降钙素基因相关肽对高氧肺损伤的影响和Sonic hedgehog信号通路的相关研究
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摘要
背景
较高浓度和较长时间的持续氧疗可导致急性肺损伤（ALI）或支气管肺发育不良（BPD），严重威胁新生儿健康。目前对于ALI和BPD国内外均无确定有效的防治方法，但研究显示其病因与肺的发育成熟及肺损伤的异常修复密切相关。肺的弥漫性非特异性炎症，进而导致气道重塑和肺泡纤维化是其最主要危险因素和最终病理特征。理想中的肺损伤修复是由原来性质的细胞来修复，并恢复原有的结构和功能，而不是由成纤维细胞增殖替代正常组织结构。肺泡Ⅱ型上皮细胞具有干细胞功能，可分化为Ⅰ型肺泡（AECⅠ）上皮细胞，但其自身增殖能力差，成为肺结构正常修复的障碍。CGRP是具有广泛调节功能的神经肽，其在肺损伤修复中的功能成为目前研究的热点。而Sonichedgehog （Shh）信号通路是与细胞增殖和分化密切相关的信号转导系统。通过神经肽干预Shh信号通路从而实现体内外诱导AECⅡ增殖重建，有可能会成为高氧肺损伤研究方面基础理论创新与解决临床实际问题相结合的新切入点。
目的
1建立实验用原代早产大鼠AECⅡ模型，进一步探索分离纯化和培养方法，为后续高氧诱导AECⅡ损伤和CGRP干预实验奠定基础。
2探讨降钙素基因相关肽（CGRP）对氧体积分数大于95%暴露下的早产大鼠AECⅡ的影响及其意义。
3研究CGRP对高氧诱导AECⅡ损伤的保护作用是否涉及Shh信号通路，观察该信号通路中Smo和Gli1信号分子的改变。
4探讨CGRP对高氧诱导的新生大鼠的肺组织损伤的保护作用。
5研究Shh信号通路中Smo和Gli1蛋白在高氧诱导的新生大鼠急性肺损伤中的表达及其意义。
方法
1孕19d SPF级SD大鼠经麻醉后，剖宫产取出胎鼠，提取肺脏，剪碎，先后用胰蛋白酶联合DNA酶和胶原酶Ⅰ消化肺组织，分离肺脏细胞。采用差速离心和差速贴壁的方法纯化AECⅡ，然后以DMEM/F12完全培养基培养(胎牛血清浓度为10%)。台盼蓝染色检测AECⅡ活力，改良巴氏染色检测AECⅡ纯度，透射电镜鉴定AECⅡ，倒置相差显微镜观察AECⅡ生长情况。
2早产大鼠原代AECⅡ分离纯化后，随机分为空气组、空气+CGRP组、空气+CGRP+CGRP8-37组、高氧组、高氧+CGRP组、高氧+CGRP+CGRP8-37组。空气组室内空气条件培养，高氧组给予氧体积分数大于95%的氧气培养。培养24h后观察AECⅡ的形态变化，检测细胞凋亡率,细胞内活性氧（ROS）、丙二醛（MDA）和超氧化物歧化酶（SOD）的活性，以及实时荧光定量PCR（RT-qPCR）检测表面活性蛋白C（SPC）mRNA表达。
3分离纯化的早产大鼠原代AECⅡ，随机分为空气组、高氧组、高氧+CGRP组、高氧+CGRP+CGRP8-37组。培养24h后RT-qPCR检测培养细胞的Smo和Gli1mRNA表达水平，用Western blot方法检测Smo和Gli1蛋白表达水平。
4新生大鼠随机分为正常空气组，高氧组和高氧+CGRP组。氧气干预：空气组处理吸入正常室内空气，高氧组和高氧+CGRP组给以氧体积分数大于95%的氧气吸入。CGRP干预：空气组和高氧组腹腔注射生理盐水，高氧+CGRP组给以αCGRP腹腔注射。共持续14d后，取肺组织做HE染色，试剂盒检测肺内MDA， SOD。实时荧光定量PCR法(RT-qPCR)检测肿瘤坏死因子（TNF-α）和白细胞介素6（IL-6），内源性CGRP mRNA表达。转化生长因子1（TGF-β1）蛋白表达通过Western blot检测。
5通过持续吸入高浓度氧气，建立新生大鼠高氧肺损伤模型。实验组吸入氧体积分数大于95%的医用氧气，对照组吸入空气。分别留取第3d、7d和14d的肺组织，切片HE染色观察肺脏病理改变，应用免疫组织化学和Western blot技术检测肺组织Smo和Gli1蛋白的动态表达情况。
结果
1原代培养的AECⅡ产量较好，每只早产大鼠肺组织可获得(8.1±1.1)×106AECⅡ，细胞活力为(95.5±1.6)％；细胞纯度鉴定为（95.6±2.1）％。电镜可见细胞膜表面特征微绒毛样结构，胞浆内可见板层小体。AECⅡ体外培养12h左右开始贴壁，至18h已绝大部分贴壁伸展，至24h～48h生长良好，增殖活跃，密度适中，达最佳状态，72h后细胞状态转差，开始死亡。
2与空气组比较，高氧暴露24h AECⅡ凋亡率明显增加，ROS和MDA显著升高，SOD活性明显下降，SPC mRNA蛋白表达显著降低（P＜0.05）； CGRP干预后可明显降低AECⅡ凋亡率，下调ROS和MDA，上调SOD，促进SPC mRNA表达（P＜0.05）。CGRP8-37能阻断CGRP的生物学作用。
