氢气在高氧肺损伤修复中的作用及相关机制研究

发布时间：2018-05-03 05:41

本文选题：高氧肺损伤 + 肺泡Ⅱ型上皮细胞　；参考：《重庆医科大学》2013年博士论文

【摘要】：背景氧疗是临床常用的一种治疗手段，但持续高浓度氧疗易引起机体氧中毒，在新生儿特别是早产儿易导致慢性肺疾病（CLD）或支气管肺发育不良（BPD）的发生，严重影响患儿健康，目前尚无确切有效的防治方法。肺泡上皮损伤的正常修复主要依赖肺泡Ⅱ型上皮细胞（AECⅡ）的增殖与分化，高氧导致AECⅡ氧化应激性损伤，并抑制AECⅡ增殖是BPD发生的主要机制之一。传统抗氧化剂在减轻高氧肺损伤的同时干扰了正常肺发育。氢气（H_2）的选择性抗氧化作用和相对安全性使H_2成为治疗多种疾病的研究热点，但其具体分子机制不清。叉头框蛋白O（FoxO）是一类与氧化应激、凋亡、增殖、发育等密切相关的转录因子，其活性受丝裂原活化蛋白激酶（MAPKs）和磷酸酰肌醇3激酶/蛋白激酶B（PI3K/Akt）等多种信号途径调控，而业已证实H_2对MAPKs信号通路中重要的关键酶细胞外调节蛋白激酶（ERK）、c-Jun氨基末端激酶（JNK）、P38等的活性和PI3K/Akt信号通路中Akt活性均具有调控作用。我们推测H_2可能通过调控FoxO信号途径对高氧肺损伤发挥保护作用。深入研究H_2在高氧肺损伤中的作用及其FoxO信号机制可能为临床防治BPD带来新突破，为H_2的机制研究带来新进展。目的 1.分离培养高纯度、高活力的原代早产大鼠AECⅡ细胞；建立高氧致AECⅡ细胞损伤模型；建立高氧致新生鼠肺损伤动物模型。为后续H_2干预实验及机制研究奠定基础。 2.观察H_2对高氧致AECⅡ细胞损伤的作用，探讨其作用是否与FoxO信号途径有关。 3.观察H_2对高氧致新生鼠肺损伤的作用，探讨FoxO信号途径在其中的可能机制。方法 1. SPF级孕19d Sprague-Dawley(SD)大鼠，水合氯醛麻醉后剖宫产取出胎鼠，分离肺脏，剪碎，胰蛋白酶联合胶原酶消化肺组织细胞，制成细胞悬液，差速离心和反复贴壁纯化AECⅡ，含10%胎牛血清（FCS）的DMEM/F12培养基培养细胞。台盼蓝染色法检测细胞活力，改良巴氏染色法检测细胞纯度，透射电镜鉴定细胞，倒置相差显微镜下观察细胞的生长情况。 2.原代AECⅡ体外培养24h后，随机分为空气组和高氧组，高氧组细胞置于氧体积分数为95%（95%浓度氧）的细胞氧仓中，氧仓与空气组细胞一并放于细胞培养箱中。24h后观察细胞形态，MTT检测细胞增殖，流式细胞仪检测细胞的凋亡及存活情况。 3. SD新生大鼠随机分为空气组和高氧组，高氧组大鼠置于氧体积分数为95%的动物氧仓中，与空气组大鼠置于同一室内。3d，7d，14d，21d后取出肺组织行病理学检查，辐射状肺泡计数（RAC），化学比色法测定肺组织羟脯氨酸（HYP）含量。 4.原代分离培养的AECⅡ随机分为空气组、高氧组、空气+H_2组、高氧+H_2组。H_2组细胞用富氢培养基干预。24h后观察AECⅡ的形态变化；检测MTT、细胞周期和增殖细胞核抗原（PCNA）蛋白表达观察细胞增殖情况；检测细胞线粒体膜电位（△Ψ）和凋亡率观察细胞损伤情况；检测细胞内活性氧（ROS）和超氧化物阴离子（O-2）水平，细胞培养上清丙二醛（MDA）水平和超氧化物歧化酶（SOD）活性，观察细胞氧化损伤和抗氧化能力；Western Blot检测细胞总FoxO3a、β-catenin蛋白和p-FoxO3a、p-β-catenin蛋白的表达。 5. SD新生大鼠随机分为空气组、空气+富氢生理盐水组、空气+H_2组、高氧组、高氧+富氢生理盐水组和高氧+H_2组。各高氧组大鼠均置于氧体积分数为95%的动物氧仓中。H_2干预：富氢生理盐水组大鼠予腹腔注射富氢生理盐水10mL/kg，每天2次；H_2组大鼠予腹腔注射H_2气体10mL/kg，每天2次。非H_2干预组大鼠则腹腔注射等量生理盐水。14d后，取肺组织做病理学检查；检测大鼠血清MDA水平和SOD活力；测定肺组织HYP含量；免疫组化法测定肺组织α-平滑肌激动蛋白（α-SMA）表达；Western blot检测肺组织总FoxO3a、β-catenin蛋白和p-FoxO3a、p-β-catenin蛋白的表达。结果 1.原代培养的AECⅡ产量较高，每只早产大鼠肺组织可获得(8.5±1.8)×106AECⅡ，细胞活力为(95.0±2.1)％，细胞纯度为（94.3±2.5）％。电镜可见AECⅡ的特征性结构--细胞膜表面的微绒毛和胞浆内的板层小体。AECⅡ体外培养12h左右开始贴壁生长，至18h绝大部分细胞已贴壁伸展，24-48h细胞生长良好，增殖活跃，处于对数生长期，72h后细胞状态逐渐变差，丧失功能。 2. AECⅡ予95%浓度氧刺激24h后，细胞出现皱缩变形，细胞间隙增大，增殖较空气组明显受抑，凋亡率明显增加，存活率明显降低。 3. SD新生大鼠高氧暴露3d和7d后肺组织出现肺泡上皮细胞肿胀，间质充血水肿，炎性细胞浸润，肺结构紊乱，7d更明显。14d和21d可见纤维增生，肺泡间隔明显增宽，肺组织HYP含量较空气组显著增高，21d更为明显。高氧组RAC值于7d，14d，21d显著低于空气组。 4.与空气组比较，空气+H_2组细胞总FoxO3a蛋白表达增加，p-FoxO3a蛋白表达降低，其余各指标均无显著差异。与空气组比较，高氧组细胞OD492值和PCNA蛋白表达明显降低，G1期细胞比例增多而S期细胞比例减少；细胞△Ψ降低，凋亡率增加；细胞内ROS和O-2水平增高；细胞上清MDA含量增高，SOD活性下降；细胞总FoxO3a蛋白表达增加，p-FoxO3a蛋白表达降低；总β-catenin蛋白表达降低，p-β-catenin蛋白表达增高。与高氧组比较，H_2干预可减轻高氧引起的上述改变。 