【作者】 蔡成；

【导师】 常立文；

【作者基本信息】 华中科技大学 ， 儿科学， 2009， 博士

【摘要】 第一部分高氧暴露所致线粒体呼吸链功能障碍与高氧肺损伤关系的研究实验一高氧暴露对早产新生大鼠肺组织线粒体呼吸链酶复合体Ⅲ、Ⅴ表达的影响【目的】研究高浓度氧暴露对早产新生大鼠肺组织线粒体呼吸链酶复合体Ⅲ（cytochrome b，Cytb）、Ⅴ（Adenosine Triphosphate Synthase6，8，ATPase6，8）动态表达的影响。探讨高浓度氧暴露所导致的线粒体呼吸链功能障碍与高氧肺损伤的关系。【方法】参照本研究室前期研究建立的早产鼠高氧肺损伤模型。早产新生SD大鼠生后1d随机分为空气组、高氧组。高氧组持续暴露于常压氧舱中，氧浓度为85％；空气组置于同一室常压空气中。两组分别于高氧或空气暴露后1、4、7、10和14d提取肺组织总RNA和蛋白，采取半定量逆转录聚合酶链反应（reverse transcription polymerase chain reaction，RT-PCR）测定Cytb、ATPase6，8 mRNA水平的动态表达；同时应用免疫组织化学方法（SP法）检测肺组织切片中Cytb蛋白分布和表达；应用Western blot检测肺组织Cytb蛋白水平的动态表达。【结果】（1）与空气组相比，高氧暴露1、4d Cytb mRNA含量及其表达显著增强（P＜0.05）；7d后Cytb呈下降趋势，其表达较空气组减弱，但两者相比差异无统计学意义（P＞0.05）。随日龄变化高氧暴露后，肺组织Cytb免疫组织化学结果与Cytb mRNA表达相似。（2） Western blot结果显示，与空气组相比，高氧暴露1、4d肺组织Cytb蛋白表达增强但差异无统计学意义（P＞0.05）；7d后Cytb呈逐渐下降趋势，其表达较空气组减弱，但7、10d两者相比差异无统计学意义，14d极显著减弱。（3）与空气组相比，高氧暴露1d时ATPase6 mRNA表达显著增强（P＜0.05），4d虽仍增强，但两者相比差异无统计学意义（P＞0.05），7、10d时减弱，但两者相比差异无统计学意义（P＞0.05），14d又出现极显著增强（P＜0.05）；与空气组相比，高氧暴露1、4d时ATPase8 mRNA表达减弱，但两者相比差异无统计学意义（P＞0.05），7d后开始出现增强，7、14d时显著增强（P＜0.05），10d时虽仍增强，但两者相比差异无统计学意义（P＞0.05）。【结论】高氧暴露诱导线粒体呼吸链酶复合体Ⅲ、Ⅴ表达水平的改变，可能导致线粒体呼吸链氧化磷酸化解偶联、“电子漏”产生ROS引起氧化应激和能量代谢障碍，这些变化共同参与高氧肺损伤的发生发展过程，因此，高浓度氧暴露所导致的线粒体呼吸链功能障碍可能在高氧肺损伤的发病过程中起重要作用。实验二高氧暴露对人肺腺癌A549细胞线粒体呼吸链酶复合体Ⅰ、Ⅳ表达的影响【目的】研究高浓度氧暴露对人肺泡Ⅱ型上皮细胞来源的肺腺癌A549细胞线粒体呼吸链酶复合体Ⅰ（NADH dehydrogenase subunit 1，ND1）、Ⅳ（cytochromeoxidaseⅠ，COXⅠ）动态表达的影响。探讨高浓度氧暴露所导致的线粒体呼吸链功能障碍与高氧肺损伤的关系。【方法】体外传代培养A549细胞系，待其在37℃5％CO₂培养箱中生长至接近亚汇合状态时，随机分为高氧组和空气组。高氧组持续暴露于常压高浓度氧中，氧浓度＞90％，CO₂浓度为5％；空气组仍置于含5％CO₂培养箱中。两组分别于高氧或空气暴露12、24、48 h提取A549细胞总RNA，采取半定量逆转录聚合酶链反应（RT-PCR）测定ND1、COXⅠmRNA的表达。【结果】（1）与同时间点空气组相比，高氧暴露12h A549细胞ND1 mRNA的表达增强，但两者相比差异无统计学意义（P＞0.05）；高氧组24h ND1 mRNA的表达显著减弱（P＜0.05）；高氧组48h ND1 mRNA的表达减弱，但两者相比差异无统计学意义（P＞0.05）。（2）与同时间点空气组相比，高氧暴露12h A549细胞COXⅠmRNA的表达增强，但两者相比差异无统计学意义（P＞0.05）；高氧暴露24、48h COXⅠmRNA的表达显著增强（P＜0.05），高氧暴露24h以后COXⅠmRNA的表达虽比空气组增强，但呈下降趋势。【结论】高氧暴露诱导A549细胞线粒体呼吸链ND1、COXⅠ表达水平的改变，提示高氧暴露所致的线粒体呼吸链功能障碍可能在高氧肺损伤的发病过程中起重要作用。第二部分高氧暴露对人肺腺癌A549细胞中硫氧还蛋白-2及细胞色素C表达的影响【目的】研究高浓度氧暴露对人肺泡Ⅱ型上皮细胞来源的肺腺癌A549细胞中硫氧还蛋白-2（thioredoxin-2，Trx-2）及细胞色素C（cytochrome c，Cytc）表达的影响，探讨Trx-2和Cytc的表达变化在高氧肺损伤发生发展中所起的作用，Trx-2对高氧肺损伤线粒体的保护作用。【方法】体外传代培养A549细胞系，待其在37℃5％CO₂培养箱中生长至接近亚汇合状态时，随机分为高氧组和空气组。高氧组持续暴露于常压高浓度氧中，氧浓度＞90％，CO₂浓度为5％；空气组仍置于含5％CO₂培养箱中。两组分别于高氧或空气暴露12、24、48 h提取A549细胞总RNA和蛋白，采取半定量逆转录聚合酶链反应（RT-PCR）测定Trx-2、Cytc mRNA的表达；采用Westernblot检测A549细胞Trx-2蛋白的表达变化。【结果】（1）与空气组相比，高氧暴露12 h A549细胞Trx-2 mRNA表达减弱，但两者相比差异无统计学意义（P＞0.05）。24 h Trx-2 mRNA表达显著增强（P＜0.05）；48 h后Trx-2呈下降趋势，其表达较空气组减弱，但两者相比差异无统计学意义（P＞0.05）。（2）与空气组相比，高氧暴露12h Cytc mRNA的表达显著增强（P＜0.05），24h Cytc mRNA的表达减弱，但两者相比差异无统计学意义（P＞0.05），48h显著减弱（P＜0.05）。（3）与空气组相比，高氧暴露12h Trx-2蛋白的表达显著增强（P＜0.05），24h Trx-2蛋白的表达仍增强，但两者相比差异无统计学意义（P＞0.05），随后逐渐减弱，至48h显著减弱（P＜0.05）。