3与空气组比较，高氧暴露24h AECⅡ Smo和Gli1基因和蛋白表达显著降低（P＜0.05）； CGRP干预后可明显促进Smo和Gli1基因和蛋白表达（P＜0.05）。CGRP8-37阻断CGRP的生物学作用后，使Smo和Gli1的基因和蛋白表达下降（P＜0.05）。
4高氧诱导新生大鼠14d后肺组织呈现较为严重的炎症性病理改变。肺组织MDA增加，SOD减少，TNF-α和IL-6的表达升高。CGRP处理能缓解高氧对肺组织的损伤作用，能显著改善组织病理学特征，抑制MDA的激活，促进SOD激活，减少TNF-α的mRNA表达并抑制TGF-β1的产生。内源性CGRP的mRNA表达各组之间未见差异。
5高氧暴露3d肺毛细血管开始充血，渗出；7d表现为肺结构紊乱，炎性细胞浸润；14d肺泡融合，纤维增生，间隔增宽。高氧组Smo和Gli1蛋白主要分布于支气管上皮，肺泡上皮和血管内皮细胞，以及部分纤维组织。和对照组相比，随着高氧暴露时间的延长，Smo于高氧第7d表达显著增加，14d达高峰；Gli1蛋白表达于14d显著增加。
结论
1采用差速贴壁和差速离心法分离纯化的AECⅡ数量大，纯度高，活力好，可用于细胞学实验研究。对其中重要环节的重视和改进可进一步提高细胞数量、成活率和纯度。
2氧体积分数大于95%的氧气浓度可显著抑制AECⅡ增殖，并导致AECⅡ损伤，CGRP能部分对抗氧化应激并促进AECⅡ增殖。
3高氧诱导AECⅡ损伤的过程中有Shh信号通路的变化，CGRP部分对抗氧化应激并促进AECⅡ增殖的作用可能与Shh信号通路的激活有关。
4高氧诱导的急性肺损伤与氧化应激以及炎症反应相关，CGRP能部分缓解高氧诱导的肺损伤并对部分炎症因子有调节作用。
5高浓度氧可致新生大鼠泡结构紊乱和肺发育停滞，Smo和Gli1蛋白呈现高表达，这可能和高氧诱导的肺损伤和支气管肺发育不良的发生发展有关。
Background
Continuous oxygen therapy with higher concentration can induceacute lung injury (ALI) or bronchopulmonary dysplasia (BPD), whichseriously threaten to the health of children. There are not effectiveprevention and cure methods to ALI and BPD at home and abroad. Butstudies suggest that the etiologies are closely associated with thematuration and injury/repair of the lungs. The diffuse and non-specificinflammations in lungs can lead to remodeling of airway and fibrosis ofalveolar, which are the most important risk factor and the final pathologycharacteristics in ALI and BPD. The satisfactory repair of lung injury is therestoration of both the structure and function by the nature of the cellsinstead of the proliferation of fibroblasts. Alveolar epithelial cells typeⅡare stem cells and can differentiate into alveolar epithelial cells typeⅠbutlack of proliferation ability. Calcitonin gene-related peptide (CGRP) is aneuropeptide with general regulating function. Shh signaling pathway is closely related to proliferation and differentiation in cells. Shh signalingpathway probably is regulated by the neuropeptide and affect theproliferation and reconstruction of AECⅡ in vitro and in vivo, which maybecome a new entry point of the basic theoretical innovation and clinicalpractice in hyperoxic induced lung injury.
