本文選題：高氧肺損傷 + 肺泡Ⅱ型上皮細(xì)胞　； 參考：《重慶醫(yī)科大學(xué)》2013年博士論文

【摘要】：背景 氧療是臨床常用的一種治療手段，但持續(xù)高濃度氧療易引起機(jī)體氧中毒，在新生兒特別是早產(chǎn)兒易導(dǎo)致慢性肺疾�。–LD）或支氣管肺發(fā)育不良（BPD）的發(fā)生，嚴(yán)重影響患兒健康，目前尚無確切有效的防治方法。肺泡上皮損傷的正常修復(fù)主要依賴肺泡Ⅱ型上皮細(xì)胞（AECⅡ）的增殖與分化，高氧導(dǎo)致AECⅡ氧化應(yīng)激性損傷，并抑制AECⅡ增殖是BPD發(fā)生的主要機(jī)制之一。傳統(tǒng)抗氧化劑在減輕高氧肺損傷的同時(shí)干擾了正常肺發(fā)育。氫氣（H_2）的選擇性抗氧化作用和相對(duì)安全性使H_2成為治療多種疾病的研究熱點(diǎn)，但其具體分子機(jī)制不清。叉頭框蛋白O（FoxO）是一類與氧化應(yīng)激、凋亡、增殖、發(fā)育等密切相關(guān)的轉(zhuǎn)錄因子，其活性受絲裂原活化蛋白激酶（MAPKs）和磷酸酰肌醇3激酶/蛋白激酶B（PI3K/Akt）等多種信號(hào)途徑調(diào)控，而業(yè)已證實(shí)H_2對(duì)MAPKs信號(hào)通路中重要的關(guān)鍵酶細(xì)胞外調(diào)節(jié)蛋白激酶（ERK）、c-Jun氨基末端激酶（JNK）、P38等的活性和PI3K/Akt信號(hào)通路中Akt活性均具有調(diào)控作用。我們推測H_2可能通過調(diào)控FoxO信號(hào)途徑對(duì)高氧肺損傷發(fā)揮保護(hù)作用。深入研究H_2在高氧肺損傷中的作用及其FoxO信號(hào)機(jī)制可能為臨床防治BPD帶來新突破，為H_2的機(jī)制研究帶來新進(jìn)展。 目的 1.分離培養(yǎng)高純度、高活力的原代早產(chǎn)大鼠AECⅡ細(xì)胞；建立高氧致AECⅡ細(xì)胞損傷模型；建立高氧致新生鼠肺損傷動(dòng)物模型。為后續(xù)H_2干預(yù)實(shí)驗(yàn)及機(jī)制研究奠定基礎(chǔ)。 2.觀察H_2對(duì)高氧致AECⅡ細(xì)胞損傷的作用，探討其作用是否與FoxO信號(hào)途徑有關(guān)。 3.觀察H_2對(duì)高氧致新生鼠肺損傷的作用，探討FoxO信號(hào)途徑在其中的可能機(jī)制。 方法 1. SPF級(jí)孕19d Sprague-Dawley(SD)大鼠，水合氯醛麻醉后剖宮產(chǎn)取出胎鼠，分離肺臟，剪碎，胰蛋白酶聯(lián)合膠原酶消化肺組織細(xì)胞，制成細(xì)胞懸液，差速離心和反復(fù)貼壁純化AECⅡ，含10%胎牛血清（FCS）的DMEM/F12培養(yǎng)基培養(yǎng)細(xì)胞。臺(tái)盼藍(lán)染色法檢測細(xì)胞活力，改良巴氏染色法檢測細(xì)胞純度，透射電鏡鑒定細(xì)胞，倒置相差顯微鏡下觀察細(xì)胞的生長情況。 2.原代AECⅡ體外培養(yǎng)24h后，隨機(jī)分為空氣組和高氧組，高氧組細(xì)胞置于氧體積分?jǐn)?shù)為95%（95%濃度氧）的細(xì)胞氧倉中，氧倉與空氣組細(xì)胞一并放于細(xì)胞培養(yǎng)箱中。24h后觀察細(xì)胞形態(tài)，MTT檢測細(xì)胞增殖，流式細(xì)胞儀檢測細(xì)胞的凋亡及存活情況。 3. SD新生大鼠隨機(jī)分為空氣組和高氧組，高氧組大鼠置于氧體積分?jǐn)?shù)為95%的動(dòng)物氧倉中，與空氣組大鼠置于同一室內(nèi)。3d，7d，14d，21d后取出肺組織行病理學(xué)檢查，輻射狀肺泡計(jì)數(shù)（RAC），化學(xué)比色法測定肺組織羥脯氨酸（HYP）含量。 4.原代分離培養(yǎng)的AECⅡ隨機(jī)分為空氣組、高氧組、空氣+H_2組、高氧+H_2組。H_2組細(xì)胞用富氫培養(yǎng)基干預(yù)。24h后觀察AECⅡ的形態(tài)變化；檢測MTT、細(xì)胞周期和增殖細(xì)胞核抗原（PCNA）蛋白表達(dá)觀察細(xì)胞增殖情況；檢測細(xì)胞線粒體膜電位（△Ψ）和凋亡率觀察細(xì)胞損傷情況；檢測細(xì)胞內(nèi)活性氧（ROS）和超氧化物陰離子（O-2）水平，細(xì)胞培養(yǎng)上清丙二醛（MDA）水平和超氧化物歧化酶（SOD）活性，觀察細(xì)胞氧化損傷和抗氧化能力；Western Blot檢測細(xì)胞總FoxO3a、β-catenin蛋白和p-FoxO3a、p-β-catenin蛋白的表達(dá)。 5. SD新生大鼠隨機(jī)分為空氣組、空氣+富氫生理鹽水組、空氣+H_2組、高氧組、高氧+富氫生理鹽水組和高氧+H_2組。各高氧組大鼠均置于氧體積分?jǐn)?shù)為95%的動(dòng)物氧倉中。