基于生物信息学途径研究先天性膈疝肺发育不良的发生机制及干预药物

发布时间：2018-07-09 17:44

本文选题：先天性膈疝 + 肺发育不良　；参考：《南方医科大学》2016年博士论文

【摘要】：背景先天性膈疝(Congenital diaphragmatic hernia, CDH)是由于先天性发育异常而导致膈肌缺损,腹腔脏器疝入胸腔,引起一系列病理生理变化,对心肺功能、全身状况均造成不同程度的影响,是新生儿急危重症之一,其发病率为1：2000-5000。尽管近年来对CDH的认识及治疗水平有所提高,其病死率仍较高,达到40%-60%,其主要死亡原因是肺发育不良。然而,CDH肺发育不良的发病机制尚未清楚,仍缺乏行之有效的治疗手段,因此,对该方面的研究已日益成为国际小儿外科领域的热点和难点。肺的发育是一个受到精确调控的复杂过程,包括细胞发育、增殖、分化以及细胞凋亡,也是细胞与细胞、细胞与间充质以及上皮与间质之间相互作用复杂过程。这个过程涉及到众多生物活性分子,包括生长分子、转录因子、细胞外基质分子等,它们形成相互作用的强大网络,在各个发育时期调控肺的发育。最近的研究发现一类新的非编码RNA微小RNA (microRNA, miRNA)也参与肺的发生发育过程。miRNA是一类长度为18-25核苷酸的单链非编码小RNA分子,主要通过碱基互补配对的方式与特异性靶基因信使RNA(mRNA)的3'UTR非翻译区结合,从而降解靶mRNA或抑制蛋白质的翻译合成,实现对靶基因转录后水平的调控。miRNA具有高度的保守性,普遍存在于真核细胞内,几乎参与了各类生物学过程,据估计,它们调控着人类近60%的基因,在发育和疾病发生过程中均起到关键作用。miRNA分子的功能不断被挖掘、被发现,miRNA已成为基因调控体系中的重要层面。如果从单一生物活性分子来阐明CDH肺发育不良的发病机制,往往比较片面,但既往的检测手段难以帮助研究人员在短时间内进行大量生物学数据的筛选和鉴别。随着现代科技的迅猛发展,生物芯片及生物信息学的兴起,可为揭示大量而复杂的生物数据所赋予的生物学奥秘提供了新的方法,为我们全面、快速研究CDH肺发育不良提供了更有效的手段。生物芯片是生命科学领域的一项重要的技术平台,极大加速和推动疾病的致病基因发现、早期诊断、分子分型、预后评估及药物研究等领域的发展。生物芯片具有高通量和快速测量等优点,它可产生海量的、复杂的生物信息数据,但是,如何解读芯片大量基因点的杂交信息,揭示其中蕴含的生命特征和规律,己成为基因芯片技术应用和发展的主要研究内容。生物信息学的出现,为解决这些生物数据提供一种新的思路和方法。生物信息学是一门新兴的学科,它综合运用数学、计算机科学和生物学等多学科知识和工具,阐明和揭示大量数据所包含的生物学意义。随着基因芯片和生物信息学的出现,药物发现模式发生了重大变革,由既往偶然发现、工业合成有效成份的传统时代进入了一个以基因为基础的药物研发新阶段。近年来,基因表达谱在药物研究方面的应用越来越广泛,研究人员构建了与活性化合物或药物相关的基因表达谱数据库,绘制了“基因-疾病-药物”之间的关系图。这些“联系图”为药物发现及研究提供新思路,形成一种独特的药物发现新模式,即基于化合物或药物基因表达谱的药物发现模式,通过该方法可筛选到一些疾病治疗候选化合物,加快了药物发现过程,特别是对于那些临床罕见疾病的治疗药物发现有着更为重要的意义。本课题主要采用生物芯片技术,运用现行公认的生物信息学方法,检测Nitrofen诱导CDH胎肺发育不良中的差异表达基因及其相关miRNA表达模式,揭示了全转录组水平的编码基因的表达变化,结合已知的基因功能和信号通路以及miRNA靶基因预测数据库,探讨非编码基因miRNA在转录后水平对编码基因转录后的潜在调控关系和作用,多角度、全方面综合地分析和归纳miRNA所调控的靶基因的功能、信号通路及相关干预化合物,这将深化CDH肺发育不良发病机制的认识,为预防肺发育不良的发生及探索新的产前干预方法提供理论依据和实验基础。研究目的(1)利用国际上通用的CDH动物模型(Nitrofen诱导CDH大鼠模型),通过全基因组mRNA表达谱芯片检测技术,研究肺发育不良mRNA表达谱变化；(2)利用miRNA芯片的检测,通过生物信息学技术分析差异表达miRNA与mRNA,研究CDH肺发育不良中miRNA和]mRNA表达调控网络及生物功能,为进一步揭示CDH肺发育不良的机制提供更直接的科学线索。(3)通过生物信息学药物筛选工具Connectivity Map,研究CDH肺发育不良中基因与化合物分子功能的关系,为开发CDH肺发育不良的干预药物提供更直接的实验依据。研究方法1. Nitrofen诱导CDH大鼠模型构建取成年SPF级SD雌性大鼠12只,将其编号后根据随机数字表法分为对照组和CDH组,另取6只雄性SD大鼠,将雄性和雌性SD大鼠按1：2比例合笼过夜,于第二日上午在显微镜下观察雌性大鼠阴道涂片,镜下见到精子者视为妊娠,当天上午则定为妊娠第0.5天。在孕第9.5天,CDH组大鼠经胃管注入Nitrofen100 mg(溶于橄榄油,浓度100mg/ml),对照组大鼠则灌入1ml橄榄油。在妊娠第21.5天,两组分别在全身麻醉下行剖宫产术取出胎鼠,解剖胎鼠胸腔后取左肺组织。2. mRNA表达谱芯片检测采取Agilent 4×44K大鼠全基因表达谱芯片检测胎肺mRNA表达谱,分析差异化的mRNA;利用文献挖掘工具FACTA及Pubmed数据库对表达差异的mRNA进行验证。3. miRNA芯片检测及差异miRNA-mRNA表达谱信息的联合分析利用miRNA芯片(Agilent Rat miRNA 8×15K)检测Nitrofen诱导CDH大鼠模型胎肺miRNA的表达情况,通过TargetScan预测差异表达miRNA的靶基因,联合差异表达mRNA进行基因本体论(Gene Ontology)分析和信号通路(Pathway)分析,并筛选出相关的miRNA及其靶基因。4.已筛选miRNA及其靶基因的验证应用实时荧光定量PCR检测miRNA及其靶基因在Nitrofen诱导CDH大鼠模型胎肺中的表达水平,免疫组化检测靶基因在胎肺组织中的定位,双荧光素酶报告实验明确miRNA及其靶基因的调节关系。5. mRNA表达谱信息与connectivity map药物数据库的整合分析应用生物信息学工具Connectivity Map,整合差异表达的mRNA信息,推测CDH肺发育不良中基因与药物小分子功能的关系。研究结果(1) Nitrofen诱导CDH大鼠模型对照组的6只孕鼠经剖宫产得到胎鼠71只。Nitrofen诱导CDH组6只孕鼠得到胎鼠59只,其中膈疝共38只,占64.4%(38/59)：膈疝均为左侧的后外侧巨大膈疝(膈肌缺损达50%以上),疝入胸腔的腹腔脏器主要有肝脏、胃、小肠和脾脏。解剖膈疝胎鼠胸腔后可见双肺均缩小,与对照组相比,左肺明显发育不良。HE染色从组织学上证实模型胎肺发育不良。(2) mRNA表达谱在所检测的17,679个基因中,与对照组相比,CDH组共有10,121个mRNA表达上调、7,558个mRNA表达下调。利用Pubmed数据库及文献挖掘工具FACTA进行验证,共有63个相同的检测基因,其中79.4%(50/63)与mRNA表达谱芯片检测结果一致,表明mRNA芯片的实验结果可信。