Objective
1Set up the method of isolation, purification and primary cell cultureof alveolar epithelial cells typeⅡ (AECⅡ) from preterm rats for the effectstudy of CGRP on hyperoxia induced AECⅡ injury and its signal pathway.
2Explore the influence of95％oxygen and the protective effect ofcalcitonin gene-related peptide(CGRP)on alveolar epithelial cellstypeⅡ(AECⅡ).
3Investigate the effect of Calcitonin Gene-Related Peptide(CGRP) onprimary alveolar epithelial cells typeⅡ (AECⅡ) and the expressions ofSonic hedgehog (Shh) signaling pathway components，Smo and Gli1, uponthe exposure of hyperoxia.
4Study the protection of Calcitonin gene-related peptide (CGRP)treatment on hyperoxia-induced ALI symptoms in neonatal rats.
5Investigate the expressions of Smo and Gli1signaling molecules inlungs of newborn rats exposed to prolonged hyperoxia and explore the roleof Sonic hedgehog (Shh) signaling pathway in hyperoxia-induced lung injury.
Methods
1Isolated AECⅡ from the Sprague-Dawley (SD) fetal lungs of SPFrats with19days gestational age. The pregnant rats were anesthetized bychloral hydrate. Use the way of uterine incision delivery to pick out thefetal rats. The fetal lungs were isolated from the fetal rats. Digested theLung tissues by trypsin and collagenase-1, then the AECⅡ were purifiedwith the methods of different centrifugal force and repeated attachment.The dulbecco's modified eagle's medium (DMEM/F12) complemented with10%fetal calf serum were used to cultured the AECⅡ. Trypan blueexclusion was used to assessed AECⅡ viability. The cells purity wasevaluated by modified papanicolaou staining. AECⅡ were also identifiedby transmission electron microscopy. And the cellular growth status andmorphologic changes of AECⅡ were observed with inverted phasecontrast microscope.
2AECⅡ were isolated and purified from premature rats and exposedto air (21%oxygen), air+CGRP, air+CGRP+CGRP8-37, hyperoxia(95%oxygen), hyperoxia+CGRP and hyperoxia+CGRP+CGRP8-37for24hrespectively. Morphologic change of AECⅡ was observed under theinverted phase contrast microscope. The apoptosis ratio of AECⅡ,production of intracellular reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase(SOD) in AECⅡ weredetected by flow cytometry or spectrophotography. The mRNA expressionsof surfactant protein C (SPC) in AECⅡ were also detected by real-timequantitative polymerase chain reaction (RT-qPCR).
3AECⅡ were isolated and purified from premature rats and exposedto air (21%oxygen), hyperoxia (95%oxygen), hyperoxia+CGRP andhyperoxia+CGRP+CGRP8-37for24h respectively. The mRNAexpressions of Smo and Gli11in AECⅡ were detected by real-timequantitative polymerase chain reaction (RT-qPCR), and the proteinsexpression of Smo and Gli11were also detected by western blot.
4Newborn Sprague-Dawley rats were randomly divided into threeexperimental treatment groups: normoxia, hyperoxia and hyperoxia withCGRP. Hyperoxia groups were subjected to conditions of oxygen levels inexcess of95%and treated with alternating daily doses of either normalsaline or CGRP for14days. Subsequently, rat lung pathology wasexamined using hematoxylin and eosin (HE) staining under lightmicroscopy, with further studies revealing the presence of bothmalondialdehyde (MDA) and superoxide dismutase (SOD) in homogenizedlung tissues. Furthermore, tumor necrosis factor-α (TNF-α), interleukin-6(IL-6) and endogenous αCGRP mRNA were quantitated by RT-qPCR, andthe protein expression of transforming growth factor-β1(TGF-β1) wasanalyzed by western blot analysis.
