Sprague-Dawley大鼠胎肺组织miRNA差异表达谱分析
发布时间：2019-06-04 21:38
【摘要】：随着早产儿救治率及存活率的提高,呼吸窘迫综合征(Respiratory Distress Syndrome, RDS)及支气管肺发育不良(Bronchopulmonary Dysplasia, BPD)的发生率也有明显升高的趋势,现已成为新生儿临床常见的几种主要病种,因而对其发病机制和治疗靶点的探索已成为新生儿领域的研究热点。 临床上这两种疾病病因较为复杂,前者的发生是由于肺发育晚期肺泡表面活性物质分泌不足所致。同时,研究者还发现RDS的发生同胎龄及出生体重也紧密相关,这可能也暗示了肺发育成熟度同RDS之间存在着一定的关联。而后者——BPD的发生被认为主要是在基因易感性基础上由包括高氧毒性、机械通气、感染、炎症等在内的多因素造成的肺损伤。特别是在早产儿,出生前肺发育的整个过程尚未完成,肺泡数目少,肺泡结构简单,此时极易受到各种危险因素的影响,而导致肺损伤和肺发育的滞后。 MicroRNAs(miRNAs)是近年来发现的一类重要的短小内生RNA,能通过对靶向mRNA的直接降解或抑制而阻止其翻译、表达,从而在转录后水平参与基因的表达调控。目前一致认为miRNA在细胞发育时序、细胞增殖、信号转导、干细胞分化和肿瘤的发生转移等紧要的生物学进程中都起着非常重要的作用。 生物芯片技术是20世纪90年代初发展起来的新技术,具有高通量、高集成、微型化、连续化和自动化的特点,此技术现今已经成熟运用到miRNA水平基因表达检测上,是检测细胞或组织miRNA表达谱的理想方法。 本文课题分为三部分,第一部分研究大鼠胚胎发育三个关键时间点胎肺组织形态学的改变。第二部分研究大鼠胎肺发育中三个关键时间点miRNA的差异表达情况。第三部分着重探讨miRNA-126/miRNA-126*在大鼠胎肺发育中的相关作用。 本课题组通过对胎肺发育三个时间点进行miRNA筛查,试图找到与肺发育相关的miRNAs。这些新发现的miRNAs或许在肺发育中会起着一定作用,同时这一研究结果也可为未来肺发育及其相关疾病的研究提供一定的依据。 第一部分Sprague-Dawley大鼠胎肺发育形态学观察研究 目的：观察大鼠胚胎肺泡发育形态学变化,为将来课题建立连续而系统的胎肺发育形态学资料。 方法：12只健康孕鼠随机分为孕16天组(记为S1组)、孕19天组(记为S2组)和孕21天组(记为S3组),三组孕鼠分别在孕16天,孕19天,孕21天快速剖宫取出其胎鼠肺脏,行切片、H-E染色(Haemo-toxylin and Eosin, H-E)及透射电镜检测以观察胎肺组织不同发育时期形态结构差异。 结果：(1)光镜下观察：S1组见原始支气管呈树枝状分布延伸,上皮为高柱状,上皮细胞呈环形排列,间质厚,毛细血管少见,未见肺泡结构存在；S2组见初级肺泡,且肺泡上皮细胞由高柱状转化为矮柱状立方形,间质变薄,毛细血管数目增多；S3组见肺泡间隔进一步增多,肺腺泡腔体极度扩张,间质进一步变薄。 (2)透射电镜下观察：S1组未见肺泡Ⅱ型上皮细胞,只有原始的肺泡上皮细胞存在,原始上皮细胞内可见线粒体,多而密集,但在细胞内并未见板层小体。S2组中Ⅱ型肺泡上皮开始分化出现,在细胞内部此时可见板层小体,染色深且结构致密。在S3组中Ⅱ型肺泡上皮细胞进一步增多,板层小体数目也逐渐增多,且外有界膜包绕,结构更为致密,肺泡上皮绒毛结构清楚,间质毛细血管易见。结论：大鼠胎肺发育是循序渐进的生理过程,且一直延续到生后。细胞内板层小体的出现是肺泡Ⅱ型上皮细胞分化的标志,且其数目随着发育过程而增多。 第二部分Sprague-Dawley大鼠正常胎肺发育过程中miRNA的差异表达情况研究 目的：通过比较孕16天,孕19天,孕21天三组间大鼠胎肺组织miRNA表达谱的差异,筛选出有意义的miRNAs,为进一步探讨其在Sprague-Dawley大鼠胎肺发育机制中的作用提供一定的理论依据。 方法：12只健康孕鼠随机分为孕16天组(S1组)、孕19天组(S2组)和孕21天组(S3组),分别采用了包含有1891个探针的新型miRNA芯片,通过观察大鼠胎肺发育后期三个关键时间点(孕16天,孕19天,孕21天)的miRNAs表达情况从而筛选出具有差异表达的miRNAs。在这些niRNAs中随后挑选出在三个时间点都有显著差异表达的miRNAs,并且进行real-time PCR的验证。 结果：(1)在上述的三个时间点中发现了167个miRNAs呈现差异性表达。其中,有81个上调的miRNAs,86个下调的miRNAs。由于在连续三个时间点中都表现出超过2倍以上倍数变化,并呈连续上调或下调趋势,故本实验又进一步从中筛查出了7个连续差异表达的miRNAs。 (2)由于在这7个miRNAs中miRNA-125b-5p, miRNA-296, miRNA-93, miRNA-146b以及miRNA-3560等5个具有更高的连续倍数变化,所以最终被挑选出进行real-time P(R的验证,而real-time PCR的最终验证结果与miRNA芯片结果基本一致。 结论：通过基因芯片技术,课题组胎鼠肺发育三个时间点共筛选出167个差异表达miRNAs,我们推测这些具有差异表达的miRNAs在正常肺发育的调控机制中可能起着一定的作用,这有助于我们进一步阐明包含肺泡Ⅱ型上皮在内的胎肺发育机制,同时也为以后进行肺发育相关疾病的研究提供了一定的生理学基础。 第三部分miRNA-126/miRNA-126*在Sprague-Dawley大鼠正常胎肺发育中相关作用的研究 目的：通过比较孕16天,孕19天,孕21天这三个时间点间大鼠胎肺组织芯片中miRNA-126/miRNA-126*的差异表达水平,探讨miRNA-126^miRNA-126*在Sprague-Dawley大鼠胎肺发育机制中可能的作用,为今后进一步研究新生儿肺发育相关疾病提供一定的依据。 方法：12只健康孕鼠随机分为孕16天组(S1组)、孕19天组(S2组)和孕21天组(S3组),本实验采用了包含有1891个探针的新型miRNA芯片(详见本实验第二部分),观察大鼠胎肺发育后期三个关键时间点(孕16天,孕19天,孕21天)的miRNA-126及miRNA-126*的表达情况,并且随后进行了相关real-time PCR的验证。 结果：经过芯片筛查,我们发现miRNA-126在三组间均呈高水平表达,表达水平无明显差异；而miRNA-126*三组间存在差异表达,随后的real-time PCR结果也证实miRNA-126*的表达在三组间均存在显著性差异(Group SI→Group S2→Group S3),具有统计学意义(P0.05)。 结论：本研究首次报道了在胎肺发育中miRNA-126随着肺发育过程存在显著差异表达。故推测miRNA-126*可能在胎肺发育中扮演着重要的角色。对miRNA-126*的深入研究可能揭示其在大鼠胚胎肺发育中所起的相关作用,且对进一步研究与新生儿肺发育的相关疾病(如支气管肺发育不良及呼吸窘迫综合征等)提供一定的理论基础。
[Abstract]:The incidence of respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) has also increased significantly with the improvement of the treatment rate and the survival rate of the premature infants. Therefore, the study of its pathogenesis and treatment target has become a hot spot in the field of newborn. The causes of these two diseases are complicated, and the former is due to the insufficient secretion of surface active substances in the late stage of the lung development. To the same time, the researchers also found that the occurrence of RDS is closely related to the gestational age and the birth weight, which may also suggest that there is a certain correlation between the maturity of the lung and the RDS. The latter, the occurrence of the latter _ BPD, is believed to be primarily due to multiple factors, including high oxygen toxicity, mechanical ventilation, infection, inflammation, and the like, on the basis of genetic susceptibility In particular, in premature infants, the whole process of pulmonary development before birth is not completed, the number of alveoli is small, the alveolar structure is simple, and at this time, the lung injury and the development of the lung can be caused by the influence of various risk factors. MicroRNAs (miRNAs), which have been found in recent years, are an important short, endogenous RNA that can be prevented from being translated and expressed by direct degradation or inhibition of targeted mRNA, thereby participating in a table of genes at the post-transcriptional level. It is currently agreed that miRNAs play a very important role in critical biological processes such as cell development timing, cell proliferation, signal transduction, stem cell differentiation, and metastasis of tumors The biological chip technology is a new technology developed in the early 1990s, and has the characteristics of high flux, high integration, microminiaturization, continuous and automatic. The paper is divided into three parts. The first part studies the three key time point fetal lung groups of the rat embryo development. The change of weaving morphology. The second part studies three key point-in-time miRNAs in fetal lung development in rats The third part focuses on miRNA-126/ miRNA-126 * in rat fetal lung The research group conducted miRNA screening for three time points of fetal lung development, and tried to find and develop the lung. The newly discovered miRNAs may play a role in the development of lung, and the results of this study can also be used for future lung development and related diseases. The study provides a basis. The first part of Sprague-Dawley is large The purpose of the morphological observation of the development of rat fetal lung: to observe the morphological changes of the development of the alveolar development in the rat, and to establish a continuous basis for future research. Methods:12 healthy pregnant rats were randomly divided into 16 groups (recorded as S1 group),19 days of gestation (recorded as S2 group) and 21 days of gestation (recorded as S3 group), and the three groups of pregnant rats were randomly divided into two groups:16 days of gestation,19 days of gestation,21 days of gestation, and the fetal rat lungs, the row sections and the H-E staining (Haemoo- Toxylin and Eosin, H-E and transmission electron microscopy to observe the fetus The results were as follows: (1) It was observed under the light microscope: (1) In the first group, the dendritic distribution of the original bronchi was extended, the epithelium was high, the epithelial cells were in an annular arrangement, the stroma was thick, and the capillaries were rare. The alveolar structure was not found; in the S2 group, the primary alveoli were found, and the alveolar epithelial cells were transformed from a high column to a short prismatic, thin, thin, and the number of capillaries increased; in the S3 group, there was a further increase in the alveolar space, The cavity of the acinar cell was expanded and the stroma was further thinned. (2) Under the transmission electron microscope, the alveolar type 鈪，
本文编号：2493022
