【作者】 许燕萍；

【导师】 杜立中； 梁黎；

【作者基本信息】 浙江大学 ， 儿科学， 2011， 博士

【摘要】 新生儿持续肺动脉高压(persistent pulmonary hypertension of newborn, PPHN)为新生儿期的严重疾病,出生后肺动脉压力不下降,胎儿循环过渡至正常“成人’循环发生障碍,当其压力等于或超过体循环压力时,出现动脉导管及(或)卵圆孔水平的右向左分流。PPHN患儿往往存在严重的低氧血症,长期低氧致组织氧合不足,出现酸中毒,最终导致心力衰竭,造成死亡。生后成功的胎儿循环的转变依赖于肺血管阻力的下降和肺血流的快速增加。介导肺血管阻力降低的因素目前尚未被完全认识。目前研究调节肺血管张力的四条主要信号通路为：一氧化氮(nitric oxide, NO)/环鸟甘单磷酸(cyclic guanosine monophosphate, cGMP)通路、前列环素/环腺甘单磷酸(cyclic adenosine monophosphate, cAMP)通路,内皮素通路和Rho激酶途径。典型的肺发育分为五期：胚胎期,假腺体期,小管期,囊泡期和最后的肺泡期和微血管成熟期。人类肺发育在出生时几乎完成,生后只发生部分的肺泡化和微血管的成熟。大鼠在出生时只具有囊泡期的肺,相对于人类,它们只能以较不成熟阶段的肺在呼吸空气的环境中发育成熟。在此肺发育关键期,予10-12%低氧刺激新生仔鼠模拟人类宫内缺氧导致PPHN,同样可导致肺泡和肺微血管结构和功能的损害,从而为成功建立PPHN模型提供理论依据。低氧性肺动脉高压(PAH)的病理过程包括初期功能性的肺血管收缩,此后固定性的肺血管官腔狭窄,肺血管阻力提高,血栓形成和最终导致右心衰竭和死亡。一旦发生肺血管结构重建,以药物干预来逆转肺血管阻力至正常范围大多是徒劳的。因此,寻找有效的细胞信号通路药物作用靶点,早期逆转上升的肺血管阻力,干预肺动脉高压的进一步恶化,显得尤为重要。Ghrelin是在1999年由Kojima等首次发现的体内生长激素促分泌素受体的内源性配体,为一种28个氨基酸组成的具有生物学活性的多肽。Ghrelin在体内有两种存在形式：非酰基化的ghrelin和酰基化的ghrelin,后者具有生物学活性能通过GHS-R1a受体而发挥多种生物学作用。GHS-R1a分布广泛,在下丘脑弓状核表达最高,在心肌组织表达较少。越来越多的研究显示ghrelin对心血管功能有保护作用,包括降低血管阻力,扩张血管,增加微血管血流量,增加左心射血分数,心输出量和保护缺血再灌注心肌损伤。Ghrelin能减轻野百合碱诱导成年大鼠的PAH,但由野百合碱诱导的PAH机理及药物治疗效果不同于慢性低氧诱导的PAH。近年,Schwenke等提出ghrelin能减轻低氧诱导成年大鼠肺动脉高压,改善肺血管重建及右心室肥厚,但具体机理未阐明。研究显示,ghrelin模拟胰岛素的作用激活P13K-依赖的信号通路,通过直接刺激血管内皮细胞释放一氧化氮(NO)而舒张血管。在体外培养的人脐静脉内皮细胞和牛主动脉内皮细胞中,加入ghrelin能诱导内皮型一氧化氮合酶(eNOS)的磷酸化和一氧化氮(NO)的产生。Ghrelin是否直接诱导舒张血管介质的释放,还是通过GHS-R1a受体调控第二信使,本课题假设ghrelin能介导某些信号通路来改善PPHN患儿病情严重程度。基于上述已探索的PPHN的发病机制及ghrelin舒张血管的作用机理,本课题以建立常压低氧新生大鼠肺动脉高压模型,并用ghrelin进行干预,筛选PPHN及ghrelin作用PPHN后的互为相关的信号通路,探讨ghrelin作用于PPHN相关靶点蛋白的调节,深入了解细胞内信号转导过程,阐明信号刺激所造成的生物学反应过程及其机制,希望能为今后PPHN的治疗提供新的途径。第一部分低氧诱导新生大鼠肺动脉高压信号通路的筛选目的：1.建立低氧诱导新生大鼠肺动脉高压模型,观察缺氧新生鼠肺组织病理学的变化及肺小动脉的重建情况,监测血流动力学改变。2.从整体动物水平筛选低氧性肺动脉高压新生鼠肺组织基因表达改变的关键信号分子,从而进一步了解PPHN的病理生理意义。方法：1.模型建立和分组缺氧组：将新生SD大鼠置于密封有机玻璃箱中,调整氧气和氮气的比例,使箱内氧浓度维持在12%左右。对照组：吸入Fi02为0.21(即空气),具体方法及实验控制因素同缺氧组。2.评价模型是否成功测量缺氧14天后右心室收缩压的变化,并用α-SMA免疫组化染色观察肺血管的肌化程度,测量右心室与左心室加室间隔的比值[ratio of right ventricle to left ventricle plus septum, RV/(LV+S)]评价右心室肥厚情况。3.PCR基因芯片筛选肺组织信号通路表达情况结果：1.缺氧后,右心室出现了肥厚,RV/(LV+S)的比值较对照组上升(P<0.05),随着缺氧时间的延长,RV/(LV+S)的比值进一步上升。对照组右心室平均收缩压在(20.02±1.02)mmHg,缺氧14天后右心室平均收缩压明显升高达(37.29±3.15)mmHg,两者有统计学差异。小动脉的血管壁增厚,非肌型血管出现了不同程度的肌化。2.缺氧14天后与对照组相比有两条信号通路中重要基因的域值增长倍数≥3.0：分别是Wnt信号通路(Lefl,4.79; Wnt,3.87)和CREB信号通路(Cypl9al,4.87; Egrl,7.79; Fos,3.86)结论：1.成功建立低氧性新生大鼠肺动脉高压大鼠模型。2.低氧性新生大鼠肺动脉高压改变Wnt和CREB两条最为主要的信号通路。第二部分Ghrelin调控低氧性肺动脉高压信号通路的研究目的：1.采用ghrelin干预低氧性新生大鼠肺动脉高压模型,观察肺血流动力学改变和肺血管重建情况。2.分析ghrelin干预后基因芯片改变情况,进一步验证芯片结果,试阐述ghrelin调控低氧性PAH的信号途径。方法：1.模型的建立和分组缺氧组：在缺氧前生理盐水皮下注射(0.2m1),一天一次,持续14天。Ghrelin治疗组：在缺氧前用ghrelin (150μg/kg,0.2 ml)皮下注射,一天一次,持续14天。常氧组：常氧呼吸,生理盐水皮下注射(0.2m1),一天一次,持续14天。Ghrelin对照组：常氧呼吸,用ghrelin (150μg/kg,0.2 ml)皮下注射,一天一次,持续14天。2.实验指标检测比较缺氧及ghrelin干预14天后,肺小动脉重塑及右心室肥厚的变化。检测血清活性ghrelin浓度,用基因芯片方法检测,验证筛选出的Wnt信号通路,检测GHS-R1a蛋白表达变化。结果：1. Ghrelin对照组右心室平均收缩压(mRVSP) 19.19±2.12mmHg, ghrelin治疗组mRVSP 23.52±0.82 mmHg;缺氧后mRVSP明显升高达37.29±3.15mmHg,常氧组mRVSP为20.02±1.02 mmHg, ghrelin治疗后右心室平均收缩压呈显著下降(P<0.05)。同时,ghrelin治疗组RV/(LV+S)的比值0.27±0.02较缺氧组0.51±0.09显著降低(P<0.05)。大鼠缺氧后α-SMAarea/LD值升高,与常氧组比较有统计学差异(194.2±8.8,88.9±4.8,P<0.05),ghrelin治疗后α-SMAarea/LD显著下降(107.4±6.8；P=0.000)。