(3) mRNA表达谱信息与其相应miRNA的联合分析13个表达上调的miRNA共有3,126个表达下调的靶基因,共涉及716条信号通路,其中共同的信号通路是TGFβ信号通路、GnRHR信号通路：10个表达下调的miRNA共有2,729个表达上调的靶基因,共涉及564条信号通路,其中共同的信号通路是WNT信号通路、TGFβ信号通路、FGF信号通路、GnRHR信号通路。选取TGFβ信号通路中TGF-β2基因及其相应mir-141-3p、WNT信号通路中FZD8基因及其相应mir-375-3p进行下一步的验证。(4) TGFβ信号通路中TGF-β2基因及其相应mir-141-3p在Nitrofen诱导CDH大鼠模型胎肺中的表达CDH组与对照组胎肺中mir-141-3p的相对表达量分别为1.57±0.28、1.00±0.18,两组比较差异有统计学意义(p0.05)。CDH组和对照组胎肺中TGF-β2 mRNA的相对表达量分别为0.58±0.17、1.00±0.15,两组相比差异有统计学意义(p0.05)。免疫组化检测显示TGF-β2主要表达在气道上皮细胞,CDH组中TGF-β2的表达水平明显低于对照组(20.32±3.54、45.14±4.56,p0.05)。双荧光素酶报告实验显示,mir-141-3p与TGFβ-2存在直接调控关系。(5)WNT信号通路中FZD8基因及其相应mir-375-3p在Nitrofen诱导CDH大鼠模型胎肺中的表达CDH组与对照组胎肺中mir-375-3p的相对表达量分别为0.41±0.15、1.00±0.17,两组比较差异有统计学意义(p0.05)。CDH组和对照组胎肺中TGF-β2 mRNA的相对表达量分别为1.62±0.20、1.00±0.16,两组相比差异有统计学意义(p0.05)。免疫组化检测显示FZD8主要表达在气道上皮细胞,CDH组中FZD8的表达水平明显低于对照组(38.32±8.54、20.14±5.76,p0.05)。双荧光素酶报告实验显示,mir-375-3p与FZD8存在直接调控关系。(6) mRNA表达谱信息与connectivity map药物数据库的整合分析通过connectivity map药物数据库分析,曲匹地尔、维甲酸、间羟异丙肾上腺素等药物的负性富集分数较高且P0.05,提示可较好地逆转TGFβ信号通路相关基因；伊洛前列素、肾上腺酮、硫马唑等药物的富集分数较高且P0.05,提示可较好地逆转WNT信号通路相关基因。结论(1)利用高通量基因芯片检测方法可筛选出Nitrofen诱导CDH大鼠模型胎肺发育不良的发展演变过程中差异表达基因谱,结果表明CDH肺发育不良的发生是一个涉及多基因的复杂网络调控过程,其中TGFβ信号通路、WNT信号通路、FGF信号通路、GnRHR信号通路与其关系最为密切。(2)双荧光素酶报告系统证明mir-141-3p与TGF-β2者存在直接的调节关系；在CDH组中,mir-141-3p表达水平明显上升,TGF-β2表达水平明显下降,提示mir-141-3p及其靶基因TGF-β2参与了CDH肺发育不良的发生机制。(3)双荧光素酶报告系统证明mir-375-3p与FZD8者存在直接的调节关系；在CDH组中,mir-375-3p表达水平明显下降,FZD8表达水平明显上升,提示mir-375-3p及其靶基因Fzd8参与了CDH肺发育不良的发生机制。(4)通过connectivity map药物数据库分析,曲匹地尔、维甲酸、间羟异丙肾上腺素等药物可较好地逆转TGFβ信号通路相关基因,推测可作为干预CDH肺发育不良的候选药物；伊洛前列素、肾上腺酮、硫马唑等较好地逆转WNT信号通路相关基因,推测可作为干预CDH肺发育不良的候选药物。
[Abstract]:Background congenital diaphragmatic hernia (Congenital diaphragmatic hernia, CDH) is caused by congenital dysplasia of the diaphragm. Abdominal viscera hernia enters the thoracic cavity and causes a series of pathophysiological changes. It has a different degree of influence on the cardiopulmonary function and the whole body condition. It is one of the neonatal acute critical diseases, the incidence of which is 1:2000-5000. though near. The level of understanding and treatment of CDH has been improved over the years, and its mortality rate is still high, reaching 40%-60%. The main cause of death is lung dysplasia. However, the pathogenesis of CDH lung dysplasia is not clear and still lacks effective treatment methods. Therefore, the research on this aspect has become a hot and difficult problem in the field of international pediatric surgery. The development of lung is a complex process that is precisely regulated, including cell development, proliferation, differentiation, and cell apoptosis, and is also a complex process between cell and cell, cell and mesenchymal and epithelial and interstitial interaction. This process involves numerous bioactive components, including growth molecules, transcription factors, extracellular matrix components. They form a powerful network of interaction and regulate lung development at various developmental stages. Recent studies have found that a new class of non coded RNA micro RNA (microRNA, miRNA) is also involved in the development of lung,.MiRNA is a class of single strand noncoding small RNA molecules with a length