【作者】 周海；

【导师】 杨建平；

【作者基本信息】 苏州大学 ， 临床麻醉， 2012， 博士

【摘要】 目的：建立高浓度氧诱导在体大鼠肺泡细胞凋亡损伤模型，观察短时间内（4～8小时）吸入高浓度氧肺泡上皮细胞的损伤；观察一氧化氮及其合酶对急性吸入高浓度氧导致大鼠肺泡上皮细胞凋亡的影响；探讨线粒体途径和细胞酸化在氧化应激早期诱导的肺泡上皮细胞凋亡信号传导过程中的作用。方法：WISTAR大鼠放至于氧浓度为80－100％的氧箱中4、8、12、16小时，空气对照组放置于氧浓度为21％的氧箱中，实验分三部分，按实验目的不同制取样本做如下测试:1.比色法测定血浆、肺组织匀浆中丙二醛（MDA）、超氧化物歧化酶(SOD）、谷光甘肽（GSH）、过氧化氢酶（CAT）、一氧化氮（NO）、一氧化氮合酶（NOS）和总抗氧化能力（T-AOC）；HE染色观察肺组织常规病理变化，TUNEL染色法检测肺泡表面凋亡细胞；2. Western blot检测肺组织中eNOS和iNOS蛋白表达，RT-PCR检测eNOSmRNA、iNOSmRNA的表达。3. RT-PCR检测bcl-2mRNA、baxmRNA、eNOSmRNA、iNOSmRNA的表达，Western blot检测bcl-2、bax、 Akt1/磷酸化-Akt1、mTOR/磷酸化-mTOR的蛋白表达；BCECF-AM检测细胞浆pH值，流式细胞仪(Flowcytometry,FCM)检测细胞增殖指数。结果：1.与对照组相比（凋亡2.17%），吸入高浓度氧4小时即出现典型的肺泡表面凋亡细胞(9.13±3.2％)，8～12小时组凋亡细胞的数量明显增加(17.47±3.5％、19.22±4.5％)，16小时组有减少的趋势(11.03±2.8％)。HE染色切片显示4小时组肺轻度充血，8小时组肺毛细血管扩张、充血、红细胞渗出、轻度肺水肿。16小时组开始出现以中性粒细胞为主的炎症细胞,肺组织水肿明显、肺大泡和肺不张，甚至可见肺组织增生、结构紊乱。各组血清、肺组织匀浆MDA、NO、NOS显著升高（P＜0.01），而SOD、GSH、CAT、T-AOC均显著降低（P＜0.01）。2. Westenblot检测发现，高氧4h组eNOS表达开始升高，8h、12h组后eNOS蛋白质表达升高明显,16h组高表达但略有下降；高氧各组eNOSmRNA表达显著增高。对照组iNOS蛋白微量表达,与对照组比较，4h、8h、12h组表达无显著差异，表达量远低于eNOS,16h组表达略增强；iNOSmRNA的表达在16h组表达也增强。3.RT-PCR检测发现吸氧各组bcl-2mRNA表达明显降低，baxmRNA表达显著增高；Western blot检测Bax蛋白表达逐渐增强，Bcl-2蛋白、AKT-1/PhosphoAkt-1(Ser473)蛋白、mTOR/Phospho-mTOR蛋白表达逐渐减弱；高氧氧应激后大鼠肺泡上皮细胞Akt/PKB蛋白磷酸化程度都降低，随吸氧时间延长，Akt/mTOR信号转导蛋白表达降低更加明显；BCECF-AM检测细胞浆pH值显示随着吸氧时间的延长，体内ROS水平的不断升高，FL1/FL2比值不断降低，凋亡的比例也不断增加。各时段细胞酸化均较显著，各组细胞增殖改变和细胞浆酸性变化趋势相一致。结论：1．大鼠活体急性高氧氧应激模型实验发现，短时间吸入高浓度氧导致急性氧应激时即可导致肺泡上皮细胞凋亡显著增加，且随着吸氧时间的延长损伤加重，损伤转为向炎症表现发展。2．氧应激凋亡早期（4-8h）NO大幅升高由eNOS表达增强引起的。16h iNOS和iNOSmRNA表达略增强，但并不高，炎性表现不明显，说明炎性细胞功能还未被完全激活。3.线粒体膜通透性改变和线粒体通透性转变孔道的开放，BCL-2/BAX减小，AKT/mTOR及其磷酸化表达均减少，导致PI3/Akt/mTOR信号途径转导异常，说明了高氧氧应激早期线粒体途径应是肺泡上皮细胞凋亡的主要信号传导路径。4.高氧氧应激后ROS同时损伤了溶酶体膜的稳定性，溶酶体内活性物质的释放导致线粒体损伤更加严重，促使溶酶体参与的线粒体途径的细胞凋亡机制的发生。但这种早期损伤还尚未启动其他如自噬等炎性生理反应，仅凋亡现象表现的非常明显。5.通过实验观察提示氧化应激诱导肺泡上皮细胞凋亡与细胞浆酸化有关，ROS通过直接损伤了细胞质膜抑制Na+/H+交换使细胞内酸化，或者损伤溶酶体膜，释放大量H+进入细胞浆，导致细胞酸化，这有利于细胞内酸性核酸内切酶等生物活性物质发挥作用，诱导凋亡。细胞浆酸化程度与细胞凋亡发生率存在某种量效关系，细胞酸化既可能是细胞凋亡的伴随现象，同时又可能导致线粒体、溶酶体损伤，加重线粒体-溶酶体途径引起的细胞死亡，其机制有待于进一步研究。本实验利用大鼠在体活细胞实验，吸入氧浓度与临床全麻使用氧浓度相同，时间相近，实验结果提示临床使用纯氧存在安全隐患，但这种损伤尚处于早期阶段。但随着吸氧时间的延长，损伤趋于炎性化，程度会加重。其损伤机理还不十分清楚，有待进一步研究。更多还原