5.与空气组比较，空气+富氢生理盐水组和空气+H_2组大鼠肺组织均有FoxO3a与β-catenin的轻微激活，其余各指标均无显著差异；与空气组比较，高氧组肺发育受阻，RAC值降低；肺组织间隔增宽，，纤维化明显，肺组织HYP含量增高，α-SMA表达增高；血清MDA水平升高，SOD活力降低；肺组织总FoxO3a蛋白表达增加，且较H_2干预空气组显著，p-FOXO3蛋白表达降低；总β-catenin蛋白表达增加，p-β-catenin蛋白表达亦增高。与高氧组比较，高氧+富氢生理盐水组和高氧+H_2组肺损伤均有所减轻，均一定程度恢复了高氧引起的上述改变。高氧+H_2组血清MDA水平和肺组织HYP含量较高氧+富氢生理盐水组低，差异有统计学意义，其余指标两组间无差异。结论 1.采用胰酶联合胶原酶消化、差速离心和反复贴壁的方法获得的原代AECⅡ产量、纯度和活力均较高，可满足细胞学实验研究的需要。AECⅡ体外培养24-48h生长状态最佳，适合做体外研究。 2.95%浓度氧可诱导AECⅡ的损伤、凋亡并抑制其增殖，也可导致新生鼠肺损伤和肺发育受阻，成功建立高氧致AECⅡ细胞损伤模型和高氧致新生鼠肺损伤动物模型。 3.富氢培养基能一定程度减轻高氧导致的AECⅡ凋亡、氧化损伤，并促进其增殖，而对正常AECⅡ的增殖无明显作用。 4.腹腔注射富氢生理盐水和H_2气体均可有效减轻高氧导致的肺损伤，减轻肺纤维化，腹腔注射H_2气体的效果略佳。H_2对正常肺组织无明显作用。 5. H_2对高氧导致的细胞和肺损伤的保护作用可能与抑制高氧导致的FoxO3a蛋白过度活化并激活β-catenin蛋白有关。
[Abstract]:background
Oxygen therapy is a commonly used therapeutic method, but continuous high concentration oxygen therapy is easy to cause oxygen poisoning in the body. It is easy to cause chronic lung disease (CLD) or bronchopulmonary dysplasia (BPD) in newborns, especially premature infants, which seriously affect the health of children. At present, there is no effective method for prevention and treatment. The normal repair of alveolar epithelial injury is the main method. The proliferation and differentiation of pulmonary alveolar type II epithelial cells (AEC II), hyperoxia induced oxidative stress injury of AEC II and the inhibition of AEC II proliferation are one of the main mechanisms of BPD. The traditional antioxidants interfere with normal lung development while reducing hyperoxia lung injury. The selective antioxidant and relative safety of hydrogen (H_2) make H_2 a treatment. O (FoxO) is a kind of transcription factors closely related to oxidative stress, apoptosis, proliferation and development, and its activity is regulated by mitogen activated protein kinase (MAPKs) and phosphoinositide 3 kinase / egg white kinase B (PI3K/Akt) and other signaling pathways, and H has been confirmed. _2 plays a regulatory role in the activity of the key enzyme extracellular regulated protein kinase (ERK), c-Jun amino terminal kinase (JNK), P38, and the activity of Akt in the PI3K/Akt signaling pathway in the MAPKs signaling pathway. We speculate that H_2 may play a protective role in hyperoxic lung injury by regulating FoxO signaling pathway. Its role and its FoxO signaling mechanism may bring new breakthroughs in clinical prevention and treatment of BPD, and bring new progress in the research of H_2 mechanism.