5Neonatal rat pups were placed in chambers containing room air oroxygen about95%for14days after birth. The rats were sacrificed at3,7or14days and their lungs removed and subjected to hematoxylin and eosin（HE） staining. Smo and Gli11were observed by immunohistochemistry,and western blotting was utilized to assess the dynamic expressions of Smoand Gli1protein.
Results
1One of pregnant rat lungs can obtain the number of AECⅡ about(8.1±1.1)×106the survival rate is about (95.5±1.6)％.the purity is about（95.6±2.1）％. The microvillus on the cell membrane and the lamellarbodies in cytoplasm were observed under electron microscope. AECⅡattached the bottom after cultured12~18h. The exponential growth phaseof the AECⅡ was24h~48h after culture. After72h, AECⅡ becameattached cells decreased and died.
2Under the95%oxygen condition, the apoptosis ratio of AECⅡ, theproduction of ROS and MDA in AECⅡ were significantly increased, butthe levels of SOD, SPC mRNA in AECⅡ were decreased comparison withair group(P＜0.05). After the intervention of CGRP, the apoptosis ratio,production of ROS and MDA in AECⅡ were down regulated, however, theSOD was up regulated, and the SPC mRNA in AECⅡ were also increasedobviously(P＜0.05).CGRP8-37could block the biological effects CGRP.
3Under the95%hyperoxia condition, the mRNA and proteinsexpression of Smo and Gli1in AECⅡ were decreased comparison with airgroup(P＜0.05). After the intervention of CGRP, the mRNAs and proteinsof Smo and Gli11in AECⅡ were increased obviously(P＜0.05).CGRP8-37could down regulate these mRNAs and proteins by blockthe biological effects of CGRP.
4Hyperoxia could induce server histomorphological changes inneonatal lung tissue after exposed to high concentration oxygen. And theMDA was unregulated, the SOD was down regulated, the mRNAexpressions of TNF-α andIL-6were also increased. CGRP treatmentsignificantly alleviates ALI symptoms, preventing both reductions inneonatal body weight and histomorphological changes to neonatal lungtissue. CGRP treatment for exposure to hyperoxic conditions resulted in theinhibition of MDA activation, increased SOD activation, decreased mRNAexpression of TNF-α, and reduced expression of TGF-β1. The endogenousmRNA expressions of αCGRP were no difference in each group.
5Significant histo-morphologic changes were showed in thehyperoxia-exposed lungs at3,7and14days of age by HE staining. Smoand Gli1proteins were observed in bronchial epithelial cells, alveolarepithelial cells, and vascular endothelium cells, and partly showed on thefibrotic tissue in lung interstitium. The results of immunohistochemistryand western blot showed that the expression of Smo was significantly higher in the hyperoxia-exposed lungs at7and14days，and Gli1wassignificantly elevated at14days. But low Smo and Gli1were detected innormal air group.
Conclusion
1It has been known that the methods of different centrifugal force andrepeated attachment are the best ways to culture the primary AECⅡ. Itcould get the best cells number, purity and vitality of AECⅡ. These cellscould be used for the experiments in vitro about AECⅡ. The purity andvitality of AECⅡ perhaps could be further elevated by improve the keysteps in this methods.
2The hyperoxia of95%oxygen suppressed the growth of AECⅡ, andresults in dysfunction of the cells. CGRP could partly protect AECⅡ fromoxidative stress injury and promote the cells proliferation.
3The situation of Shh signal pathway maybe inhibited by hyperoxiain AECⅡ. CGRP could partly protect AECⅡ from oxidative stress injuryand promote the cells proliferation, which might be associated with theactivation of Smo and Gli1in Shh signal pathway.
4Hyperoxia-induced ALI is likely associated with both the oxidativestress and the inflammatory response induced by high oxygen conditions.CGRP treatment may play a protective role in the prevention and treatmentof hyperoxia-induced ALI in neonatal rats by inhibiting these processes.
5Exposure of neonatal rat to prolonged hyperoxia results in acutelung injury and arrested lung development. Smo and Gli1are up-regulatedat time points preceding the lung injury, and Shh signal pathway seems tobe involved in the pathogenesis of hyperoxia-induced lung injury andbronchopulmonary dysplasia.
