本文選題：先天性膈疝 + 肺發(fā)育不良　； 參考：《南方醫(yī)科大學》2016年博士論文

【摘要】：背景先天性膈疝(Congenital diaphragmatic hernia, CDH)是由于先天性發(fā)育異常而導致膈肌缺損,腹腔臟器疝入胸腔,引起一系列病理生理變化,對心肺功能、全身狀況均造成不同程度的影響,是新生兒急危重癥之一,其發(fā)病率為1：2000-5000。盡管近年來對CDH的認識及治療水平有所提高,其病死率仍較高,達到40%-60%,其主要死亡原因是肺發(fā)育不良。然而,CDH肺發(fā)育不良的發(fā)病機制尚未清楚,仍缺乏行之有效的治療手段,因此,對該方面的研究已日益成為國際小兒外科領(lǐng)域的熱點和難點。肺的發(fā)育是一個受到精確調(diào)控的復雜過程,包括細胞發(fā)育、增殖、分化以及細胞凋亡,也是細胞與細胞、細胞與間充質(zhì)以及上皮與間質(zhì)之間相互作用復雜過程。這個過程涉及到眾多生物活性分子,包括生長分子、轉(zhuǎn)錄因子、細胞外基質(zhì)分子等,它們形成相互作用的強大網(wǎng)絡,在各個發(fā)育時期調(diào)控肺的發(fā)育。最近的研究發(fā)現(xiàn)一類新的非編碼RNA微小RNA (microRNA, miRNA)也參與肺的發(fā)生發(fā)育過程。miRNA是一類長度為18-25核苷酸的單鏈非編碼小RNA分子,主要通過堿基互補配對的方式與特異性靶基因信使RNA(mRNA)的3'UTR非翻譯區(qū)結(jié)合,從而降解靶mRNA或抑制蛋白質(zhì)的翻譯合成,實現(xiàn)對靶基因轉(zhuǎn)錄后水平的調(diào)控。miRNA具有高度的保守性,普遍存在于真核細胞內(nèi),幾乎參與了各類生物學過程,據(jù)估計,它們調(diào)控著人類近60%的基因,在發(fā)育和疾病發(fā)生過程中均起到關(guān)鍵作用。miRNA分子的功能不斷被挖掘、被發(fā)現(xiàn),miRNA已成為基因調(diào)控體系中的重要層面。如果從單一生物活性分子來闡明CDH肺發(fā)育不良的發(fā)病機制,往往比較片面,但既往的檢測手段難以幫助研究人員在短時間內(nèi)進行大量生物學數(shù)據(jù)的篩選和鑒別。隨著現(xiàn)代科技的迅猛發(fā)展,生物芯片及生物信息學的興起,可為揭示大量而復雜的生物數(shù)據(jù)所賦予的生物學奧秘提供了新的方法,為我們?nèi)�、快速研究CDH肺發(fā)育不良提供了更有效的手段。生物芯片是生命科學領(lǐng)域的一項重要的技術(shù)平臺,極大加速和推動疾病的致病基因發(fā)現(xiàn)、早期診斷、分子分型、預后評估及藥物研究等領(lǐng)域的發(fā)展。生物芯片具有高通量和快速測量等優(yōu)點,它可產(chǎn)生海量的、復雜的生物信息數(shù)據(jù),但是,如何解讀芯片大量基因點的雜交信息,揭示其中蘊含的生命特征和規(guī)律,己成為基因芯片技術(shù)應用和發(fā)展的主要研究內(nèi)容。生物信息學的出現(xiàn),為解決這些生物數(shù)據(jù)提供一種新的思路和方法。生物信息學是一門新興的學科,它綜合運用數(shù)學、計算機科學和生物學等多學科知識和工具,闡明和揭示大量數(shù)據(jù)所包含的生物學意義。隨著基因芯片和生物信息學的出現(xiàn),藥物發(fā)現(xiàn)模式發(fā)生了重大變革,由既往偶然發(fā)現(xiàn)、工業(yè)合成有效成份的傳統(tǒng)時代進入了一個以基因為基礎的藥物研發(fā)新階段。近年來,基因表達譜在藥物研究方面的應用越來越廣泛,研究人員構(gòu)建了與活性化合物或藥物相關(guān)的基因表達譜數(shù)據(jù)庫,繪制了“基因-疾病-藥物”之間的關(guān)系圖。這些“聯(lián)系圖”為藥物發(fā)現(xiàn)及研究提供新思路,形成一種獨特的藥物發(fā)現(xiàn)新模式,即基于化合物或藥物基因表達譜的藥物發(fā)現(xiàn)模式,通過該方法可篩選到一些疾病治療候選化合物,加快了藥物發(fā)現(xiàn)過程,特別是對于那些臨床罕見疾病的治療藥物發(fā)現(xiàn)有著更為重要的意義。