【结论】高浓度氧暴露诱导A549细胞中Trx-2和Cytc异常表达，提示Cytc在高氧肺损伤细胞凋亡发生过程中可能起重要作用，Trx-2对高氧肺损伤线粒体可能起重要的保护作用。第三部分小分子干扰RNA抑制高氧暴露下A549细胞中Trx-2表达及其与肺细胞代谢和凋亡的关系【目的】研究小分子干扰RNA（small interference RNA，SiRNA）抑制高氧暴露下人肺腺癌A549细胞中硫氧还蛋白-2（thioredoxin-2，Trx-2）表达及其与肺细胞代谢和凋亡的关系，探讨高氧肺损伤的发病机制和防治措施。【方法】体外传代培养A549细胞系，待其在37℃5％CO₂培养箱中生长至接近亚汇合状态时，将体外化学合成Trx-2序列特异性的SiRNA通过Lipofectamine 2000转染A549细胞。随机分为Ⅰ空气组，Ⅱ高氧组，Ⅲ小分子干扰RNA空气组，Ⅳ小分子干扰RNA高氧组。高氧组持续暴露于常压高浓度氧中，氧浓度为95％，CO₂浓度为5％；空气组仍置于含5％CO₂培养箱中。四组分别于高氧或空气暴露12、24、48h提取A549细胞总RNA和蛋白。采用半定量逆转录聚合酶链反应（RT-PCR）测定Trx-2、三磷酸腺苷合成酶6，8（ATPase6，8）及细胞色素C（cytochrome c，Cytc）mRNA的表达，采用Westernblot方法检测Trx-2蛋白的表达，流式细胞术检测高氧暴露和沉默Trx-2基因对A549细胞凋亡的影响。【结果】（1）序列特异性SiRNA可显著抑制Trx-2表达，Western blot检测显示Trx-2蛋白表达明显下降，SiRNA-1、SiRNA-2、SiRNA-3三组中Trx-2蛋白表达均受到抑制，SiRNA-1组抑制效率最高（79.51％）。（2）与空气组相比，高氧组24h Trx-2和Cytc mRNA的表达显著增高，但在48h Cytc mRNA的表达显著降低（P＜0.05）。与小分子干扰RNA空气组相比，小分子干扰RNA高氧组24、48h Trx-2mRNA的表达显著减弱但在12、24h Cytc mRNA表达水平显著升高（P＜0.05）。与高氧组相比，小分子干扰RNA高氧组48h Trx-2mRNA的表达显著减弱，而24h Cytc mRNA表达显著升高（P＜0.05）。（3）与空气组相比，高氧组12、48h ATPase6 mRNA的表达显著增高（P＜0.05），但在24、48h ATPase8 mRNA的表达显著增高（P＜0.05）。与小分子干扰RNA空气组相比，小分子干扰RNA高氧组24、48h ATPase6，8 mRNA的表达显著减弱（P＜0.05）。与高氧组相比，小分子干扰RNA高氧组12、48h ATPase6 mRNA的表达显著减弱，而12、48h ATPase8 mRNA表达显著升高（P＜0.05）。（4）与高氧组相比，小分子干扰RNA高氧组24h A549细胞凋亡百分数明显增高（P＜0.05），分别为（31.78±4.52）％VS（13.57±0.69）％；小分子干扰RNA高氧组24、48h A549细胞凋亡百分数明显高于小分子干扰RNA空气组（P＜0.05），分别为（31.78±4.52）％MS（15.47±0.57）％，（38.51±4.08）％VS（25.84±1.01）％。【结论】高氧暴露和沉默Trx-2基因导致A549细胞ATPase6，8表达异常和Cytc表达升高，细胞凋亡百分率显著增高，提示能量代谢障碍和细胞凋亡参与高氧肺损伤发病过程，Trx-2对高氧肺损伤线粒体起重要的保护作用，保护线粒体DNA（mtDNA）可能是Trx系统抗高氧肺损伤的重要机制。小结1本研究成功建立早产鼠高氧肺损伤动物模型和高氧暴露A549细胞损伤模型，为从线粒体呼吸链酶复合体结构和功能改变的角度探讨高氧肺损伤的发病机制奠定基础。2高氧暴露诱导线粒体呼吸链酶复合体Ⅰ、Ⅲ、Ⅳ、Ⅴ表达水平的改变，可能导致线粒体呼吸链氧化磷酸化解偶联、“电子漏”产生ROS引起氧化应激和能量代谢障碍，这些变化共同参与高氧肺损伤的发生发展过程，因此，高浓度氧暴露所导致的线粒体呼吸链功能障碍可能在高氧肺损伤发病过程中起重要作用。3高浓度氧暴露诱导A549细胞中Trx-2和Cytc异常表达，提示Cytc在高氧肺损伤细胞凋亡发生过程中可能起重要作用，Trx-2对高氧肺损伤线粒体可能起重要的保护作用。4高氧暴露和沉默Trx-2基因导致A549细胞ATPase6，8表达异常和Cytc表达升高，细胞凋亡百分率显著增高，提示能量代谢障碍和细胞凋亡参与高氧肿损伤发病过程，Trx-2对高氧肺损伤线粒体起重要的保护作用，保护线粒体DNA可能是硫氧还蛋白系统抗高氧肺损伤的重要机制。更多还原

【Abstract】 Part 1 An Investigation of the relationship of functional impairmentof mitochondrial respiration chain induced by hyperoxia andhyperoxia induced lung injuryExperiment 1 Effects of Hyperoxia on Mitochondrial MultienzymeComplexⅢandⅤin the Premature Newborn Rat Lung【Objective】The aim of this study is to investigate the effects of hyperoxia onmitochondrial multienzyme complexⅢ（cytochrome, Cytb） andⅤ（ATPase6, 8）in premature newborn rat lung. To explore the relationship of functional impairmentof mitochondrial respiration chain induced by hyperoxia and hyperoxia-inducedlung injury.