objective
1. the primary preterm AEC II cells of high purity and high vitality were isolated and cultured, and the damage model of AEC II cells induced by hyperoxia was established, and the animal model of lung injury induced by hyperoxia was established, which laid the foundation for the subsequent H_2 intervention experiment and mechanism research.
2. to observe the effect of H_2 on the injury of AEC II cells induced by hyperoxia, and to find out whether it is related to the FoxO signaling pathway.
3. to observe the effect of H_2 on lung injury induced by hyperoxia in neonatal rats, and explore the possible mechanism of FoxO signaling pathway.
Method
1. SPF grade pregnant 19d Sprague-Dawley (SD) rats were treated with chloral hydrate and caesarean section after caesarean section. The lungs were separated, cut and broken, and trypsin combined with collagenase to digest the lung tissue cells to make cell suspension, differential centrifugation and repeated adherence to AEC II, and DMEM/F12 culture medium containing 10% fetal bovine serum (FCS). Trypan blue staining method was used to detect the cells. The cell purity was detected by modified PAP staining, the cells were identified by transmission electron microscope, and the growth of cells was observed under inverted phase contrast microscope.
2. primary AEC II was cultured for 24h in vitro. The cells were randomly divided into air group and hyperoxia group. The cells in the hyperoxic group were placed in the oxygen chamber of 95% (95% oxygen). The oxygen storehouse and the air group cells were placed in the cell culture box to observe the cell morphology, the cell proliferation was detected by MTT, and the apoptosis and survival of the cells were detected by flow cytometry.
3. SD neonatal rats were randomly divided into the air group and the hyperoxia group. The rats in the hyperoxic group were placed in the oxygen barn of 95% of the oxygen volume fraction, and the rats in the air group were placed in the same indoor.3d, 7d, 14d, and 21d. The lung tissue was taken out for pathological examination, radiated alveolar count (RAC), and chemical colorimetry was used to determine the content of hydroxyproline (HYP) in the lung tissue.
4. AEC II primary isolation and culture were randomly divided into air group, hyperoxia group, air +H_2 group, and.H_2 group of hyperoxic +H_2 group to observe the morphological changes of AEC II after.24h rich medium in hydrogen rich medium; detect MTT, cell cycle and proliferating cell nuclear antigen (PCNA) protein expression to observe cell proliferation; detect cell mitochondrial membrane potential (delta) and apoptosis Rate of cell damage and detection of intracellular reactive oxygen species (ROS) and superoxide anion (O-2), cell culture and superoxide dismutase (MDA) level and superoxide dismutase (SOD) activity, cell oxidative damage and antioxidant capacity were observed, and Western Blot was used to detect the total FoxO3a, beta -catenin protein and p-FoxO3a, p- beta -catenin protein. Expression.
5. SD neonatal rats were randomly divided into air group, air + rich saline group, air +H_2 group, hyperoxia group, high oxygen + hydrogen rich physiological saline group and hyperoxic +H_2 group. The rats in each hyperoxic group were given.H_2 intervention in the oxygen volume fraction of the oxygen volume fraction of the animal oxygen storage group: the hydrogen rich saline group rats were injected with the hydrogen rich physiological saline 10mL/kg, 2 times a day, H_. The 2 groups of rats were intraperitoneally injected with H_2 gas 10mL/kg, 2 times a day. The rats in the non H_2 intervention group were intraperitoneally injected with the same amount of normal saline.14d, the lung tissue was taken for pathological examination, the serum MDA level and SOD activity were detected, the HYP content in the lung tissue was measured, the expression of alpha smooth muscle agonist (alpha -SMA) in lung tissue was determined by immunohistochemistry; Western blo was measured. T was used to detect the expression of total FoxO3a, beta -catenin protein, p-FoxO3a and p- beta -catenin in lung tissue.