较高浓度和较长时间的持续氧疗可导致急性肺损伤（ALI）或支气管肺发育不良（BPD），严重威胁新生儿健康。目前对于ALI和BPD国内外均无确定有效的防治方法，但研究显示其病因与肺的发育成熟及肺损伤的异常修复密切相关。肺的弥漫性非特异性炎症，进而导致气道重塑和肺泡纤维化是其最主要危险因素和最终病理特征。理想中的肺损伤修复是由原来性质的细胞来修复，并恢复原有的结构和功能，而不是由成纤维细胞增殖替代正常组织结构。肺泡Ⅱ型上皮细胞具有干细胞功能，可分化为Ⅰ型肺泡（AECⅠ）上皮细胞，但其自身增殖能力差，成为肺结构正常修复的障碍。CGRP是具有广泛调节功能的神经肽，其在肺损伤修复中的功能成为目前研究的热点。而Sonichedgehog （Shh）信号通路是与细胞增殖和分化密切相关的信号转导系统。通过神经肽干预Shh信号通路从而实现体内外诱导AECⅡ增殖重建，有可能会成为高氧肺损伤研究方面基础理论创新与解决临床实际问题相结合的新切入点。
目的
1建立实验用原代早产大鼠AECⅡ模型，进一步探索分离纯化和培养方法，为后续高氧诱导AECⅡ损伤和CGRP干预实验奠定基础。
2探讨降钙素基因相关肽（CGRP）对氧体积分数大于95%暴露下的早产大鼠AECⅡ的影响及其意义。
3研究CGRP对高氧诱导AECⅡ损伤的保护作用是否涉及Shh信号通路，观察该信号通路中Smo和Gli1信号分子的改变。
4探讨CGRP对高氧诱导的新生大鼠的肺组织损伤的保护作用。
5研究Shh信号通路中Smo和Gli1蛋白在高氧诱导的新生大鼠急性肺损伤中的表达及其意义。
方法
1孕19d SPF级SD大鼠经麻醉后，剖宫产取出胎鼠，提取肺脏，剪碎，先后用胰蛋白酶联合DNA酶和胶原酶Ⅰ消化肺组织，分离肺脏细胞。采用差速离心和差速贴壁的方法纯化AECⅡ，然后以DMEM/F12完全培养基培养(胎牛血清浓度为10%)。台盼蓝染色检测AECⅡ活力，改良巴氏染色检测AECⅡ纯度，透射电镜鉴定AECⅡ，倒置相差显微镜观察AECⅡ生长情况。
2早产大鼠原代AECⅡ分离纯化后，随机分为空气组、空气+CGRP组、空气+CGRP+CGRP8-37组、高氧组、高氧+CGRP组、高氧+CGRP+CGRP8-37组。空气组室内空气条件培养，高氧组给予氧体积分数大于95%的氧气培养。培养24h后观察AECⅡ的形态变化，检测细胞凋亡率,细胞内活性氧（ROS）、丙二醛（MDA）和超氧化物歧化酶（SOD）的活性，以及实时荧光定量PCR（RT-qPCR）检测表面活性蛋白C（SPC）mRNA表达。
3分离纯化的早产大鼠原代AECⅡ，随机分为空气组、高氧组、高氧+CGRP组、高氧+CGRP+CGRP8-37组。培养24h后RT-qPCR检测培养细胞的Smo和Gli1mRNA表达水平，用Western blot方法检测Smo和Gli1蛋白表达水平。
4新生大鼠随机分为正常空气组，高氧组和高氧+CGRP组。氧气干预：空气组处理吸入正常室内空气，高氧组和高氧+CGRP组给以氧体积分数大于95%的氧气吸入。CGRP干预：空气组和高氧组腹腔注射生理盐水，高氧+CGRP组给以αCGRP腹腔注射。共持续14d后，取肺组织做HE染色，试剂盒检测肺内MDA， SOD。实时荧光定量PCR法(RT-qPCR)检测肿瘤坏死因子（TNF-α）和白细胞介素6（IL-6），内源性CGRP mRNA表达。转化生长因子1（TGF-β1）蛋白表达通过Western blot检测。
5通过持续吸入高浓度氧气，建立新生大鼠高氧肺损伤模型。实验组吸入氧体积分数大于95%的医用氧气，对照组吸入空气。分别留取第3d、7d和14d的肺组织，切片HE染色观察肺脏病理改变，应用免疫组织化学和Western blot技术检测肺组织Smo和Gli1蛋白的动态表达情况。
结果
1原代培养的AECⅡ产量较好，每只早产大鼠肺组织可获得(8.1±1.1)×106AECⅡ，细胞活力为(95.5±1.6)％；细胞纯度鉴定为（95.6±2.1）％。电镜可见细胞膜表面特征微绒毛样结构，胞浆内可见板层小体。AECⅡ体外培养12h左右开始贴壁，至18h已绝大部分贴壁伸展，至24h～48h生长良好，增殖活跃，密度适中，达最佳状态，72h后细胞状态转差，开始死亡。
2与空气组比较，高氧暴露24h AECⅡ凋亡率明显增加，ROS和MDA显著升高，SOD活性明显下降，SPC mRNA蛋白表达显著降低（P＜0.05）； CGRP干预后可明显降低AECⅡ凋亡率，下调ROS和MDA，上调SOD，促进SPC mRNA表达（P＜0.05）。CGRP8-37能阻断CGRP的生物学作用。
3与空气组比较，高氧暴露24h AECⅡ Smo和Gli1基因和蛋白表达显著降低（P＜0.05）； CGRP干预后可明显促进Smo和Gli1基因和蛋白表达（P＜0.05）。CGRP8-37阻断CGRP的生物学作用后，使Smo和Gli1的基因和蛋白表达下降（P＜0.05）。