of 18-25 nucleotides, mainly by the complementary pairs of base pairs. The combination of the 3'UTR non translation region of the specific target gene messenger RNA (mRNA) is combined to degrade the target mRNA or inhibit the translation synthesis of protein. It is highly conserved for the regulation of.MiRNA after the target gene transcriptional level. It is ubiquitous in eukaryotic cells and almost participates in various biological processes. It is estimated that they regulate nearly 60% of human beings. Genes, the functions of.MiRNA molecules, which play a key role in development and disease, have been continuously excavated. MiRNA has been found to be an important aspect of the gene regulation system. It is often more one-sided than a single bioactive molecule to elucidate the pathogenesis of CDH lung dysplasia, but previous detection methods are difficult to help study. With the rapid development of modern science and technology, the rise of biochip and bioinformatics provides a new way to reveal the biological mysteries given by a large number of complex biological data, which provides a more effective method for the comprehensive and rapid study of CDH lung dysplasia. Biochip is an important technical platform in the field of life science, which greatly accelerates and promotes the development of disease pathogenic gene discovery, early diagnosis, molecular typing, prognostic evaluation and drug research. Biochips have the advantages of high throughput and rapid measurement, which can produce massive, complex biological information data. It is, how to interpret the hybridization information of a large number of genes on the chip, and reveal the characteristics and laws contained in it, has become the main research content of the application and development of the gene chip technology. The emergence of bioinformatics provides a new way of thinking and method for solving these biological data. Bioinformatics is a new subject and it is integrated into a new subject. Using multidisciplinary knowledge and tools, such as mathematics, computer science and biology, to elucidate and reveal the biological significance of a large number of data. With the emergence of genetic chips and bioinformatics, major changes have taken place in the drug discovery model. The traditional era of past accidental discovery and industrial synthesis of effective components has entered a genetic basis. In recent years, the application of gene expression profiles to drug research has become more and more widely used. Researchers have constructed a database of gene expression profiles related to active compounds or drugs and drew a diagram of the relationship between "gene diseases and drugs". These "linkage maps" provide new ideas for drug discovery and research. A unique drug discovery model, the drug discovery model based on the gene expression profiles of compounds or drugs, can be used to screen candidate compounds for some disease treatment, speeding up the drug discovery process, especially for the treatment of rare diseases. Using biochip technology, the present accepted bioinformatics