转录因子CASZ1和前B细胞集落增强因子在高氧致新生鼠支气管肺发育不良中的表达及意义

发布时间：2018-05-31 01:00

本文选题：支气管肺发育不良 + 转录因子CASZ1　；参考：《南方医科大学》2016年硕士论文

【摘要】：研究背景支气管肺发育不良(Bronchopulmonary dysplasia, BPD)是早产儿疾病中的一种常见并发症,近年来在新生儿重症监护病房中此种疾病的发病率和死亡率大大增加。在过去的几十年里,尽管新生儿医疗水平在不断进步,如先进的机械通气模式、产后使用肺表面活性物质和产前使用激素等,然后新生儿支气管肺发育不良的发生率却没有下降。在国,每年有10,000—15,000例患儿出现此病,这些患儿大多是出生体重1250 g的早产儿。此种疾病的呼吸和神经系统后遗症会一直持续到成年。1967年,William (Bill) Northway和他的同事首次报道并命名这一疾病。支气管肺发育不良主要出现在伴有急性呼吸衰竭并接受机械通气和氧疗的早产儿,正压机械通气引起的气压伤和容量伤加剧了早产儿的肺损伤。此外,氧毒性和肺部炎症阻碍了新生儿出生后肺的发育。支气管肺发育不良的主要病理特征为肺泡和肺微血管发育受阻,表现为肺泡结构简单化,肺毛细血管生成减少等。支气管肺发育不良是婴幼儿最常见的慢性呼吸系统疾病,胎肺发育过程有五个不同的阶段分别是：胚胎期、假腺管期、微管期、囊泡期和肺泡期,胚胎肺发育在微管期和囊泡早期出生的早产儿容易发生BPD。原始肺泡、肺泡毛细血管和Ⅰ型、Ⅱ型肺泡壁细胞主要在微管后期形成和发育,囊泡早期主要发育特点是肺表面活性物质的产生,肺血管的形成分化和终末气道扩张等。早产婴儿胎肺的正常发育中断,在加上可能由感染、机械通气或高氧引起的炎症,使胎儿肺泡化受阻从而导致BPD的发生。支气管肺发育不良确切的病因和发病机制仍不十分明确,目前认为主要是在遗传易感性的基础上,气压伤、容量伤、氧化应激、宫内感染、炎症反应等因素共同作用于发育未成熟的肺组织而导致的肺泡和肺微血管发育障碍和肺组织损伤后异常修复的结果。有研究发现肺微血管发育障碍与BPD的发生发展密切相关,调控肺微血管发育的基因表达失衡会干扰肺微血管的正常发育以及减慢肺泡化进程,其在BPD的发病机制中起了关键作用。在肺发育过程中,肺血管的生成可以促进肺泡的发育,并维持肺泡的正常结构；另外肺泡分化的同时肺血管网随之扩张,从而形成有效的气血屏障,早产儿由于肺尚未发育完善,辅助通气等治疗可能使肺血管床减少肺泡发育受阻,导致气血交换不足引起一系列临床症状,调控肺微血管发育的基因和蛋白也成为近年来国内外研究的热点。亦有研究发现,早产儿吸入高浓度氧或长期机械通气时,肺组织大量的氧自由基及炎症介质的产生,引起炎症细胞在肺内聚集,最终导致的BPD的发生。可见,氧化应激及其诱发的炎症反应在BPD的发生发展中起了至关重要的作用。转录因子CASZ1又称ZNF693,是新近发现的一种转录因子,属于锌指基因家族的成员之一,其蛋白产物只要通过与DNA结合或聚合而起转录因子的重要作用,参与正常细胞的增值、凋亡和分化过程。Charpentier等研究表明CASZ1主要在血管内皮细胞中表达,CASZ1通过EGFL7/RhoA通路调控内皮细胞的行为促进血管网的形成和分支,另外CASZ1还可通过RhoA调节内皮细胞的黏附从而促进血管生芽,这些研究显示CASZ1在微血管的出芽和重建中起着重要作用,为血管的生成和形态发生所必须。前B细胞集落增强因子(pre-B cell colony enhancing factor, PBEF)又称为内脂素或烟酰胺磷酸核糖转移酶,是近年来发现的主要由成熟脂肪细胞和激活的炎症细胞分泌,全身多器官均可表达的炎症因子,与炎症、代谢综合征、肿瘤、自身免疫性疾病等密切相关具有调控炎症、细胞生长、血管生成和凋亡等多种作用的蛋白。目前有体外细胞实验研究发现炎症刺激可以使肺泡上皮细胞、肺微动脉内皮细胞等大量表PBEF,而减少PBEF的表达则可减轻上述细胞由炎症刺激引起的相关损害,这表明PBEF可能涉及包括肺微血管损伤、肺泡上皮细胞损伤、通透性增加等在内有关呼吸系统疾病的多个病理生理过程。本研究采用80%的高体积分数氧制作新生鼠BPD模型,观察各组新生鼠肺组织的病理学变化,微血管密度测定,转录因子CASZ1和炎症因子PBEF的基因的蛋白表达,探讨转录因子CASZ1在高氧致支气管肺发育不良新生大鼠肺组织中的表达及其对肺微血管发育的关系和前B细胞集落增强因子(PBEF)在高氧致新生大鼠支气管肺发育不良中的表达及意义。研究目的1、用80%的高体积分数氧制作新生鼠BPD模型,观察各组新生鼠肺组织的病理学变化,微血管密度测定,以及CASZ1和PBEF的基因和蛋白表达情况。2、分析高氧组新生大鼠肺组织中CASZ1的蛋白表达水平与肺微血管密度之间的关系,探讨CASZ1在BPD发生发展过程的可能机制。3、观察肺组织中PBEF的表达在BPD发生发展中的动态变化规律,分析其在BPD发病过程中的可能作用。研究方法1、实验动物模型的建立：以足月新生鼠为研究对象,将48只新生鼠在出生24小时内随机分为实验组和对照组,每组均为24只。实验组持续暴露于80%左右的氧气中,制作新生鼠BPD模型。对照组置于同一室内的空气中,其他实验控制因素和实验组相同。2、分别于实验开始后3天、7天、14天观察高氧对新生鼠一般状况的影响,并取肺组织用HE染色方法光镜下观察各实验组肺组织病理学变化并进行放射状肺泡计数(RAC),采用IPP6图像分析系统测量肺泡呼吸膜厚度(ST)。3、应用免疫组化方法检测各时间点两组新生鼠肺组织中CD31、CASZ1、 PBEF蛋白的分布和表达；以肺组织CD31免疫染色阳性的内皮细胞所占的面积与肺实质细胞总面积的百分比计算肺微血管密度。4、应用ELISA方法检测各时间点新生鼠肺泡灌洗液中PBEF的蛋白浓度。5、Real-Time PCR方法检测各组各时间点新生鼠肺组织中CASZ1、PBEF的基因表达水平。6、Western blot方法检测各组各时间点新生鼠肺组织中CASZ1、PBEF的蛋白表达水平。7、实验数据用均数±标准差(X±SD)表示,应用SPSS 17.0软件对数据进行分析, 同一时间点两组比较采用t检验,各组间比较采用单因素方差分析,相关分析采用Pearson相关分析, P0.05为差异有统计学意义。结果1、随着日龄的增加对照组新生鼠肺组织逐渐发育成熟,肺泡大小均匀,结构规整,放射状肺泡计数逐渐增加,呼吸膜较薄；而高氧组新生鼠肺组织肺泡逐渐出现融合,肺间隔断裂,肺泡大小不均,放射状肺泡计数明显减少,呼吸膜增厚,肺间质中有大量炎性细胞浸润,肺组织结构发生类似BPD的病理改变。2、两组新生鼠肺组织RAC和ST,3天时无明显差异,7天和14天时高氧组RAC明显减少、ST显著增厚与对照组相比差异均有统计学意义(P0.05)。3、免疫组织化学染色结果显示：1)CD31广泛表达于肺微血管内皮细胞的胞质中,随日龄的增加对照组CD31表达水平逐渐升高,高氧组和对照组相比,各时间点微血管密度均显著下降(P0.05)；2)肺组织中CASZ1主要表达于支气管上皮细胞、肺泡上皮细胞和肺血管内皮细胞中。高氧组3天、7天、14天CASZ1阳性细胞的积分光密度值均显著低于相应对照组(P0.01)；3)肺组织中PBEF的表达于支气管上皮细胞、肺泡上皮细胞和炎症细胞中,高氧组和对照组PBEF的表达都有逐渐升高的趋势,高氧组3天、7天、14天PBEF阳性细胞的积分光密度值均高于相应对照组,差异有统计学意义(P0.05)。4、3天、7天两组新生鼠肺组织支气管肺泡灌洗液中PBEF蛋白浓度无统计学差异,14天时高氧组显著高于对照组(P0.01)。5、与对照组相比,各时间点高氧组CASZ1基因和蛋白表达水平降低(P0.01)；而高氧组PBEF基因和蛋白表达平水平较对照组升高(P0.05)。6、高氧组新生鼠肺组织中CASZ1蛋白表达水平与肺微血管密度呈正相关(r=0.519,P=0.0090.01)。结论1、新生大鼠持续吸入80%高氧后,肺泡逐渐出现融合,肺间隔断裂,肺泡大小不均,放射状肺泡计数明显减少,呼吸膜增厚,肺间质中有大量炎性细胞浸润,肺组织结构发生类似BPD的病理改变。2、高氧组新生鼠CASZ1 mRNA和蛋白表达随着高氧暴露时间的延长而逐渐下降,各时间点均显著低于对照组,提示高浓度氧可以抑制转录因子CASZ1的表达。3、高氧组新生大鼠肺组织中CASZ1的蛋白表达水平与肺微血管密度呈正相关,推测高氧可能通过抑制CASZ1的表达,从而阻碍肺血管的发育,导致BPD的发生。4、研究结果显示BPD实验组PBEF的表达显著高于正常对照组(P0.05),表明PBEF参与了支气管肺发育不良的发生发展,但PBEF参与支气管肺发育不良发生的具体机制尚不清楚,有待进一步研究。
