Sprague-Dawley大鼠胎肺組織miRNA差異表達譜分析
發(fā)布時間：2019-06-04 21:38
【摘要】：隨著早產(chǎn)兒救治率及存活率的提高,呼吸窘迫綜合征(Respiratory Distress Syndrome, RDS)及支氣管肺發(fā)育不良(Bronchopulmonary Dysplasia, BPD)的發(fā)生率也有明顯升高的趨勢,現(xiàn)已成為新生兒臨床常見的幾種主要病種,因而對其發(fā)病機制和治療靶點的探索已成為新生兒領域的研究熱點。 臨床上這兩種疾病病因較為復雜,前者的發(fā)生是由于肺發(fā)育晚期肺泡表面活性物質分泌不足所致。同時,研究者還發(fā)現(xiàn)RDS的發(fā)生同胎齡及出生體重也緊密相關,這可能也暗示了肺發(fā)育成熟度同RDS之間存在著一定的關聯(lián)。而后者——BPD的發(fā)生被認為主要是在基因易感性基礎上由包括高氧毒性、機械通氣、感染、炎癥等在內的多因素造成的肺損傷。特別是在早產(chǎn)兒,出生前肺發(fā)育的整個過程尚未完成,肺泡數(shù)目少,肺泡結構簡單,此時極易受到各種危險因素的影響,而導致肺損傷和肺發(fā)育的滯后。 MicroRNAs(miRNAs)是近年來發(fā)現(xiàn)的一類重要的短小內生RNA,能通過對靶向mRNA的直接降解或抑制而阻止其翻譯、表達,從而在轉錄后水平參與基因的表達調控。目前一致認為miRNA在細胞發(fā)育時序、細胞增殖、信號轉導、干細胞分化和腫瘤的發(fā)生轉移等緊要的生物學進程中都起著非常重要的作用。 生物芯片技術是20世紀90年代初發(fā)展起來的新技術,具有高通量、高集成、微型化、連續(xù)化和自動化的特點,此技術現(xiàn)今已經(jīng)成熟運用到miRNA水平基因表達檢測上,是檢測細胞或組織miRNA表達譜的理想方法。 本文課題分為三部分,第一部分研究大鼠胚胎發(fā)育三個關鍵時間點胎肺組織形態(tài)學的改變。第二部分研究大鼠胎肺發(fā)育中三個關鍵時間點miRNA的差異表達情況。第三部分著重探討miRNA-126/miRNA-126*在大鼠胎肺發(fā)育中的相關作用。 本課題組通過對胎肺發(fā)育三個時間點進行miRNA篩查,試圖找到與肺發(fā)育相關的miRNAs。這些新發(fā)現(xiàn)的miRNAs或許在肺發(fā)育中會起著一定作用,同時這一研究結果也可為未來肺發(fā)育及其相關疾病的研究提供一定的依據(jù)。 第一部分Sprague-Dawley大鼠胎肺發(fā)育形態(tài)學觀察研究 目的：觀察大鼠胚胎肺泡發(fā)育形態(tài)學變化,為將來課題建立連續(xù)而系統(tǒng)的胎肺發(fā)育形態(tài)學資料。 方法：12只健康孕鼠隨機分為孕16天組(記為S1組)、孕19天組(記為S2組)和孕21天組(記為S3組),三組孕鼠分別在孕16天,孕19天,孕21天快速剖宮取出其胎鼠肺臟,行切片、H-E染色(Haemo-toxylin and Eosin, H-E)及透射電鏡檢測以觀察胎肺組織不同發(fā)育時期形態(tài)結構差異。 結果：(1)光鏡下觀察：S1組見原始支氣管呈樹枝狀分布延伸,上皮為高柱狀,上皮細胞呈環(huán)形排列,間質厚,毛細血管少見,未見肺泡結構存在；S2組見初級肺泡,且肺泡上皮細胞由高柱狀轉化為矮柱狀立方形,間質變薄,毛細血管數(shù)目增多；S3組見肺泡間隔進一步增多,肺腺泡腔體極度擴張,間質進一步變薄。 (2)透射電鏡下觀察：S1組未見肺泡Ⅱ型上皮細胞,只有原始的肺泡上皮細胞存在,原始上皮細胞內可見線粒體,多而密集,但在細胞內并未見板層小體。S2組中Ⅱ型肺泡上皮開始分化出現(xiàn),在細胞內部此時可見板層小體,染色深且結構致密。在S3組中Ⅱ型肺泡上皮細胞進一步增多,板層小體數(shù)目也逐漸增多,且外有界膜包繞,結構更為致密,肺泡上皮絨毛結構清楚,間質毛細血管易見。結論：大鼠胎肺發(fā)育是循序漸進的生理過程,且一直延續(xù)到生后。細胞內板層小體的出現(xiàn)是肺泡Ⅱ型上皮細胞分化的標志,且其數(shù)目隨著發(fā)育過程而增多。 