【Methods】Based on the animal model of hyperoxia induced lung injury set upby our preliminary research, the 1-day-old preterm SD rats were randomly assignedto hyperoxia group and air group. The rats in hyperoxia group were continuouslyexposed to 85% oxygen and those in air group to room air. After 1, 4, 7, 10, 14 day（s）of exposure, these rats were killed, total lung RNA was extracted and Cytb, ATPase6,8 mRNA were detected by reverse transcription polymerase chain reaction （RT-PCR）.Immunohistochemistry was used to detect the distribution and expression of Cytb protein on lung sections. Western blot was used to detect the expression of Cytbprotein in lung tissue.【Results】（1） Compared with air group, Cytb mRNA expression wassignificantly increased after 1, 4 day（s） of exposure （P＜0.05）. The general tendencydecreased after 7 days, and its expression became weak but the difference of CytbmRNA expression was not significant between the two groups （P＞0.05）.（2） Western blot showed that Cytb protein expression was increased after 1,4day（s） of exposure, but the difference between the two groups was not significant.The general tendency was decreased after 7 days, and its expression became weakbut difference was not significant after 7, 10 days of exposure （P＞0.05）. At day 14its expression became significantly weak （P＜0.05）.（3） ATPase6 mRNA expression was significantly increased after 1 day ofexposure （P＜0.05） and did not show any significant change after 4, 7, 10 days ofexposure （P＞0.05）. At the 14th day, ATPase6 mRNA expression was significantlyincreased （P＜0.05）. ATPase8 mRNA expression did not show any significant changeafter 1, 4, 10 day（s） of exposure （P＞0.05）. At the 7th and 14th day, ATPase8 mRNAexpression was significantly increased （P＜0.05）.【Conclusion】We are led to conclude that exposure to high concentrations ofoxygen can significantly change the expression of Cytb and ATPase6,8 in prematurerat lung, which results in uncoupling of oxidative phosphorylation in mitochondrialrespiration chain, oxidative stress caused by ROS from "electron leakage", energymetabolism impairment, this indicated that these changes may participate in thephysiological and pathology processes of hyperoxia-induced lung injury. Functionalimpairment of mitochondrial respiration chain induced by hyperoxia palys animportant role in the development of hyperoxia-induced lung injury. Experiment 2 Effects of Hyperoxia on Mitochondrial MultienzymeComplexⅠandⅣin human adenocarcinoma of lung A549 cell【Objective】The aim of this study is to investigate the effects of hyperoxia onmitochondrial multienzyme complexⅠ（NADH dehydrogenase subunit 1, ND1） andⅣ（cytochrome oxidaseⅠ, COXⅠ）in human adenocarcinoma of lung A549 cell.To explore the relationship of functional impairment of mitochondrial respirationchain induced by hyperoxia and hyperoxia-induced lung injury.