸(NAC)對(duì)高氧致新生大鼠肺損傷肺組織中ROS、p-JNK及細(xì)胞凋亡的影響,并初步探討其可能的作用機(jī)制,為NAC成為早期防治新生兒高氧肺損傷的藥物提供實(shí)驗(yàn)依據(jù)。方法:1.實(shí)驗(yàn)分組:將72只生后第3d的SD大鼠隨機(jī)分為以下3組(每組n=24):空氣+生理鹽水組(空氣組對(duì)照組)、高氧+生理鹽水組(高氧暴露組)和高氧+N-乙酰半胱氨酸組(NAC干預(yù)組)。2.模型制作及藥物干預(yù):自制高氧氧箱,氧箱內(nèi)持續(xù)通入100%氧氣,將高氧暴露組和NAC干預(yù)組的新生大鼠置于高氧氧箱中,空氣對(duì)照組置于同一室內(nèi)的常壓空氣中。NAC組于實(shí)驗(yàn)第1d開始腹腔注射NAC 150mg/kg,連續(xù)14d,空氣對(duì)照組和高氧暴露組于相同時(shí)間點(diǎn)腹腔注射等容積的生理鹽水。3.標(biāo)本的采集與檢測(cè)指標(biāo):分別在實(shí)驗(yàn)第3d,7d,14d從各組中取8只新生鼠斷頭處死,開胸取肺組織。將右側(cè)肺組織置于少量的PBS中,然后置于液氮中速凍,用Elisa法檢測(cè)肺組織中ROS濃度;將左側(cè)肺組織置于中性甲醛溶液中固定后石蠟包埋,用HE染色觀察肺組織病理學(xué)變化,用免疫組化法檢測(cè)p-JNK蛋白表達(dá)并計(jì)算累積光密度值(IOD),用TUNEL法檢測(cè)肺組織細(xì)胞凋亡并計(jì)算凋亡指數(shù)(AI)。結(jié)果:1.肺組織病理學(xué)改變:在光鏡下觀察,空氣對(duì)照組肺組織結(jié)構(gòu)正常。高氧暴露組3d可見肺泡間隔充血水腫,少量炎癥細(xì)胞滲出。高氧暴露組7d可見肺泡間隔增寬,充血水腫加重,大量炎癥細(xì)胞浸潤,部分肺泡腔內(nèi)可見滲出液,肺組織結(jié)構(gòu)紊亂。高氧暴露組14d可見肺泡間隔充血水腫減輕,炎癥細(xì)胞減少,肺組織結(jié)構(gòu)紊亂。NAC干預(yù)組各時(shí)間點(diǎn)較高氧暴露組肺泡間隔充血水腫減輕,炎癥細(xì)胞減少,肺泡間隔厚度變薄,肺組織結(jié)構(gòu)未見明顯紊亂。2.肺組織中ROS濃度變化:高氧暴露組、NAC干預(yù)組各時(shí)間點(diǎn)的ROS濃度均高于空氣對(duì)照組(P0.01);NAC干預(yù)組各時(shí)間點(diǎn)ROS濃度均低于高氧暴露組(P0.01)�？諝鈱�(duì)照組中各時(shí)間點(diǎn)之間的ROS濃度比較無統(tǒng)計(jì)學(xué)意義(P0.05);高氧暴露組中3d的ROS濃度開始升高,7d達(dá)到高峰,14d ROS濃度下降,各時(shí)間點(diǎn)兩兩比較差異均有顯著統(tǒng)計(jì)學(xué)意義(P0.01);NAC干預(yù)組中3d ROS濃度開始升高,7d達(dá)到高峰,14d ROS濃度下降,各時(shí)間點(diǎn)兩兩比較差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01)。3.肺組織中p-JNK蛋白變化及分布:高氧暴露組、NAC干預(yù)組各時(shí)間點(diǎn)的p-JNK蛋白表達(dá)均高于空氣對(duì)照組(P0.01),肺泡上皮細(xì)胞、血管內(nèi)皮細(xì)胞、巨噬細(xì)胞及炎癥細(xì)胞中均可見大量陽性細(xì)胞的表達(dá);NAC干預(yù)組各時(shí)間點(diǎn)的p-JNK蛋白表達(dá)低于高氧暴露組(P0.01)。空氣對(duì)照組中各時(shí)間點(diǎn)之間的p-JNK蛋白表達(dá)比較無統(tǒng)計(jì)學(xué)意義(P0.05);高氧暴露組中3d的p-JNK蛋白表達(dá)開始升高,7d達(dá)到高峰,14d p-JNK蛋白表達(dá)下降,各時(shí)間點(diǎn)兩兩比較差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01);NAC干預(yù)組中3d的p-JNK蛋白表達(dá)開始升高,7d達(dá)到高峰,14d p-JNK蛋白表達(dá)下降,各時(shí)間點(diǎn)兩兩比較差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01)。4.肺組織細(xì)胞凋亡的變化及分布:高氧暴露組、NAC干預(yù)組的凋亡細(xì)胞明顯增加,肺泡上皮細(xì)胞、血管內(nèi)皮細(xì)胞,巨噬細(xì)胞、炎癥細(xì)胞中均可見大量凋亡細(xì)胞,各時(shí)間點(diǎn)凋亡指數(shù)與空氣對(duì)照組相比顯著升高(P0.01);NAC干預(yù)組各時(shí)間點(diǎn)凋亡指數(shù)均低于高氧暴露組(P0.01)�？諝鈱�(duì)照組中各時(shí)間點(diǎn)之間的凋亡指數(shù)比較無統(tǒng)計(jì)學(xué)意義(P0.05);高氧暴露組中3d的凋亡指數(shù)開始升高,7d達(dá)到高峰,14d凋亡指數(shù)下降,各時(shí)間點(diǎn)兩兩比較差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01);NAC干預(yù)組中3d的凋亡指數(shù)開始升高,7d達(dá)到高峰,14d凋亡指數(shù)下降,各時(shí)間點(diǎn)兩兩比較差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01)。5.ROS、p-JNK蛋白、AI的之間相關(guān)分析:高氧暴露組新生大鼠肺組織中ROS、p-JNK、AI兩兩均呈正相關(guān)(P0.01)。結(jié)論:1.我們已成功建立新生大鼠高氧肺損傷模型,高氧暴露后新生大鼠肺組織病理可見肺泡間膈增寬,充血水腫,炎癥反應(yīng),肺組織結(jié)構(gòu)紊亂等。2.高氧暴露后新生大鼠肺組織中ROS、p-JNK蛋白表達(dá)及細(xì)胞凋亡增加,提示ROS-JNK-細(xì)胞凋亡通路可能是高氧致新生大鼠肺損傷原因之一。3.高氧暴露組新生大鼠肺組織中ROS、p-JNK及凋亡指數(shù)兩兩相關(guān)性分析均成正相關(guān),提示ROS、p-JNK和細(xì)胞凋亡可能在高氧致新生大鼠肺損傷中起協(xié)同作用。4.NAC可能通過抑制ROS的產(chǎn)生,減少p-JNK蛋白的表達(dá),最終減輕細(xì)胞凋亡,從而發(fā)揮對(duì)高氧致新生大鼠肺損傷的保護(hù)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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R722.1

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