methods were used to detect the differentially expressed genes and related miRNA expression patterns in Nitrofen induced CDH fetal lung dysplasia, revealing the changes in the expression of the encoding genes in the whole transcriptional group, and combining the known gene function and signal pathways and the miRNA target gene prediction database. To discuss the potential regulation relationship and function of the non coding gene miRNA after transcriptional gene transcription after transcriptional gene transcription, multi angle, comprehensive analysis and induction of the function of target genes regulated by miRNA, signal pathways and related interfering compounds, which will deepen the understanding of the mechanism of CDH lung dysplasia and prevent the occurrence of pulmonary dysplasia. And explore new prenatal intervention methods to provide theoretical basis and experimental basis. (1) using the international common CDH animal model (Nitrofen induced CDH rat model), through the whole genome mRNA expression spectrum chip detection technology to study the changes of mRNA expression profiles of lung dysplasia; (2) using miRNA chip detection, through bioinformatics. Technical analysis of differential expression of miRNA and mRNA to study the regulatory network and biological function of miRNA and]mRNA expression in CDH pulmonary dysplasia and provide a more direct scientific clue to further reveal the mechanism of CDH lung dysplasia. (3) the study of genes and compounds in CDH pulmonary dysplasia by bioinformatics screening tool Connectivity Map The relationship of function provides more direct experimental basis for the development of CDH pulmonary dysplasia intervention drugs. 1. Nitrofen induced CDH rats were induced to construct 12 adult SPF SD female rats, and their numbers were divided into the control group and the CDH group according to the random number table method, and 6 male SD rats were taken, and the male and female SD rats were divided into 1:2 rats and 1:2 rats. In the morning of second, the vaginal smear of female rats was observed under the microscope on the morning of second. The sperm was seen as pregnancy on the morning of the microscope. On the day of the 9.5 day of pregnancy, the rats in group CDH were injected with Nitrofen100 Mg (dissolved in olive oil and 100mg/ml) by gastric tube, and the rats in the control group were injected with 1ml olive oil. In pregnancy 21.5. In the two groups, the two groups were taken from the caesarean section under general anesthesia, and the fetal rat was dissected and the left lung tissue was dissected to detect the mRNA expression profile of the fetal lung by Agilent 4 x 44K rat full gene expression chip, and the differential mRNA was analyzed, and the FACTA and Pubmed database of the document mining tools were used to improve the expression of mRNA. The joint analysis of the detection of.3. miRNA chip and the difference of miRNA-mRNA expression profiles using miRNA chip (Agilent Rat miRNA 8 x 15K) was used to detect the expression of fetal lung miRNA in the Nitrofen induced CDH rat model, and the gene ontology was