Result
1. primary culture AEC II produced a higher yield. The lung tissue of each preterm rat could obtain (8.5 + 1.8) x 106AEC II, cell viability was (95 + 2.1)%, and the cell purity was (94.3 + 2.5)%. The microvilli on the surface of AEC II and the lamellar body.AEC II in the cytoplasm of the cell membrane began to adhere to the wall and began to adhere to the wall and began to adhere to the wall and began to adhere to the wall, to 18h Most of the cells had been adhered to the wall. 24-48h cells grew well, proliferated, and were in logarithmic phase. After 72h, the cell state gradually became worse and lost function.
After 2. AEC II to 95% concentration of oxygen to stimulate 24h, the cells were wrinkled and the cell space was increased, the proliferation was obviously suppressed in the air group, the rate of apoptosis increased obviously, and the survival rate decreased obviously.
3. SD newborn rats exposed to 3D and 7d of hyperoxia, pulmonary alveolar epithelial cells swelling, interstitial congestion edema, inflammatory cell infiltration, lung structure disorder, 7d more obvious.14d and 21d visible fiber proliferation, alveolar septum obviously widened, lung tissue HYP content is significantly higher than the air group, 21d more obvious. RAC in 7d, 14d, 21d significantly lower than the high oxygen group. Air group.
4. compared with the air group, the expression of total FoxO3a protein in the air +H_2 group increased, the expression of p-FoxO3a protein decreased, and the other indexes had no significant difference. Compared with the air group, the OD492 and PCNA protein expression in the hyperoxic group decreased obviously, the proportion of the cells in the G1 phase increased and the proportion of the S phase decreased; the cell delta decreased and the apoptosis rate increased; the intracellular rate of apoptosis was increased. The level of ROS and O-2 increased, the content of MDA in cell supernatant increased, the activity of SOD decreased, the expression of total FoxO3a protein in cell increased, the expression of p-FoxO3a protein decreased, the expression of total beta -catenin protein decreased, and the expression of p- beta -catenin protein increased. Compared with the hyperoxia group, the H_2 intervention could reduce the above changes caused by hyperoxia.
5. compared with the air group, there was a slight activation of FoxO3a and beta -catenin in the lung tissue of the air + rich saline group and the air +H_2 group, and there was no significant difference in the other indexes. Compared with the air group, the lung development was blocked, the RAC value was reduced, the pulmonary tissue interval was broadened, the fibrination was obvious, the HYP content of the lung tissue was increased and the expression of alpha -SMA was increased. The serum MDA level increased, the activity of SOD decreased, the expression of total FoxO3a protein in the lung increased, and the expression of p-FOXO3 protein was lower than that of the H_2 intervention air group, the expression of the total beta -catenin protein was increased and the expression of p- beta -catenin protein increased. Compared with the hyperoxia group, the lung injury in the hyperoxic + rich saline group and the hyperoxic +H_2 group were all relieved, and the homogenization of lung injury was all The level of hyperoxia caused by hyperoxia was restored. The level of serum MDA in the hyperoxic +H_2 group and the higher HYP content in the lung tissue were lower in the oxygen + rich saline group, and there was no difference between the two groups.
conclusion
1. the production of primary AEC II was obtained by trypsin combined with collagenase digestion, differential centrifugation and repeated adherence, with high purity and vitality, which could meet the needs of.AEC II in vitro culture of 24-48h and be suitable for in vitro study.
2.95% concentration of oxygen can induce AEC II damage, apoptosis and inhibit its proliferation, and can also lead to lung injury and lung development in newborn rats. The model of hyperoxia induced AEC II cell damage and hyperoxia induced lung injury in neonatal rats were successfully established.
3. hydrogen rich medium can reduce the apoptosis and oxidative damage of AEC II induced by hyperoxia to a certain extent, and promote its proliferation, but has no obvious effect on the proliferation of normal AEC II.
4. the intraperitoneal injection of hydrogen rich saline and H_2 gas can effectively reduce the lung injury caused by hyperoxia, reduce pulmonary fibrosis, and the effect of intraperitoneal injection of H_2 gas is slightly better.H_2 has no obvious effect on normal lung tissue.
The protective effect of 5. H_2 on hyperoxia induced cell and lung injury may be related to the inhibition of hyperoxia induced FoxO3a protein overactivation and activation of beta -catenin protein.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R726.1

【参考文献】