[Abstract]:Background Bronchopulmonary dysplasia (BPD) is a common complication of premature infant disease. In recent years, the incidence and mortality of this disease have increased greatly in the neonatal intensive care unit. In the past few decades, although the medical level of newborn infants is progressing, such as advanced mechanical ventilation Patterns, postpartum use of pulmonary surfactant and prenatal hormone, and then the incidence of bronchopulmonary dysplasia in the newborn did not decline. In the country, 10000 to 15000 children have this disease each year, most of which are premature babies born with birth weight of 1250 g. The respiratory and nervous system sequelae of this disease will continue to continue. By the age of.1967, William (Bill) Northway and his colleagues first reported and named the disease. Bronchopulmonary dysplasia mainly appeared in premature infants accompanied by acute respiratory failure and undergoing mechanical ventilation and oxygen therapy. Barometric and volumetric injuries caused by positive mechanical ventilation combined with lung injury in preterm infants. Inflammation hinders the development of the lung after birth. The main pathological features of bronchopulmonary dysplasia are the obstruction of alveolar and pulmonary microvascular development, characterized by the simplification of the alveolar structure and the decrease of pulmonary capillary formation. Bronchopulmonary dysplasia is the most common chronic respiratory system disease in infants and five different fetal lung development processes. The stages are: embryonic stage, false glandular tube stage, microtubule stage, vesicle stage and alveolar period. Embryonic lung development is prone to BPD. primordial alveoli, alveolar capillaries and type I, type I alveolar wall cells in the early stage of microtubule and early birth of vesicles. The main development characteristics of the early vesicle are pulmonary surface activity. The formation of substance, the formation and differentiation of the pulmonary vessels, and the dilatation of the terminal airway. The normal development of the fetal lung in premature infants is interrupted by the addition of inflammation that may be caused by infection, mechanical ventilation or hyperoxia, causing fetal pulmonary alveolar obstruction to cause BPD. The exact etiology and pathogenesis of bronchopulmonary dysplasia are still not very clear, currently recognized. The results of abnormal repair of pulmonary alveolar and pulmonary microvascular development and lung tissue damage caused by factors such as air pressure injury, volume injury, oxidative stress, intrauterine infection, inflammatory reaction and other factors, mainly on the basis of genetic susceptibility. It is closely related that the imbalance of gene expression in the regulation of pulmonary microvascular development will interfere with the normal development of pulmonary microvessels and slow the process of alveolar degeneration. It plays a key role in the pathogenesis of BPD. In the process of lung development, the formation of pulmonary vessels can promote the development of the alveoli, and maintain the normal structure of the alveoli; in