carried out by the differential expression of the target gene by the differential expression of the TargetScan prediction. Analysis and signal pathway (Pathway) analysis, and screening related miRNA and its target gene.4. have screened miRNA and its target genes to verify the application of real-time fluorescent quantitative PCR detection miRNA and its target gene in Nitrofen induced CDH rat model fetal lung expression level, immunohistochemical detection target gene in fetal lung tissue localization, double luciferase reporter The relationship between miRNA and its target gene regulation relationship between.5. mRNA expression profiles and connectivity map drug database integrated analysis and application of bioinformatics tool Connectivity Map to integrate the differential expressed mRNA information to speculate on the relationship between gene and small molecular function in CDH pulmonary dysplasia. The results of the study (1) Nitrofen induction CDH 6 pregnant rats in the control group were obtained by cesarean section and 59.Nitrofen induced 6 pregnant rats in the CDH group were induced by caesarean section. The diaphragmatic hernia was 38, accounting for 64.4% (38/59). The diaphragmatic hernia was the left posterolateral giant diaphragmatic hernia (more than 50%). The abdominal viscera of the hernia into the thoracic cavity was mainly liver, stomach, small intestine and spleen. Dissecting diaphragmatic hernia fetus. Both lungs were reduced in the rat's thoracic cavity. Compared with the control group, the obvious dysplasia of the left lung.HE staining showed that the model fetal lung dysplasia was histologically proved. (2) in the 17679 genes detected by the 17679 genes, the expression of 10121 mRNA expressions in the CDH group and the 7558 mRNA expressions were down. The Pubmed database and the literature were dug. FACTA was verified with 63 identical detection genes, of which 79.4% (50/63) was in agreement with the mRNA expression spectrum chip detection results, indicating that the experimental results of mRNA chips were credible. (3) the combined analysis of mRNA expression profiles with corresponding miRNA and 13 up regulated miRNA had 3126 down regulated target genes, involving 716 signals. The common signaling pathway is TGF beta signaling pathway, GnRHR signaling pathway: 10 down regulated miRNA has 2729 up regulated target genes, involving 564 signal pathways, of which the common signal pathways are WNT signaling pathway, TGF beta signaling pathway, FGF signaling pathway, GnRHR signaling pathway, and TGF- beta 2 in the TGF beta signaling pathway. The gene and its corresponding mir-141-3p, the FZD8 gene in the WNT signaling pathway and its corresponding mir-375-3p were tested next. (4) the TGF- beta 2 gene in the TGF beta signaling pathway and its corresponding mir-141-3p in the Nitrofen induced CDH rat model fetal lung, the relative expression of mir-141-3p in the fetal lung of the control group was 1.57 + 0.18, respectively. The comparative difference between the two groups was statistically significant (P0.05) in the.CDH group and the control group, the relative