[Abstract]:The incidence of respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) has also increased significantly with the improvement of the treatment rate and the survival rate of the premature infants. Therefore, the study of its pathogenesis and treatment target has become a hot spot in the field of newborn. The causes of these two diseases are complicated, and the former is due to the insufficient secretion of surface active substances in the late stage of the lung development. To the same time, the researchers also found that the occurrence of RDS is closely related to the gestational age and the birth weight, which may also suggest that there is a certain correlation between the maturity of the lung and the RDS. The latter, the occurrence of the latter _ BPD, is believed to be primarily due to multiple factors, including high oxygen toxicity, mechanical ventilation, infection, inflammation, and the like, on the basis of genetic susceptibility In particular, in premature infants, the whole process of pulmonary development before birth is not completed, the number of alveoli is small, the alveolar structure is simple, and at this time, the lung injury and the development of the lung can be caused by the influence of various risk factors. MicroRNAs (miRNAs), which have been found in recent years, are an important short, endogenous RNA that can be prevented from being translated and expressed by direct degradation or inhibition of targeted mRNA, thereby participating in a table of genes at the post-transcriptional level. It is currently agreed that miRNAs play a very important role in critical biological processes such as cell development timing, cell proliferation, signal transduction, stem cell differentiation, and metastasis of tumors The biological chip technology is a new technology developed in the early 1990s, and has the characteristics of high flux, high integration, microminiaturization, continuous and automatic. The paper is divided into three parts. The first part studies the three key time point fetal lung groups of the rat embryo development. The change of weaving morphology. The second part studies three key point-in-time miRNAs in fetal lung development in rats The third part focuses on miRNA-126/ miRNA-126 * in rat fetal lung The research group conducted miRNA screening for three time points of fetal lung development, and tried to find and develop the lung. The newly discovered miRNAs may play a role in the development of lung, and the results of this study can also be used for future lung development and related diseases. The study provides a basis. The first part of Sprague-Dawley is large The purpose of the morphological observation of the development of rat fetal lung: to observe the morphological changes of the development of the alveolar development in the rat, and to establish a continuous basis for future research. Methods:12 healthy pregnant rats were randomly divided into 16 groups (recorded as S1 group),19 days of gestation (recorded as S2 group) and 21 days of gestation (recorded as S3 group), and the three groups of pregnant rats were randomly divided into two groups:16 days of gestation,19 days of gestation,21 days of gestation, and the fetal rat lungs, the row sections and the H-E staining (Haemoo- Toxylin and Eosin, H-E and transmission electron microscopy to observe the fetus The results were as follows: (1) It was observed under the light microscope: (1) In the first group, the dendritic distribution of the original bronchi was extended, the epithelium was high, the epithelial cells were in an annular arrangement, the stroma was thick, and the capillaries were rare. The alveolar structure was not found; in the S2 group, the primary alveoli were found, and the alveolar epithelial cells were transformed from a high column to a short prismatic, thin, thin, and the number of capillaries increased; in the S3 group, there was a further increase in the alveolar space, The cavity of the acinar cell was expanded and the stroma was further thinned. (2) Under the transmission electron microscope, the alveolar type 鈪，
本文编号：2493022
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