addition, the alveolar differentiation is simultaneously differentiated. The pulmonary vascular network expands, thus forming an effective air blood barrier, the premature infant has not developed the lung yet, and the auxiliary ventilation may cause the pulmonary vascular bed to reduce the pulmonary alveolar development and cause a series of clinical symptoms. The basic causes and proteins that regulate the development of the pulmonary microvessel have also become a hot spot of research at home and abroad in recent years. It is also found that a large number of oxygen free radicals and inflammatory mediators in the lung tissue are produced in preterm infants when high concentrations of oxygen or long term mechanical ventilation are inhaled, resulting in the accumulation of inflammatory cells in the lungs and the occurrence of BPD. Oxidative stress and its induced inflammatory response have played a vital role in the development of BPD. CASZ1, also known as ZNF693, is a newly discovered transcription factor, one of the members of the zinc finger gene family. Its protein product is an important role of the transcription factor by combining or polymerizing with DNA. It participates in the increment of normal cells, the process of apoptosis and the process of differentiation,.Charpentier and so on, which indicate that CASZ1 is mainly expressed in vascular endothelial cells, CASZ 1 the regulation of endothelial cells through the EGFL7/RhoA pathway promotes the formation and branching of vascular networks, and CASZ1 also regulates the adhesion of endothelial cells by RhoA to promote vascular buds. These studies show that CASZ1 plays an important role in the buds and reconstructions of microvessels, which is necessary for the formation and morphogenesis of blood vessels. The colony of the former B cells Pre-B cell colony enhancing factor (PBEF), also known as endogenous lipoprotein or nicotinamide phosphonotinase, is found in recent years mainly secreted by mature adipocytes and activated inflammatory cells, and can be expressed in multiple organs. It is closely related to inflammation, metabolic syndrome, tumor, autoimmune disease and so on. There is a variety of proteins that regulate inflammation, cell growth, angiogenesis, and apoptosis. In vitro cell experiments have found that inflammatory stimulation can make the alveolar epithelial cells, pulmonary microarterial endothelial cells and so on a large number of PBEF, and the reduction of PBEF expression can reduce the related damage caused by the inflammatory stimulation, which indicates that PBEF can be used. It can involve multiple pathophysiological processes related to respiratory diseases, including pulmonary microvascular injury, alveolar epithelial cell damage, and increased permeability. This study used 80% high body integral oxygen to make the BPD model of neonatal rats, to observe the pathological changes of lung tissue, microvessel density, transcription factor CASZ1 and inflammatory causes in the newborn rats. Protein expression of the subPBEF gene and the expression of transcription factor CASZ1 in the lung tissue of neonatal rats with hyperoxic bronchopulmonary dysplasia and its relationship to pulmonary microvascular development and the expression and significance of B cell colony enhancement factor (PBEF) in hyperoxic neonatal rats with bronchopulmonary dysplasia. Objective 1, with a high volume of 80% The BPD model of neonatal rats was