H_2干預(yù)：富氫生理鹽水組大鼠予腹腔注射富氫生理鹽水10mL/kg，每天2次；H_2組大鼠予腹腔注射H_2氣體10mL/kg，每天2次。非H_2干預(yù)組大鼠則腹腔注射等量生理鹽水。14d后，取肺組織做病理學(xué)檢查；檢測大鼠血清MDA水平和SOD活力；測定肺組織HYP含量；免疫組化法測定肺組織α-平滑肌激動(dòng)蛋白（α-SMA）表達(dá)；Western blot檢測肺組織總FoxO3a、β-catenin蛋白和p-FoxO3a、p-β-catenin蛋白的表達(dá)。 結(jié)果 1.原代培養(yǎng)的AECⅡ產(chǎn)量較高，每只早產(chǎn)大鼠肺組織可獲得(8.5±1.8)×106AECⅡ，細(xì)胞活力為(95.0±2.1)％，細(xì)胞純度為（94.3±2.5）％。電鏡可見AECⅡ的特征性結(jié)構(gòu)--細(xì)胞膜表面的微絨毛和胞漿內(nèi)的板層小體。AECⅡ體外培養(yǎng)12h左右開始貼壁生長，至18h絕大部分細(xì)胞已貼壁伸展，24-48h細(xì)胞生長良好，增殖活躍，處于對(duì)數(shù)生長期，72h后細(xì)胞狀態(tài)逐漸變差，喪失功能。 2. AECⅡ予95%濃度氧刺激24h后，細(xì)胞出現(xiàn)皺縮變形，細(xì)胞間隙增大，增殖較空氣組明顯受抑，凋亡率明顯增加，存活率明顯降低。 3. SD新生大鼠高氧暴露3d和7d后肺組織出現(xiàn)肺泡上皮細(xì)胞腫脹，間質(zhì)充血水腫，炎性細(xì)胞浸潤，肺結(jié)構(gòu)紊亂，7d更明顯。14d和21d可見纖維增生，肺泡間隔明顯增寬，肺組織HYP含量較空氣組顯著增高，21d更為明顯。高氧組RAC值于7d，14d，21d顯著低于空氣組。 4.與空氣組比較，空氣+H_2組細(xì)胞總FoxO3a蛋白表達(dá)增加，p-FoxO3a蛋白表達(dá)降低，其余各指標(biāo)均無顯著差異。與空氣組比較，高氧組細(xì)胞OD492值和PCNA蛋白表達(dá)明顯降低，G1期細(xì)胞比例增多而S期細(xì)胞比例減少；細(xì)胞△Ψ降低，凋亡率增加；細(xì)胞內(nèi)ROS和O-2水平增高；細(xì)胞上清MDA含量增高，SOD活性下降；細(xì)胞總FoxO3a蛋白表達(dá)增加，p-FoxO3a蛋白表達(dá)降低；總β-catenin蛋白表達(dá)降低，p-β-catenin蛋白表達(dá)增高。與高氧組比較，H_2干預(yù)可減輕高氧引起的上述改變。 5.與空氣組比較，空氣+富氫生理鹽水組和空氣+H_2組大鼠肺組織均有FoxO3a與β-catenin的輕微激活，其余各指標(biāo)均無顯著差異；與空氣組比較，高氧組肺發(fā)育受阻，RAC值降低；肺組織間隔增寬，，纖維化明顯，肺組織HYP含量增高，α-SMA表達(dá)增高；血清MDA水平升高，SOD活力降低；肺組織總FoxO3a蛋白表達(dá)增加，且較H_2干預(yù)空氣組顯著，p-FOXO3蛋白表達(dá)降低；總β-catenin蛋白表達(dá)增加，p-β-catenin蛋白表達(dá)亦增高。與高氧組比較，高氧+富氫生理鹽水組和高氧+H_2組肺損傷均有所減輕，均一定程度恢復(fù)了高氧引起的上述改變。高氧+H_2組血清MDA水平和肺組織HYP含量較高氧+富氫生理鹽水組低，差異有統(tǒng)計(jì)學(xué)意義，其余指標(biāo)兩組間無差異。 結(jié)論 1.采用胰酶聯(lián)合膠原酶消化、差速離心和反復(fù)貼壁的方法獲得的原代AECⅡ產(chǎn)量、純度和活力均較高，可滿足細(xì)胞學(xué)實(shí)驗(yàn)研究的需要。AECⅡ體外培養(yǎng)24-48h生長狀態(tài)最佳，適合做體外研究。 2.95%濃度氧可誘導(dǎo)AECⅡ的損傷、凋亡并抑制其增殖，也可導(dǎo)致新生鼠肺損傷和肺發(fā)育受阻，成功建立高氧致AECⅡ細(xì)胞損傷模型和高氧致新生鼠肺損傷動(dòng)物模型。 3.富氫培養(yǎng)基能一定程度減輕高氧導(dǎo)致的AECⅡ凋亡、氧化損傷，并促進(jìn)其增殖，而對(duì)正常AECⅡ的增殖無明顯作用。 4.腹腔注射富氫生理鹽水和H_2氣體均可有效減輕高氧導(dǎo)致的肺損傷，減輕肺纖維化，腹腔注射H_2氣體的效果略佳。H_2對(duì)正常肺組織無明顯作用。 5. H_2對(duì)高氧導(dǎo)致的細(xì)胞和肺損傷的保護(hù)作用可能與抑制高氧導(dǎo)致的FoxO3a蛋白過度活化并激活β-catenin蛋白有關(guān)。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R726.1

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