4高氧诱导新生大鼠14d后肺组织呈现较为严重的炎症性病理改变。肺组织MDA增加，SOD减少，TNF-α和IL-6的表达升高。CGRP处理能缓解高氧对肺组织的损伤作用，能显著改善组织病理学特征，抑制MDA的激活，促进SOD激活，减少TNF-α的mRNA表达并抑制TGF-β1的产生。内源性CGRP的mRNA表达各组之间未见差异。
5高氧暴露3d肺毛细血管开始充血，渗出；7d表现为肺结构紊乱，炎性细胞浸润；14d肺泡融合，纤维增生，间隔增宽。高氧组Smo和Gli1蛋白主要分布于支气管上皮，肺泡上皮和血管内皮细胞，以及部分纤维组织。和对照组相比，随着高氧暴露时间的延长，Smo于高氧第7d表达显著增加，14d达高峰；Gli1蛋白表达于14d显著增加。
结论
1采用差速贴壁和差速离心法分离纯化的AECⅡ数量大，纯度高，活力好，可用于细胞学实验研究。对其中重要环节的重视和改进可进一步提高细胞数量、成活率和纯度。
2氧体积分数大于95%的氧气浓度可显著抑制AECⅡ增殖，并导致AECⅡ损伤，CGRP能部分对抗氧化应激并促进AECⅡ增殖。
3高氧诱导AECⅡ损伤的过程中有Shh信号通路的变化，CGRP部分对抗氧化应激并促进AECⅡ增殖的作用可能与Shh信号通路的激活有关。
4高氧诱导的急性肺损伤与氧化应激以及炎症反应相关，CGRP能部分缓解高氧诱导的肺损伤并对部分炎症因子有调节作用。
5高浓度氧可致新生大鼠泡结构紊乱和肺发育停滞，Smo和Gli1蛋白呈现高表达，这可能和高氧诱导的肺损伤和支气管肺发育不良的发生发展有关。
Background
Continuous oxygen therapy with higher concentration can induceacute lung injury (ALI) or bronchopulmonary dysplasia (BPD), whichseriously threaten to the health of children. There are not effectiveprevention and cure methods to ALI and BPD at home and abroad. Butstudies suggest that the etiologies are closely associated with thematuration and injury/repair of the lungs. The diffuse and non-specificinflammations in lungs can lead to remodeling of airway and fibrosis ofalveolar, which are the most important risk factor and the final pathologycharacteristics in ALI and BPD. The satisfactory repair of lung injury is therestoration of both the structure and function by the nature of the cellsinstead of the proliferation of fibroblasts. Alveolar epithelial cells typeⅡare stem cells and can differentiate into alveolar epithelial cells typeⅠbutlack of proliferation ability. Calcitonin gene-related peptide (CGRP) is aneuropeptide with general regulating function. Shh signaling pathway is closely related to proliferation and differentiation in cells. Shh signalingpathway probably is regulated by the neuropeptide and affect theproliferation and reconstruction of AECⅡ in vitro and in vivo, which maybecome a new entry point of the basic theoretical innovation and clinicalpractice in hyperoxic induced lung injury.