made by fractional oxygen. The pathological changes of lung tissue, microvessel density, and gene and protein expression of CASZ1 and PBEF were observed in each group. The relationship between the protein expression level of CASZ1 in the lung tissue of the hyperoxic group and the relationship between the microvascular density of the lung was analyzed, and the development of CASZ1 in BPD was discussed. Energy mechanism.3, observe the dynamic change rule of PBEF expression in the development of BPD in the lung tissue and analyze its possible role in the pathogenesis of BPD. Method 1, the establishment of experimental animal model: a term newborn rat was used as the study object, and the 48 newborn rats were randomly divided into the experimental group and the control group within 24 hours of birth, each group was 24. The experimental group was exposed to about 80% oxygen to make the BPD model of newborn rats. The control group was placed in the same indoor air. The other experimental control factors were the same as the experimental group.2. The effects of hyperoxia on the general condition of the newborn rats were observed on the 3 day, the 7 day and the 14 day after the experiment, and the lung tissues were observed under the HE staining method under the light microscope. The pathological changes of lung tissue and radiate alveolar count (RAC) were carried out. The pulmonary alveolar membrane thickness (ST).3 was measured by IPP6 image analysis system. The distribution and expression of CD31, CASZ1, PBEF protein in the lung tissues of two new groups of newborn rats were detected by immunohistochemical method, and the surface of the endothelial cells positive for CD31 immunoreactivity in lung tissue was detected. The percentage of lung microvascular density.4 was calculated with the percentage of the total area of lung parenchyma. The protein concentration of PBEF in the alveolar lavage fluid of newborn rats at every time point was detected by ELISA method. The Real-Time PCR method was used to detect the CASZ1, PBEF gene expression level.6 in the lung tissue of each time point of each group, and the Western blot method was used to detect the new generation of each time point. The protein expression level of CASZ1 and PBEF in rat lung tissue was.7, and the experimental data were expressed with mean standard deviation (X + SD). The data were analyzed with SPSS 17 software and t test was used in two groups at the same time point. The single factor analysis of variance was used in each group. The correlation analysis was analyzed with Pearson correlation. P0.05 was statistically significant. Fruit 1, with the increase of day age, the lung tissues of the control group gradually developed and mature, the size of the alveoli was uniform, the structure was regular, the number of radiate alveoli increased gradually, and the respiratory membrane was thinner, while the pulmonary alveolus of the newborn rats were gradually fused, the pulmonary septum was broken, the size of alveoli was uneven, the number of radiate alveoli decreased obviously, the respiratory membrane thickened and lung was thickened. There were a large number of inflammatory cells infiltrating in the interstitial tissue and the pathological changes of lung tissue similar to BPD,.2. There was no significant difference between the two groups of newborn rats' lung tissue RAC and ST. The RAC in the hyperoxia group was significantly reduced at the 7 and 14 days, and the significant thickening of ST was statistically significant compared with the control group (P0.05).3, and the immunohistochemical staining results showed 1) CD31 wide. In the cytoplasm of the pulmonary microvascular endothelial cells, the expression level of CD31 in the control group increased gradually with the increase of day age. Compared with the control group, the microvascular density decreased significantly at every time point (P0.05). 