2. Ghrelin治疗组血清活性ghrelin浓度为43.75±4.94 pg/ml与缺氧组27.38±5.29 pg/ml相比有显著性差异(P<0.05)3.缺氧后经ghrelin (?)台疗组与缺氧组相比也有两条信号通路中重要基因的阈值增长倍数≥3.0：分别是Wnt信号通路(Birc5,5.05; Myc,3.09; Pparg,4.82)和PI3K/Akt信号通路(Fn1,3.60)。缺氧组与常氧组相比,Wnt信号通路(Birc5,-8.76; Myc,1.12; Pparg,-1.59), PI3K/Akt信号通路(Fnl,-2.19)。4.与缺氧组相比,ghrelin治疗组的P-GSK3β/GSK3β和β-catenin蛋白表达水平显著上升(P<0.05),在ghrelin治疗组和ghrelin对照组中均发现GHSR-la蛋白表达量显著高于相应对照组(P<0.05)结论：1. Ghrelin连续干预14天对缺氧新生大鼠肺动脉高压、肺血管重塑、右心室肥厚有改善作用。2. PI3-K/Akt/GSK-3β与Wnt信号通路参与ghrelin改善新生大鼠肺动脉高压发病机制。3. GHSR-la作为中介受体参与ghrelin通过PI3-K/Akt/GSK-3β信号通路发挥生物作用。第三部分Ghrelin影响低氧诱导肺微血管内皮细胞凋亡及其p-GSK3β/β-catenin信号通路的研究目的：1.建立原代肺微血管内皮细胞,观察缺氧的不同时间点,内皮细胞的增殖凋亡。2.观察GHS-R1a的变化,PI3-K/Akt/GSK-3β蛋白表达变化,以及β-catenin的分布；评价ghrelin干预后ghrelin和上述指标之间的关系。方法：1.原代大鼠肺微血管内皮细胞(Rat pulmonary microvascular endothelial cells, RPMECs)的制备和鉴定。2. RPMECs细胞MTT试验和细胞凋亡检测。3.免疫荧光染色方法检测GHSR-1a和(3-catenin蛋白在RPMECs的表达情况；Western blot法检测p-Akt和p-GSK3p/GSK3β蛋白在RPMECs的表达情况。4.细胞转染及荧光素酶报告基因检测TOPFLASH质粒表达改变。结果：1. RPMECs呈鹅卵石镶嵌状排列,状如梭形或多角形,大小均匀。胞核清晰,呈卵圆孔,胞浆丰富。CD31鉴定95%以上2. TUNEL结果显示：Hypoxia组、Normoxia+ghrel in组和Hypoxia+ghrelin组的RPMECs凋亡率分别为Normoxia组的：2.77±0.28,0.92±0.07,1.63±0.19RPMECs经ghrelin处理后,凋亡细胞数显著下降(P<0.05)。3. Ghrelin刺激p-Akt表达呈时间剂量依赖性。以相同浓度作用0,5,15,30,60,120 min, p-GSK3β/GSK3β蛋白也出现逐渐上升趋势,30min后上升,持续120min。用ghrelin受体阻断剂[D-Lys3]-GHRP-6干预后发现,可以降低p-Akt的激活。同样用特异性Akt抑制剂Ly294002也能降低GSK3β磷酸化表达。4. ghrelin在低氧条件下诱导RPMECs的β-catenin蛋白转录入核,ghrelin激活β-catenin/TCF的转录活性。结论：1. PI3-K/Akt/GSK-3β与Wnt信号通路参与ghrelin保护低氧状态下RPMECs凋亡。2.在RPMECs中GHSR-1a作为中介受体参与ghrelin通过p-GSK3β/GSK3β信号通路发挥生物学作用。3.成功建立基于Wnt/β-catenin途径的报告基因模型,发现ghrelin可以上调β-catenin/TCF转录活性。更多还原

【Abstract】 Persistent pulmonary hypertension of the newborn (PPHN) is a clinical syndrome characterized by abnormal pulmonary vascular tone, reactivity, and structure. A sustained elevation of pulmonary vascular resistance at birth leads to extrapulmonary right-to-left shunting of blood, and severe hypoxemia. PPHN patients are usually full-term or post-term infants who have had perinatal asphyxia, meconium aspiration, diaphragmatic hernia, pneumonia, or sepsis. Further research is required to fully understand the mechanism(s) behind these types of lung disorders to enable the development of effective treatments.Lung development during the saccular period occurs between embryonic day 17.5 and postnatal day 5 and the alveolar stage occurs during the postnatal weaning period in mouse and rat. During this time alveolar septation initiates and leads to a tremendous increase in the surface area of the lung. Exposure to hypoxia during this critical period can impair both alveolar and pulmonary vascular structure and function. Postnatal exposure to 10-15% oxygen during the first 3 weeks of life impairs lung development, characterized by decreased alveolarization and reduced lung vascular development. Deruelle et al. found that exposure of infant rats to hypoxia for 14 days impairs alveolarization as reflected by reduced radial alveolar counts, and decreased vascular volume density. The pathogenesis of pulmonary hypertension (PH) appears to involve an initial, active