Objective
1Set up the method of isolation, purification and primary cell cultureof alveolar epithelial cells typeⅡ (AECⅡ) from preterm rats for the effectstudy of CGRP on hyperoxia induced AECⅡ injury and its signal pathway.
2Explore the influence of95％oxygen and the protective effect ofcalcitonin gene-related peptide(CGRP)on alveolar epithelial cellstypeⅡ(AECⅡ).
3Investigate the effect of Calcitonin Gene-Related Peptide(CGRP) onprimary alveolar epithelial cells typeⅡ (AECⅡ) and the expressions ofSonic hedgehog (Shh) signaling pathway components，Smo and Gli1, uponthe exposure of hyperoxia.
4Study the protection of Calcitonin gene-related peptide (CGRP)treatment on hyperoxia-induced ALI symptoms in neonatal rats.
5Investigate the expressions of Smo and Gli1signaling molecules inlungs of newborn rats exposed to prolonged hyperoxia and explore the roleof Sonic hedgehog (Shh) signaling pathway in hyperoxia-induced lung injury.
Methods
1Isolated AECⅡ from the Sprague-Dawley (SD) fetal lungs of SPFrats with19days gestational age. The pregnant rats were anesthetized bychloral hydrate. Use the way of uterine incision delivery to pick out thefetal rats. The fetal lungs were isolated from the fetal rats. Digested theLung tissues by trypsin and collagenase-1, then the AECⅡ were purifiedwith the methods of different centrifugal force and repeated attachment.The dulbecco's modified eagle's medium (DMEM/F12) complemented with10%fetal calf serum were used to cultured the AECⅡ. Trypan blueexclusion was used to assessed AECⅡ viability. The cells purity wasevaluated by modified papanicolaou staining. AECⅡ were also identifiedby transmission electron microscopy. And the cellular growth status andmorphologic changes of AECⅡ were observed with inverted phasecontrast microscope.
2AECⅡ were isolated and purified from premature rats and exposedto air (21%oxygen), air+CGRP, air+CGRP+CGRP8-37, hyperoxia(95%oxygen), hyperoxia+CGRP and hyperoxia+CGRP+CGRP8-37for24hrespectively. Morphologic change of AECⅡ was observed under theinverted phase contrast microscope. The apoptosis ratio of AECⅡ,production of intracellular reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase(SOD) in AECⅡ weredetected by flow cytometry or spectrophotography. The mRNA expressionsof surfactant protein C (SPC) in AECⅡ were also detected by real-timequantitative polymerase chain reaction (RT-qPCR).
3AECⅡ were isolated and purified from premature rats and exposedto air (21%oxygen), hyperoxia (95%oxygen), hyperoxia+CGRP andhyperoxia+CGRP+CGRP8-37for24h respectively. The mRNAexpressions of Smo and Gli11in AECⅡ were detected by real-timequantitative polymerase chain reaction (RT-qPCR), and the proteinsexpression of Smo and Gli11were also detected by western blot.
4Newborn Sprague-Dawley rats were randomly divided into threeexperimental treatment groups: normoxia, hyperoxia and hyperoxia withCGRP. Hyperoxia groups were subjected to conditions of oxygen levels inexcess of95%and treated with alternating daily doses of either normalsaline or CGRP for14days. Subsequently, rat lung pathology wasexamined using hematoxylin and eosin (HE) staining under lightmicroscopy, with further studies revealing the presence of bothmalondialdehyde (MDA) and superoxide dismutase (SOD) in homogenizedlung tissues. Furthermore, tumor necrosis factor-α (TNF-α), interleukin-6(IL-6) and endogenous αCGRP mRNA were quantitated by RT-qPCR, andthe protein expression of transforming growth factor-β1(TGF-β1) wasanalyzed by western blot analysis.