2) CASZ1 in the lung tissue was mainly expressed in the bronchial epithelial cells, alveolar epithelial cells and pulmonary vascular endothelial cells. The hyperoxia group was 3. The integral light density values of CASZ1 positive cells in days, 7 days and 14 days were significantly lower than that in the corresponding control group (P0.01), and 3) the expression of PBEF in the lung tissue was gradually increased in the bronchial epithelial cells, alveolar epithelial cells and inflammatory cells, and the expression of PBEF in the hyperoxia group and the control group was gradually increased, and the integral light density of the PBEF positive cells of the hyperoxia group for 3 days, 7 days and 14 days of PBEF positive cells. The difference was statistically significant (P0.05).4,3 days, and there was no statistical difference in the concentration of PBEF protein in the bronchoalveolar lavage fluid of the two groups of neonatal rats on the 7 day, and at 14 days, the hyperoxia group was significantly higher than the control group (P0.01).5. Compared with the control group, the CASZ1 gene and protein expression level in the hyperoxia group were decreased (P0.01). The level of PBEF gene and protein expression in the hyperoxic group was higher than that of the control group (P0.05).6, and the expression level of CASZ1 protein in the lung tissue of the hyperoxic group was positively correlated with the pulmonary microvascular density (r=0.519, P=0.0090.01). Conclusion 1, after the newborn rats inhaled 80% hyperoxia, the alveoli gradually appeared to fuse, the pulmonary septum fracture, the alveolar size uneven, and the radiate alveoli. The count decreased obviously, the respiratory membrane was thickened, a large number of inflammatory cells were infiltrated in the pulmonary interstitium and the pathological changes of the lung tissue were similar to that of BPD. The expression of CASZ1 mRNA and protein in the hyperoxic group decreased gradually with the prolongation of the exposure time of hyperoxia, and the time points were significantly lower than those in the group, suggesting that the high concentration of oxygen could inhibit the transcription factor CASZ1. Expression of.3, the protein expression level of CASZ1 in the lung tissue of the hyperoxic group was positively correlated with the lung microvascular density. It is suggested that hyperoxia may inhibit the expression of CASZ1, thus hindering the development of pulmonary vessels and causing the occurrence of.4 in BPD. The results of the study showed that the expression of PBEF in the BPD experimental group was significantly higher than that of the normal control group (P0.05), indicating that PBEF was involved in the study. The pathogenesis of bronchopulmonary dysplasia is unclear, but the specific mechanism of PBEF involvement in bronchopulmonary dysplasia is unclear.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R722.1

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