本課題主要采用生物芯片技術(shù),運用現(xiàn)行公認的生物信息學方法,檢測Nitrofen誘導CDH胎肺發(fā)育不良中的差異表達基因及其相關(guān)miRNA表達模式,揭示了全轉(zhuǎn)錄組水平的編碼基因的表達變化,結(jié)合已知的基因功能和信號通路以及miRNA靶基因預測數(shù)據(jù)庫,探討非編碼基因miRNA在轉(zhuǎn)錄后水平對編碼基因轉(zhuǎn)錄后的潛在調(diào)控關(guān)系和作用,多角度、全方面綜合地分析和歸納miRNA所調(diào)控的靶基因的功能、信號通路及相關(guān)干預化合物,這將深化CDH肺發(fā)育不良發(fā)病機制的認識,為預防肺發(fā)育不良的發(fā)生及探索新的產(chǎn)前干預方法提供理論依據(jù)和實驗基礎。研究目的(1)利用國際上通用的CDH動物模型(Nitrofen誘導CDH大鼠模型),通過全基因組mRNA表達譜芯片檢測技術(shù),研究肺發(fā)育不良mRNA表達譜變化；(2)利用miRNA芯片的檢測,通過生物信息學技術(shù)分析差異表達miRNA與mRNA,研究CDH肺發(fā)育不良中miRNA和]mRNA表達調(diào)控網(wǎng)絡及生物功能,為進一步揭示CDH肺發(fā)育不良的機制提供更直接的科學線索。(3)通過生物信息學藥物篩選工具Connectivity Map,研究CDH肺發(fā)育不良中基因與化合物分子功能的關(guān)系,為開發(fā)CDH肺發(fā)育不良的干預藥物提供更直接的實驗依據(jù)。研究方法1. Nitrofen誘導CDH大鼠模型構(gòu)建取成年SPF級SD雌性大鼠12只,將其編號后根據(jù)隨機數(shù)字表法分為對照組和CDH組,另取6只雄性SD大鼠,將雄性和雌性SD大鼠按1：2比例合籠過夜,于第二日上午在顯微鏡下觀察雌性大鼠陰道涂片,鏡下見到精子者視為妊娠,當天上午則定為妊娠第0.5天。在孕第9.5天,CDH組大鼠經(jīng)胃管注入Nitrofen100 mg(溶于橄欖油,濃度100mg/ml),對照組大鼠則灌入1ml橄欖油。在妊娠第21.5天,兩組分別在全身麻醉下行剖宮產(chǎn)術(shù)取出胎鼠,解剖胎鼠胸腔后取左肺組織。2. mRNA表達譜芯片檢測采取Agilent 4×44K大鼠全基因表達譜芯片檢測胎肺mRNA表達譜,分析差異化的mRNA;利用文獻挖掘工具FACTA及Pubmed數(shù)據(jù)庫對表達差異的mRNA進行驗證。3. miRNA芯片檢測及差異miRNA-mRNA表達譜信息的聯(lián)合分析利用miRNA芯片(Agilent Rat miRNA 8×15K)檢測Nitrofen誘導CDH大鼠模型胎肺miRNA的表達情況,通過TargetScan預測差異表達miRNA的靶基因,聯(lián)合差異表達mRNA進行基因本體論(Gene Ontology)分析和信號通路(Pathway)分析,并篩選出相關(guān)的miRNA及其靶基因。4.已篩選miRNA及其靶基因的驗證應用實時熒光定量PCR檢測miRNA及其靶基因在Nitrofen誘導CDH大鼠模型胎肺中的表達水平,免疫組化檢測靶基因在胎肺組織中的定位,雙熒光素酶報告實驗明確miRNA及其靶基因的調(diào)節(jié)關(guān)系。5. mRNA表達譜信息與connectivity map藥物數(shù)據(jù)庫的整合分析應用生物信息學工具Connectivity Map,整合差異表達的mRNA信息,推測CDH肺發(fā)育不良中基因與藥物小分子功能的關(guān)系。研究結(jié)果(1) Nitrofen誘導CDH大鼠模型對照組的6只孕鼠經(jīng)剖宮產(chǎn)得到胎鼠71只。Nitrofen誘導CDH組6只孕鼠得到胎鼠59只,其中膈疝共38只,占64.4%(38/59)：膈疝均為左側(cè)的后外側(cè)巨大膈疝(膈肌缺損達50%以上),疝入胸腔的腹腔臟器主要有肝臟、胃、小腸和脾臟。解剖膈疝胎鼠胸腔后可見雙肺均縮小,與對照組相比,左肺明顯發(fā)育不良。HE染色從組織學上證實模型胎肺發(fā)育不良。(2) mRNA表達譜在所檢測的17,679個基因中,與對照組相比,CDH組共有10,121個mRNA表達上調(diào)、7,558個mRNA表達下調(diào)。利用Pubmed數(shù)據(jù)庫及文獻挖掘工具FACTA進行驗證,共有63個相同的檢測基因,其中79.4%(50/63)與mRNA表達譜芯片檢測結(jié)果一致,表明mRNA芯片的實驗結(jié)果可信。