5Neonatal rat pups were placed in chambers containing room air oroxygen about95%for14days after birth. The rats were sacrificed at3,7or14days and their lungs removed and subjected to hematoxylin and eosin（HE） staining. Smo and Gli11were observed by immunohistochemistry,and western blotting was utilized to assess the dynamic expressions of Smoand Gli1protein.
Results
1One of pregnant rat lungs can obtain the number of AECⅡ about(8.1±1.1)×106the survival rate is about (95.5±1.6)％.the purity is about（95.6±2.1）％. The microvillus on the cell membrane and the lamellarbodies in cytoplasm were observed under electron microscope. AECⅡattached the bottom after cultured12~18h. The exponential growth phaseof the AECⅡ was24h~48h after culture. After72h, AECⅡ becameattached cells decreased and died.
2Under the95%oxygen condition, the apoptosis ratio of AECⅡ, theproduction of ROS and MDA in AECⅡ were significantly increased, butthe levels of SOD, SPC mRNA in AECⅡ were decreased comparison withair group(P＜0.05). After the intervention of CGRP, the apoptosis ratio,production of ROS and MDA in AECⅡ were down regulated, however, theSOD was up regulated, and the SPC mRNA in AECⅡ were also increasedobviously(P＜0.05).CGRP8-37could block the biological effects CGRP.
3Under the95%hyperoxia condition, the mRNA and proteinsexpression of Smo and Gli1in AECⅡ were decreased comparison with airgroup(P＜0.05). After the intervention of CGRP, the mRNAs and proteinsof Smo and Gli11in AECⅡ were increased obviously(P＜0.05).CGRP8-37could down regulate these mRNAs and proteins by blockthe biological effects of CGRP.
4Hyperoxia could induce server histomorphological changes inneonatal lung tissue after exposed to high concentration oxygen. And theMDA was unregulated, the SOD was down regulated, the mRNAexpressions of TNF-α andIL-6were also increased. CGRP treatmentsignificantly alleviates ALI symptoms, preventing both reductions inneonatal body weight and histomorphological changes to neonatal lungtissue. CGRP treatment for exposure to hyperoxic conditions resulted in theinhibition of MDA activation, increased SOD activation, decreased mRNAexpression of TNF-α, and reduced expression of TGF-β1. The endogenousmRNA expressions of αCGRP were no difference in each group.
5Significant histo-morphologic changes were showed in thehyperoxia-exposed lungs at3,7and14days of age by HE staining. Smoand Gli1proteins were observed in bronchial epithelial cells, alveolarepithelial cells, and vascular endothelium cells, and partly showed on thefibrotic tissue in lung interstitium. The results of immunohistochemistryand western blot showed that the expression of Smo was significantly higher in the hyperoxia-exposed lungs at7and14days，and Gli1wassignificantly elevated at14days. But low Smo and Gli1were detected innormal air group.
Conclusion
1It has been known that the methods of different centrifugal force andrepeated attachment are the best ways to culture the primary AECⅡ. Itcould get the best cells number, purity and vitality of AECⅡ. These cellscould be used for the experiments in vitro about AECⅡ. The purity andvitality of AECⅡ perhaps could be further elevated by improve the keysteps in this methods.
2The hyperoxia of95%oxygen suppressed the growth of AECⅡ, andresults in dysfunction of the cells. CGRP could partly protect AECⅡ fromoxidative stress injury and promote the cells proliferation.
3The situation of Shh signal pathway maybe inhibited by hyperoxiain AECⅡ. CGRP could partly protect AECⅡ from oxidative stress injuryand promote the cells proliferation, which might be associated with theactivation of Smo and Gli1in Shh signal pathway.
4Hyperoxia-induced ALI is likely associated with both the oxidativestress and the inflammatory response induced by high oxygen conditions.CGRP treatment may play a protective role in the prevention and treatmentof hyperoxia-induced ALI in neonatal rats by inhibiting these processes.
5Exposure of neonatal rat to prolonged hyperoxia results in acutelung injury and arrested lung development. Smo and Gli1are up-regulatedat time points preceding the lung injury, and Shh signal pathway seems tobe involved in the pathogenesis of hyperoxia-induced lung injury andbronchopulmonary dysplasia.
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