【Methods】A549 cells were gained by serial subcultivation in vitro. Whenapproaching to the condition of confluence in CO₂ culture chamber（37℃, 5% CO₂）,the cells were randomly divided into air group and hyperoxia group. Hyperoxiagroup was continuously exposed to oxygen （oxygen＞90%, carbon dioxide=5%）While air group still in specific room air （carbon dioxide=5%）. After exposure tooxygen or room air for 12, 24 and 48 hours, those cells ’RNA was isolated, ND1 andCOXⅠmRNA were detected by reverse transcription polymerase chainreaction（RT-PCR）.【Results】（1） Compared with air group, the expression of ND1 mRNA in A549cell was increased after 12 hours of exposure, but the difference of the two groupswas not significant （P＞0.05）. the expression of ND1 mRNA significantly decreasedafter 24 hours of exposure （P＜0.05）, ND1 mRNA expression decreased after 48hours of exposure, but the difference of the two groups was not significant （P＞0.05）.（2） Compared with air group, the expression of COXⅠmRNA in A549 cellwas increased after 12 hours of exposure, but the difference of the two groups wasnot significant （P＞0.05）. the expression of COXⅠmRNA was significantlyincreased after 24, 48 hours of exposure （P＜0.05）.【Conclusion】Exposure to high concentrations of oxygen can significantlychange the expression of ND1 and COXⅠin A549 Cell, which results in functionalimpairment of mitochondrial respiration chain induced by hyperoxia palys animportant role in the development of hyperoxia-induced lung injury. Part 2 Effects of Hyperoxia on the Expression of Thioredoxin-2 andCytochrome c in A549 Cell Exposure to Hyperoxia【Objective】To investigate the effects of the expression of thioredoxin-2 andcytochrome c in human adenocarcinoma of lung A549 cell from human alveolarepithelial cellⅡexposure to high concentrations of oxygen. And to explore thatwhat Trx-2 and Cytc have changed plays an important role in hyperoxia lung injury,Trx-2 likely protects mitochondria of A549 cell in hyperoxia lung injury.【Methods】A549 cells were gained by serial subcultivation in vitro. Whenapproaching to the condition of confluence in CO₂ culture chamber（37℃,5% CO₂）,the cells were randomly divided into air group and hyperoxia group. Hyperoxiagroup was continuously exposed to oxygen （oxygen＞90%, carbon dioxide=5%）While air group still in specific room air （carbon dioxide=5%）. After exposure tooxygen or room air for 12, 24 and 48 hours, those cells ’RNA and protein wereisolated, Trx-2 and Cytc mRNA were detected by reverse transcription polymerasechain reaction （RT-PCR）. Western-blot was used to detect Trx-2 protein in A549cells.