vasoconstriction of pulmonary resistance vessels that may progress to fixed luminal narrowing, elevated pulmonary vascular resistance (PVR), thrombi formation, and, ultimately, right heart failure and death. After the structural remodeling of pulmonary vessels has occurred, therapeutic interventions to restore PVR to normal levels are largely ineffective. Therefore, drug therapies targeting cellular pathways that mediate the PH and reversible rise in PVR may be advantageous.Ghrelin is a 28 amino acid peptide originally isolated from rat stomach as an endogenous ligand for the growth hormone (GH) secretagogue receptor (GHS-R). The ghrelin gene peptides include acylated ghrelin, unacylated ghrelin., and obestatin. Acylated ghrelin, exerts its central and peripheral effects through the GHS-Rla. Indeed, acylated ghrelin was demonstrated to act as an autocrine/paracrine factor, regulating cell proliferation and survival, apoptosis, inflammation, cardiovascular and gastric functions, metabolism, angiogenesis, development, and reproduction. Ghrelin is able to attenuate the development of pulmonary artery hypertension in a monocrotaline-treated adult animal model. However, this monocrotaline-induced PH model, including the response to potential therapeutic treatments, differs considerably from PH induced by chronic hypoxia (CH). A previous study has shown that ghrelin can directly stimulate the production of nitric oxide (NO) from vascular endothelial cells through PI 3-kinase-dependent signaling pathways that mimic the effects of insulin. PPHN in utero causes sustained alteration of fetal pulmonary artery endothelial cell (PAEC) phenotypes, as determined in vitro. Also, endothelial nitric oxide synthetase (eNOS) protein expression was decreased in PPHN PAECs and NO enhanced growth and tube formation was observed in PPHN PAECs. There is increasing evidence that ghrelin has a potent vasodilator effect. We hypothesize that ghrelin would prevent PPHN by altering signal transduction pathways.In this study, using a CH-induced PPHN model in rats, we first explored the molecular signaling cascades and gene expression patterns in lung tissue. We then addressed whether ghrelin protected neonatal rats from hypoxia-induced PH, including its effects on hemodynamics and pulmonary vasculature. Finally, we analyzed signaling transduction pathways regulated by ghrelin.Part I Multiplexed profiling of candidate genes for hypoxic pulmonary hypertension in neonatal rats using a PCR Array assay Aim:1. To establish PPHN animal model and assess pulmonary hemodynamics and morphometry.2. To screen multiplexed profiling of candidate genes for lung tissues of hypoxic pulmonary hypertension in neonatal rats by PCR Array assay.Methods:1. PPHN model and groupsHypoxic group:neonatal rats were exposed to 12% oxygen in a forced-air environmental chamber for 14 days.Normoxic group:rats were exposed to 21% oxygen (room air) for 14 days.2. Assessment of pulmonary hypertensionThe right ventricular systolic pressure (RVSP) was detected in rats after 14 days’ hypoxic exposure. The degree of muscularization of pulmonary vessels was assessed by immunohistochemistry staining of a-SMA. The ratio of the right ventricle (RV) to the left ventricle plus septum (LV+S) was used to evaluate the hypertrophy of right ventricle. 