第二部分Sprague-Dawley大鼠正常胎肺發(fā)育過程中miRNA的差異表達情況研究 目的：通過比較孕16天,孕19天,孕21天三組間大鼠胎肺組織miRNA表達譜的差異,篩選出有意義的miRNAs,為進一步探討其在Sprague-Dawley大鼠胎肺發(fā)育機制中的作用提供一定的理論依據(jù)。 方法：12只健康孕鼠隨機分為孕16天組(S1組)、孕19天組(S2組)和孕21天組(S3組),分別采用了包含有1891個探針的新型miRNA芯片,通過觀察大鼠胎肺發(fā)育后期三個關鍵時間點(孕16天,孕19天,孕21天)的miRNAs表達情況從而篩選出具有差異表達的miRNAs。在這些niRNAs中隨后挑選出在三個時間點都有顯著差異表達的miRNAs,并且進行real-time PCR的驗證。 結果：(1)在上述的三個時間點中發(fā)現(xiàn)了167個miRNAs呈現(xiàn)差異性表達。其中,有81個上調的miRNAs,86個下調的miRNAs。由于在連續(xù)三個時間點中都表現(xiàn)出超過2倍以上倍數(shù)變化,并呈連續(xù)上調或下調趨勢,故本實驗又進一步從中篩查出了7個連續(xù)差異表達的miRNAs。 (2)由于在這7個miRNAs中miRNA-125b-5p, miRNA-296, miRNA-93, miRNA-146b以及miRNA-3560等5個具有更高的連續(xù)倍數(shù)變化,所以最終被挑選出進行real-time P(R的驗證,而real-time PCR的最終驗證結果與miRNA芯片結果基本一致。 結論：通過基因芯片技術,課題組胎鼠肺發(fā)育三個時間點共篩選出167個差異表達miRNAs,我們推測這些具有差異表達的miRNAs在正常肺發(fā)育的調控機制中可能起著一定的作用,這有助于我們進一步闡明包含肺泡Ⅱ型上皮在內的胎肺發(fā)育機制,同時也為以后進行肺發(fā)育相關疾病的研究提供了一定的生理學基礎。 第三部分miRNA-126/miRNA-126*在Sprague-Dawley大鼠正常胎肺發(fā)育中相關作用的研究 目的：通過比較孕16天,孕19天,孕21天這三個時間點間大鼠胎肺組織芯片中miRNA-126/miRNA-126*的差異表達水平,探討miRNA-126^miRNA-126*在Sprague-Dawley大鼠胎肺發(fā)育機制中可能的作用,為今后進一步研究新生兒肺發(fā)育相關疾病提供一定的依據(jù)。 方法：12只健康孕鼠隨機分為孕16天組(S1組)、孕19天組(S2組)和孕21天組(S3組),本實驗采用了包含有1891個探針的新型miRNA芯片(詳見本實驗第二部分),觀察大鼠胎肺發(fā)育后期三個關鍵時間點(孕16天,孕19天,孕21天)的miRNA-126及miRNA-126*的表達情況,并且隨后進行了相關real-time PCR的驗證。 結果：經(jīng)過芯片篩查,我們發(fā)現(xiàn)miRNA-126在三組間均呈高水平表達,表達水平無明顯差異；而miRNA-126*三組間存在差異表達,隨后的real-time PCR結果也證實miRNA-126*的表達在三組間均存在顯著性差異(Group SI→Group S2→Group S3),具有統(tǒng)計學意義(P0.05)。 結論：本研究首次報道了在胎肺發(fā)育中miRNA-126隨著肺發(fā)育過程存在顯著差異表達。故推測miRNA-126*可能在胎肺發(fā)育中扮演著重要的角色。對miRNA-126*的深入研究可能揭示其在大鼠胚胎肺發(fā)育中所起的相關作用,且對進一步研究與新生兒肺發(fā)育的相關疾病(如支氣管肺發(fā)育不良及呼吸窘迫綜合征等)提供一定的理論基礎。
[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，
本文編號：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，
本文編號：2493022
本文鏈接：http://www.lk138.cn/yixuelunwen/eklw/2493022.html
最近更新
教材專著