(3) mRNA表達譜信息與其相應miRNA的聯(lián)合分析13個表達上調(diào)的miRNA共有3,126個表達下調(diào)的靶基因,共涉及716條信號通路,其中共同的信號通路是TGFβ信號通路、GnRHR信號通路：10個表達下調(diào)的miRNA共有2,729個表達上調(diào)的靶基因,共涉及564條信號通路,其中共同的信號通路是WNT信號通路、TGFβ信號通路、FGF信號通路、GnRHR信號通路。選取TGFβ信號通路中TGF-β2基因及其相應mir-141-3p、WNT信號通路中FZD8基因及其相應mir-375-3p進行下一步的驗證。(4) TGFβ信號通路中TGF-β2基因及其相應mir-141-3p在Nitrofen誘導CDH大鼠模型胎肺中的表達CDH組與對照組胎肺中mir-141-3p的相對表達量分別為1.57±0.28、1.00±0.18,兩組比較差異有統(tǒng)計學意義(p0.05)。CDH組和對照組胎肺中TGF-β2 mRNA的相對表達量分別為0.58±0.17、1.00±0.15,兩組相比差異有統(tǒng)計學意義(p0.05)。免疫組化檢測顯示TGF-β2主要表達在氣道上皮細胞,CDH組中TGF-β2的表達水平明顯低于對照組(20.32±3.54、45.14±4.56,p0.05)。雙熒光素酶報告實驗顯示,mir-141-3p與TGFβ-2存在直接調(diào)控關(guān)系。(5)WNT信號通路中FZD8基因及其相應mir-375-3p在Nitrofen誘導CDH大鼠模型胎肺中的表達CDH組與對照組胎肺中mir-375-3p的相對表達量分別為0.41±0.15、1.00±0.17,兩組比較差異有統(tǒng)計學意義(p0.05)。CDH組和對照組胎肺中TGF-β2 mRNA的相對表達量分別為1.62±0.20、1.00±0.16,兩組相比差異有統(tǒng)計學意義(p0.05)。免疫組化檢測顯示FZD8主要表達在氣道上皮細胞,CDH組中FZD8的表達水平明顯低于對照組(38.32±8.54、20.14±5.76,p0.05)。雙熒光素酶報告實驗顯示,mir-375-3p與FZD8存在直接調(diào)控關(guān)系。(6) mRNA表達譜信息與connectivity map藥物數(shù)據(jù)庫的整合分析通過connectivity map藥物數(shù)據(jù)庫分析,曲匹地爾、維甲酸、間羥異丙腎上腺素等藥物的負性富集分數(shù)較高且P0.05,提示可較好地逆轉(zhuǎn)TGFβ信號通路相關(guān)基因；伊洛前列素、腎上腺酮、硫馬唑等藥物的富集分數(shù)較高且P0.05,提示可較好地逆轉(zhuǎn)WNT信號通路相關(guān)基因。結(jié)論(1)利用高通量基因芯片檢測方法可篩選出Nitrofen誘導CDH大鼠模型胎肺發(fā)育不良的發(fā)展演變過程中差異表達基因譜,結(jié)果表明CDH肺發(fā)育不良的發(fā)生是一個涉及多基因的復雜網(wǎng)絡調(diào)控過程,其中TGFβ信號通路、WNT信號通路、FGF信號通路、GnRHR信號通路與其關(guān)系最為密切。(2)雙熒光素酶報告系統(tǒng)證明mir-141-3p與TGF-β2者存在直接的調(diào)節(jié)關(guān)系；在CDH組中,mir-141-3p表達水平明顯上升,TGF-β2表達水平明顯下降,提示mir-141-3p及其靶基因TGF-β2參與了CDH肺發(fā)育不良的發(fā)生機制。(3)雙熒光素酶報告系統(tǒng)證明mir-375-3p與FZD8者存在直接的調(diào)節(jié)關(guān)系；在CDH組中,mir-375-3p表達水平明顯下降,FZD8表達水平明顯上升,提示mir-375-3p及其靶基因Fzd8參與了CDH肺發(fā)育不良的發(fā)生機制。(4)通過connectivity map藥物數(shù)據(jù)庫分析,曲匹地爾、維甲酸、間羥異丙腎上腺素等藥物可較好地逆轉(zhuǎn)TGFβ信號通路相關(guān)基因,推測可作為干預CDH肺發(fā)育不良的候選藥物；伊洛前列素、腎上腺酮、硫馬唑等較好地逆轉(zhuǎn)WNT信號通路相關(guān)基因,推測可作為干預CDH肺發(fā)育不良的候選藥物。
[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)
【學位授予單位】：南方醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R726.5

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