3. PCR Array assay to screen multiplexed profiling of candidate genes for lung tissues of hypoxic pulmonary hypertension in neonatal rats.Results:1. RVSP increased significantly in neonatal rats after 14 days’hypoxic exposure than that of normoxic group (37.29±3.15 mmHg vs 20.02±1.02 mmHg, P<0.05). The number of muscular pulmonary vessels increased significantly in rats following 14 days’hypoxic exposure than in normoxic group. The ratio of RV/(LV+S) was also significantly increased after hypoxic exposure (P<0.01).2. Two signal transduction pathways were significantly upregulated by a threshold of≥3.0 when hypoxic group compared with the normoxic group:Wnt pathway (lefl, 4.79; Wnt,3.87) and CREB pathway (Cypl9al,4.87; Egrl,7.79; Fos,3.86).Conclusions:1. We established PPHN model successfully.2. Wnt and CREB signaling pathway are most important signaling pathways in hypoxic pulmonary hypertension in newborns.PartⅡThe mechanism of ghrelin ameliorates hypoxia-induced pulmonary hypertensionAim:1. To investigate the change of pulmonary vascular remodeling and right ventricle hypertrophy in neonatal rats after 14 days’hypoxic exposure after the ghrelin administration.2. Confirmatory real-time PCR assay for lung tissues of hypoxic pulmonary hypertension in neonatal rats after ghrelin administration.Methods:1. PPHN model and groups Rats were divided into four groups (n=10/each group):hypoxia and vehicle treatment (sc,0.2ml) daily for 14 days;hypoxia and ghrelin (Phoenix Biotech Co., Ltd., Beijing, China) treatment (sc,150μg/kg (Schwenke, et al.2008),0.2ml) daily for 14 days;Sham control, sham chamber was not subjected to hypoxia and was treated with the vehicle control (sc,0.2ml) daily for the same period.Ghrelin control (sc,150μg/kg,0.2ml) ghrelin treatment daily for 14 days in normoxic rats.2. The morphological changes of the small pulmonary arteries were examined by media wall thickness (a-SMAarea/LD) after ghrelin administration. The ratio of RV/(LV+S) was calculated to evaluate the hypertrophy of right ventricle. Serum active ghrelin were determined by ELISA. Wnt Pathway verification by qRT-PCR analysis and Western blot analysisResults:1. In the hypoxia group, a significant increase in mean RVSP (mRVSP), above the control value, was observed (37.29±3.15 mmHg versus 20.02±1.02 mmHg, P<0.05). Daily administration of ghrelin during hypoxia significantly attenuated the development of neonatal PH. There was also a significant increase in the RV/(LV+S) ratio in hypoxia group (0.51±0.09 versus 0.19±0.01, P<0.05), indicating RVH. Therefore, Ghrelin treatment reduced both the magnitude of PH and the RV/(LV+S) ratio. A significant decrease in the a-SMAarea/LD ratio was observed for ghrelin-treated rats (107.4±6.8; P=0.000) versus vehicle-treated hypoxia.2. The levels of active ghrelin measured in hypoxia+ghrelin animals versus hypoxia group were 