【Abstract】 Oxidants play an important role in the pathogenesis of various airway diseases，and ithas been shown to induce two distinct types of cell death: programmed cell death(apoptosis) and necrosis. Genes controlls programmed cell death in two sorts, one isinhibiting cell death, the other is promoting cell death. The two sorts of genes interact witheach other in order to control cell normal death. ROS is increased in oxidative stress,which results in injury widely. However, whether and how hyperoxia induce apoptosis inalveolartype cells when respired high concentration oxygen in short-term remains unclear.To address these questions, we make use of respiring high concentration oxygen to inducealveolar epithelial Cell oxidative stress and test the matter of cytosolic acidification, andthen to observe the mechanisms of oxidative stress in cell apoptosis.Methods: WISTER-mice， weighing150-200g， were studied as follows:theexperimental mice were devided into four groups randomly and exposed to85-100％oxygen in a self-made chamber and the control animals were kept in normal room air.（1）The MDA、SOD、GSH、CAT、T-AOC、NO、NOS were detected in plasmaand lung homogenates in experimental4h、8h、12h、16h. Alveolar type cells apoptosis wasdetected by TUNEL technique.（2）To detect the effects of eNOS and iNOS in early stage of Alveolar epithelial cellsapoptosis induced by oxydative stress, RT-PCR and Western blot analysis were used.（3）To investigate the mechanisms of apoptosis in Alveolar epithelial cells inducedby oxydative stress, RT-PCR and Western blot test to detect Bax/Bcl-2mRNA、Bax/Bcl-2and AKT/mTOR protein changing were used.（4）To observe the role of intracellular acidification in alveolar epithelial Cellsinjured by oxydative stress, BCECF-AM Flowcytometry analysis was used. Results:（1）Compared with the control group, the levels of MDA、NO、NOS in plasm andlung tissue increased markedly in all hyperoxia groups (P＜0.01), and at the same timeSOD、GSH、CAT、T-AOC fell down accordingly(P＜O.O1). Correspondly, the number ofapoptosis cell increased to9.13±3.2％,17.47±3.5％,19.22±4.5%and11.03±2.8％in4hgroup,8h.group,12h.group and16h group respectively compared with the control group.（2）eNOSmRNA/protein expression was clear in control group, increased in4hgroup, increased obviously in8h.group,12h.group and16h group. iNOSmRNA/proteinexpression was far lower than that of eNOS in the control group, slightly increase in16hgroup. Index of plasma and lung tissue and the number of cell apoptosis had a significantpositive correlation.（3）During the ROS-induced Alveolar epithelial cells injuries，the apoptosis rate wasappeared correspond with the increased level of ROS. The ratio of Bax/Bcl-2mRNA andprotein was increased severely. The Akt/phospho-Akt at Ser-473andmTOR/phospho-mTOR at Ser-2481protein expressions decreased obviously.（4）Compared with control group, the pH-sensitive fluorescent probe BCECF-AMappeared cytoplasmic acidification in hyperoxia groups. FL1/FL2ratio decreased and CellProliferation Index increased significantly.Conclusions:1. By observing the apoptosis cellular nuclear morphology induced by oxydativestress,we considered that just for short-term respiring of high concentrate oxygen caninduce apoptosis in alveolar epithelial Cells and result in acute lung injury,and with thetime going,the injure aggravate.2. Our test results suggest that NO/NOS may play an very important role in apoptosisof rat alveolar epithelial Cells when exposed to hyperoxia. Especially at the early stage,eNOS is of prime inportance.3. In our test, BaxmRNA and Bax protein level was increased, Bcl-2mRNA and Bcl-2protein level was decreased, it suggested that ROS was related to apoptosis through Mitochondria injuring.By observing that the Akt/phospho-Akt at Ser-473protein，mTOR/phospho-mTOR at Ser-2481protein expressions obviously decreased, it indicatesthat Akt/mTOR signal pathway may be involved in apoptosis induced by Oxidative Stres.All of these suggested that Mitochondrial pathway may be the fundamental deathmechanism induced by oxydative stress at the early stage.4. By observing the changing of bcl-2/Bax and Akt/mTOR, it suggesteed that with themitochondria be injured,the lysosomal appeareed instability, and mitochondrial deathpathway maybe the main mechanism in ROS-induced alveolar epithelial Cells death. Onthe other hand, the lysosomal instability may aggravate the injury which happened onmitochondria in the early stage cauded by ROS.The balance between mitochondria andlysosome is important to cell survival.5.Lately,study on apoptosis suggested that there was intracelluar abnormal signalingtransduction. Intracellular acidification in these abnormal signaling, is a character ofapoptosis. Intracellular pH adjust maybe tightly related to apoptosis. FL1/FL2ratio andintracellular pH were all decreased, and cell number with acidic intracellular pH increasedinduced by Oxidative Stres. It suggests that oxydative stress induces alveolar epithelialCells death may be related to cytosolic acidification. Cytosolic acidification maybe alongwith apoptosis, moreover also result in mitochondrial and lysosomal injury, aggravatemitochondrial-lysosome cell death pathway,which mechanism remain be furtherinvestigated.更多还原