急性高氧氧应激过程中大鼠肺泡上皮细胞凋亡的损伤机制探讨

发布时间：2018-03-28 02:17

本文选题：高浓度氧　切入点：肺泡上皮细胞　出处：《苏州大学》2012年博士论文

【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。
[Abstract]:Objective: to establish a high concentration of oxygen induced apoptosis in rat alveolar injury model, observed in a short period of time (4~8 hours) high concentration oxygen inhalation injury were observed in alveolar epithelial cells; nitric oxide and nitric oxide synthase on acute inhalation of high concentration of oxygen leads to apoptosis of rat alveolar epithelial cells; to investigate the mitochondrial pathway and cell apoptosis in acidizing the signal transduction process of alveolar epithelial cells induced by oxidative stress in the early stage.
Methods: WISTAR rats as the oxygen concentration of oxygen in the 4,8,12,16 box 80 - 100% hours, air control group placed on the oxygen concentration of oxygen tank 21%, the experiment was divided into three parts, the following test according to different experimental purposes: 1. samples for colorimetric determination of plasma and lung homogenate (C two aldehyde MDA), superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), nitric oxide (NO), nitric oxide synthase (NOS) and total antioxidant capacity (T-AOC); observe the pathological changes of lung tissue HE staining, TUNEL staining method to detect lung surface global cell apoptosis; detection of lung tissue 2. Western blot expression of eNOS and iNOS protein, RT-PCR eNOSmRNA detection, RT-PCR detection of bcl-2mRNA expression of.3., iNOSmRNA baxmRNA, eNOSmRNA iNOSmRNA, Western blot expression, bcl-2 detection, Bax, phosphorylation of Akt1/ -Akt1 expression, mTOR/ phosphorylation of -mTOR protein was detected by BCECF-AM; The pH value of plasma and Flowcytometry (FCM) were used to detect the cell proliferation index.
Results: 1. compared with the control group (2.17% apoptosis), high concentration oxygen inhalation for 4 hours, the cell apoptosis of the alveolar surface (9.13 + 3.2%), the number of apoptotic cells in 8~12 hour group increased significantly (17.47 + 3.5%, 19.22 + 4.5%), 16 hours group decreased (11.03 + 2.8%).HE the 4 hour group lung staining showed mild congestion, 8 hours group of pulmonary capillary dilation, hyperemia, extravasation of red blood cells and mild pulmonary edema.16 hours group began to appear in neutrophils in inflammatory cells, pulmonary edema, pulmonary bulla and atelectasis, disorder or even visible lung tissue, structure. Serum. Lung tissue homogenate MDA, NO, NOS were significantly increased (P < 0.01), while SOD, GSH, CAT, T-AOC were significantly decreased (P < 0.01).2. Westenblot detection, high oxygen group 4h eNOS expression began to increase, 8h, 12h group after the expression of eNOS protein in 16h group increased significantly, but slightly higher expression The high oxygen groups decreased; the expression of eNOSmRNA was significantly increased. The control group iNOS protein expression, compared with the control group, 4h, 8h, 12h group was no significant difference, the expression is much lower than that of eNOS, 16h expression was slightly enhancement; the expression of iNOSmRNA in group 16h also enhanced.3.RT-PCR detected oxygen group bcl-2mRNA decreased obviously. The expression of baxmRNA was significantly increased; to detect the expression of Bax protein Western blot gradually increased, Bcl-2 protein, AKT-1/PhosphoAkt-1 (Ser473) protein, mTOR/Phospho-mTOR protein expression decreased gradually; high oxygen stress in rat alveolar epithelial cells Akt/PKB protein phosphorylation are decreased with the oxygen time prolonged, the expression of Akt/mTOR signal transduction protein significantly decreased; BCECF-AM detection cytoplasm pH value showed that with prolonging the time of oxygen inhalation, the level of serum ROS increased, FL1/FL2 ratio decreased, apoptosis ratio is also increasing The acidification of cells in each period was significant, and the changes of cell proliferation and plasma acidity were the same in each group.
Conclusion: 1. rats in acute hyperoxia induced oxidative stress model experiment, short time inhalation of high concentration of oxygen leads to acute oxidative stress can lead to apoptosis of alveolar epithelial cells was significantly increased, and with the time prolonging oxygen damage, damage to the development of inflammation.2. oxygen stress early apoptosis (4-8h) NO significantly increased iNOS induced.16h and iNOSmRNA expression was slightly enhanced by eNOS expression, but not high, inflammatory performance is not obvious, that inflammatory cell function has not been fully activated.3. mitochondrial membrane permeability and mitochondrial permeability transition pore opening, BCL-2/BAX decreased, AKT/mTOR expression and phosphorylation were reduced, resulting in abnormal PI3/Akt/mTOR signal transduction pathway. The high oxygen stress early mitochondrial pathway is the main signal transduction of apoptosis of alveolar epithelial cells in high oxygen stress path.4. ROS and damage the fibrinolytic enzyme The stability of membrane and soluble active substance in vivo enzyme release leads to mitochondrial damage is more serious, the apoptosis mechanism to promote the participation of the lysosomal mitochondrial pathway. But the early damage have yet to start other inflammatory reactions such as autophagy physiology, only apoptosis phenomenon is very obvious at the table.5. through the experimental observation that oxidative stress and induction the cytoplasm of alveolar epithelial cell apoptosis of ROS by direct acidification, damage the cell membrane to inhibit Na+/H+ exchange of intracellular acidification, or damage the lysosomal membrane and release large amounts of H+ into cells, leading to cell acidification, which facilitates intracellular acidic endonucleases and other bioactive substances play a role in inducing apoptosis and cell cytoplasm acidification. The occurrence of apoptosis there is a dose-response relationship, intracellular acidification can be associated with apoptosis, also may lead to mitochondrial, The damage of lysosome aggravates the cell death caused by the mitochondrial lysosome pathway, and its mechanism remains to be further studied.
This experiment using living cells of rats in vivo experiment, inhaled oxygen concentration and clinical anesthesia using the same oxygen concentration, similar to the time, the experimental results suggest that the clinical use of pure oxygen, there are security risks, but this damage is still in its early stages. With prolonging the time of oxygen inhalation injury, more inflammatory, will increase the degree of the damage mechanism also. Not very clear, needs further research.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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