expression of TGF- beta 2 mRNA in the fetal lung was 0.58 + 0.17,1.00 0.15, respectively, and there was a significant difference in the two groups (P0.05). The immunohistochemical detection showed that TGF- beta 2 was mainly expressed in the airway epithelial cells, and the expression level of TGF- beta 2 in CDH group was significantly lower than that of the control group (20.32 3.54,45.14 + 4.56, P0.05). The double Luciferase Report showed that there was a direct regulation relationship between mir-141-3p and TGF beta -2. (5) the expression of FZD8 gene and its corresponding mir-375-3p in WNT signaling pathway in Nitrofen induced CDH rat model fetal lung, the relative expression of mir-375-3p in the fetal lung was 0.41 + 0.17, two, respectively. Two The comparative differences were statistically significant (P0.05) in group.CDH and control group, the relative expression of TGF- beta 2 mRNA in the fetal lung was 1.62 + 0.20,1.00 0.16, respectively, and there was a significant difference between the two groups (P0.05). The immunohistochemical detection showed that FZD8 was mainly expressed in the airway epithelial cells, and the expression level of FZD8 in CDH group was significantly lower than that of the control group (38.32 + 8.54,20). .14 + 5.76, P0.05). The double Luciferase Report experiment showed that there was a direct regulation relationship between mir-375-3p and FZD8. (6) the integration of mRNA expression profiles and connectivity map drug database analysis through the connectivity map drug database analysis, the negative concentration fraction of trapix, retinoic acid, isoadrenaline and other drugs was higher and P0.0 5, hints can better reverse TGF beta signaling related genes; the concentration of iloprost, adrenone, thiazolazole and other drugs is higher and P0.05, suggesting that WNT signaling pathway related genes can be better reversed. Conclusion (1) high throughput gene chip detection method can be used to screen Nitrofen induced fetal lung dysplasia in CDH rat model. The differential expression gene spectrum in the evolution process indicates that the occurrence of CDH pulmonary dysplasia is a complex network regulation process involving multiple genes, in which the TGF beta signaling pathway, WNT signaling pathway, FGF signaling pathway, and GnRHR signaling pathway are most closely related. (2) double Luciferase Report system proves that mir-141-3p and TGF- beta 2 exist. In the CDH group, the expression level of mir-141-3p was obviously increased and the expression level of TGF- beta 2 decreased significantly, suggesting that mir-141-3p and its target gene TGF- beta 2 were involved in the pathogenesis of CDH pulmonary dysplasia. (3) the dual luciferase reporter system demonstrated the direct regulatory relationship between mir-375-3p and FZD8, and the mir-375-3p table in the CDH group. The level of FZD8 significantly increased, indicating that mir-375-3p and its target gene Fzd8 were involved in the pathogenesis of CDH lung dysplasia. (4)
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R726.5

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