43.75±4.94 and 27.38±5.29 pg/ml (P<0.05), respectively.3. We also found that the Wnt pathway (Birc5,5.05; Myc,3.09; Pparg,4.82) and the PI3K/Akt pathway (Fn1,3.60) were upregulated in hypoxia+ghrelin rats compared with hypoxia group. Wnt pathway (Birc5,-8.76; Myc,1.12; Pparg,-1.59) and the PI3K/ Akt pathway (Fn1,-2.19) when hypoxic group compared with the normoxic group.4. Notably, a significant increase in expression of p-GSK3β/GSK3βandβ-catenin was detected in ghrelin treated CH rats. Surprisingly, significant differences in the ID of GHSR-1a were observed between the groups with and without ghrelin treatment in the pulmonary arteriole. The results were also verified by western blot analysis (hypoxia: 0.7±0.2 versus hypoxia+ghrelin group:1.6±0.3; P<0.05).Conclusions:1. Ghrelin ameliorates hypoxia-induced pulmonary hypertension, pulmonary vascular remodeling and right ventricular hypertrophy.2. The mechanisms of ghrelin ameliorates hypoxia-induced pulmonary hypertension involving PI3-K/Akt/GSK-3βand Wnt signaling pathway.3. GHSR-la receptor is involved as an intermediary through PI3-K/Akt/GSK-3βsignaling pathway.Part III Ghrelin inhibits hypoxic pulmonary microvascular endothelial cell apoptosis and its p-GSK3β/GSK3βsignaling pathwayAim:1. To observe the impact of ghrelin on apoptosis induced by hypoxia.2. To observe the changes in PI3-K/Akt/GSK-3βprotein expression and distribution ofβ-catenin after ghrelin intervention.Methods:1. Rat pulmonary microvascular endothelial cells (RPMECs) were isolated using a modification of the technique.2. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labelling (TUNEL) assays. 3. Administration of Ghrelin Receptor Antagonist and PI3K/AKT Inhibitor, then p-Akt and p-GSK3β/7GSK3βPathway verification by Western blot analysis.4. Luciferase activity was assayed 48 hours after transfection, using a dual-luciferase reporter assay system.Results:1. RPMECs formed a confluent monolayer of cobble stone-like cells, EC markers such as labeled anti-mouse CD31 antibody accounted for 95%.2. TUNEL showed that the hypoxia group, normoxia+ghrelin group and hypoxia+ ghrelin apoptosis rate were 2.77±0.28,0.92±0.07,1.63±0.19 respectively (P<0.05)3. Treatment of RPMECs with ghrelin rapidly activated p-Akt in a time and dose-dependent manner. GSK3βphosphorylation was increased after 30min of ghrelin treatment and lasted for 120min. The inhibition of ghrelin receptor with [D-Lys3]-GHRP-6 reduced ghrelin-induced Akt phosphorylation and Ly294002 also reduced GSK3βphosphorylation.4. We found ghrelin induced phosphorlation of GSK3βis associated withβ-catenin translocation to the nucleus under hypoxia condition.Conclusions:1. Ghrelin inhibits hypoxic pulmonary microvascular endothelial cell apoptosis involving PI3-K/Akt/GSK-3βand Wnt signaling pathway.2. GHSR-la receptor is involved as an intermediary through p-GSK3p/GSK3p signaling pathway in RPMECs.3. We built a Wnt/β-catenin signaling pathway reporter gene model. Activities ofβ-catenin/TCF reporter gene was significantly up-regulated by ghrelin analyzed by using the reporter gene model.更多还原