【Results】（1） Compared with air group, Trx-2 mRNA expression became weakafter 12h of exposure （P＞0.05）, Trx-2 mRNA expression was significantlyincreased after 24 hours of exposure （P＜0.05）; its expression became weak after 48hours of exposure, but both were not significantly different （P＞0.05）（2） Compared with air group, Cytc mRNA expression was significantlyincreased after 12 hours of exposure （P＜0.05）; its expression became weak after 24hours of exposure, but both were not significantly different （P＞0.05）. itsexpression was significantly decreased after 48 hours （P＜0.05）.（3） Compared with air group, Trx-2 protein expression was significantly increased after 12 hours of exposure （P＜0.05）; the general tendency increasedafter 24 hours, but both were not significantly different （P＞0.05）.its expressionwas significantly decreased after 48 hours （P＜0.05）.【Conclusion】Exposure to high concentrations oxygen can significantly changethe expression of Trx-2 and Cytc in A549 cells, which indicates that Cytc plays animportant role in the process of cell apoptosis in hyperoxic lung injury, and Trx-2likely protects mitochondria of A549 cell in hyperoxic lung injury.Part 3 Correlation between Expression of Thioredoxin-2 Suppressedby Small Interference RNA in A549 Cells Exposed to Hyperoxia andthe Lung Cell Metabolism and Apoptosis【Objective】The aim of this study was to explore the expression ofthioredoxin-2 suppressed by SiRNA in A549 cells exposed to hyperoxia, and thecorrelation between with the lung cell metabolism and apoptosis, and to discuss thepathogenesis and control measures of hyperoxia induced lung injury.【Methods】A549 cells were gained by serial subcultivation in vitro. Whenapproaching to the condition of confluence in CO₂ culture chamber（37℃,5% CO₂）,A549 cells were transfected with synthetic Trx-2 sequence -specific siRNA byLipofectamine 2000. These cells were randomly divided into four groups:Ⅰairgroup,Ⅱhyperoxia group,Ⅲair group with small interference RNA, andⅣhyperoxia group with small interference RNA. Hyperoxia group was continuouslyexposed to oxygen （oxygen=95%, carbon dioxide=5%） While air group still inspecific room air （carbon dioxide=5%）. After exposure to oxygen or room air for 12, 24 and 48 hours, those cells ’RNA and protein were isolated, Trx-2, ATPase6,8 andCytc mRNA were detected by reverse transcription polymerase chain reaction（RT-PCR）,Trx-2 protein was detected by Western blot, Flow cytometry was used todetect the apoptosis of A549 cells suppressed by Trx-2 SiRNA.【Results】（1）Sequence-specific SiRNA targeting Trx-2 significantly down-regulatedits expression in A549 cells, the results of Western blot assay revealedTrx-2 protein levels were inhibited after transfection in different groups （SiRNA-1group、SiRNA-2 group、SiRNA-3 group）, and the highest inhibitory rate wasSiRNA-1 group （79.51%）.（2）Compared with air group, the expression of Trx-2 and Cytc mRNA weresignificantly increased after 24 hours exposure （P＜0.05）, but the expression of CytcmRNA became weak significantly after 48 hours exposure in hyperoxia group （P＜0.05）.Compared with air group with small interference RNA, the expression of Trx-2mRNA became weak significantly after 24、48 hours exposure （P＜0.05）, but theexpression of Cytc mRNA was significantly increased after 12、24 hours exposure inhyperoxia group with small interference RNA （P＜0.05）Compared with hyperoxia group, the expression of Trx-2 mRNA wassignificantly decreased after 48 hours exposure, but the expression of Cytc mRNAwas significantly increased after 24 hours exposure in hyperoxia group with smallinterference RNA （P＜0.05）（3）Compared with air group, the expression of ATPase6 mRNA wassignificantly increased after 12、48 hours exposure （P＜0.05）, but the expression ofATPase8 mRNA was significantly increased after 24、48 hours exposure in hyperoxiagroup （P＜0.05）.Compared with air group with small interference RNA, the expression ofATPase6,8 mRNA became weak significantly after 24、48 hours exposure in hyperoxia group with small interference RNA （P＜0.05）Compared with hyperoxia group, the expression of ATPase6 mRNA wassignificantly decreased after12, 48 hours exposure, but the expression of ATPase8mRNA was significantly increased after 12, 48 hours exposure in hyperoxia groupwith small interference RNA （P＜0.05）.（4） Compared with hyperoxia group, The apoptosis percent of A549 cells inhyperoxia group with small interference RNA was signifcantly higher after 24 hoursof exposure（P＜0.05）, they were（31.78±4.52）% VS （13.57±0.69）% respectively.The apoptosis percent of A549 cells in hyperoxia group with small interferenceRNA was signifcantly higher than air group with small interference RNA after 24、48 hours of exposure （P＜0.05）,they were （31.78±4.52） % VS （15.47±0.57）%, （38.51±4.08） %VS （25.84±1.01） %respectively.【Conclusion】Exposed to high concentrations oxygen can significantly changethe expression of Trx-2、ATPase6,8 and Cytc, the apoptosis percent of 549 cells wassignificantly higher, which indicates that dysmetabolism and cell apoptosisparticipate the development of hyperoxia induced lung injury, Trx-2 likely protectsmitochondria of A549 cells in hyperoxia lung injury. Protection of mitochondrialDNA （mtDNA） may be an important mechanism of anti-hyperoxic lung injury bythioredoxin system.更多还原