發(fā)布時(shí)間：2018-04-26 12:59

  本文選題：青少年特發(fā)性脊柱側(cè)彎 + 長鏈非編碼RNA��； 參考：《第二軍醫(yī)大學(xué)》2016年博士論文

【摘要】：背景:青少年特發(fā)性脊柱側(cè)彎(Adolescent Idiopathic Scoliosis,AIS)屬于特發(fā)性脊柱側(cè)彎(Idiopathic Scoliosis,IS)中最常見的一類,多為女性患者,發(fā)病年齡一般在10-18歲,并會(huì)隨著身體的成長呈現(xiàn)進(jìn)行性加重,不僅會(huì)影響到患者的外觀,更有甚者影響到患者的心臟和肺臟功能。盡管在臨床上、流行病學(xué)及分子生物學(xué)分析AIS的發(fā)病因素有很多,但目前為止對于其發(fā)病機(jī)理仍然不清楚。關(guān)于研究Harrington的相關(guān)報(bào)道,發(fā)生了cobb角度超過了15度的患者之中,其后代有27%可能會(huì)發(fā)生脊柱側(cè)彎。孿生子女的AIS相關(guān)基礎(chǔ)及臨床研究中,提示單卵雙生的共同發(fā)生率約為73%,而雙卵雙生的發(fā)生可能率只有27%。Wynne通過對AIS患者隨訪研究發(fā)現(xiàn),其具有明顯的家族性發(fā)病傾向,但機(jī)制仍不明確。由此可見,基因遺傳因素在發(fā)病機(jī)制中具有重要作用。隨著基因技術(shù)的發(fā)展,基因組計(jì)劃的完善,基因功能的發(fā)展或?qū)⒊蔀槿藗冄芯克P(guān)注的焦點(diǎn)�；谵D(zhuǎn)錄組學(xué)(Transcriptome)的AIS分子分型逐漸成為研究的熱點(diǎn)。其用于闡明AIS的發(fā)病機(jī)理及如何治療方式的研究目前還是比較度卻尚處于初始階段,但臨床應(yīng)用具有很廣闊的前景。隨著基因技術(shù)的成熟和發(fā)展,二代基因測序技術(shù)(RNA-seq)以及及全基因組的表達(dá)譜芯片(Gene Expression Microarray)的轉(zhuǎn)錄組學(xué)分析在功能基因組學(xué)的研究中發(fā)揮了非常重要的作用,不僅僅可以用于檢測編碼蛋白質(zhì)的mRNA,更能夠檢測各種的不同調(diào)節(jié)性非編碼RNA(microRNA和lncRNA等)。轉(zhuǎn)錄組學(xué)的研究對于如何進(jìn)一步篩選AIS發(fā)病可能相關(guān)的分子,揭示其發(fā)生發(fā)展機(jī)制和尋求新的AIS治療分子靶點(diǎn)等具有重要的應(yīng)用前景和臨床價(jià)值。長鏈非編碼RNA(Long noncoding RNA,lncRNA)是當(dāng)前研究的熱點(diǎn),它能通過表觀遺傳、轉(zhuǎn)錄水平、轉(zhuǎn)錄后水平等多個(gè)方面對基因表達(dá)產(chǎn)生調(diào)控作用。在許多復(fù)雜的疾病如腫瘤、神經(jīng)退行性疾病、心血管疾病等中發(fā)現(xiàn)lncRNA存在表達(dá)異常,部分lncRNA具有促進(jìn)或抑制疾病發(fā)生的作用。但至今為止,關(guān)于AIS相關(guān)的lncRNA表達(dá)等方面的報(bào)道仍很少。本研究通過應(yīng)用RNA-Seq測序技術(shù)得到AIS患者和正常人的lncRNA表達(dá)譜,同時(shí)得到mRNA表達(dá)譜,并進(jìn)一步探討AIS患者lncRNA表達(dá)存在的差異,為進(jìn)一步研究提供方向。本次研究的主要內(nèi)容概述如下:第一部分:正常青少年及AIS患者外周血樣本內(nèi)lncRNA和mRNA的表達(dá)譜研究。在這本部分研究中我們選取10例外周血樣本(5例正常樣本,5例AIS樣本)。標(biāo)準(zhǔn)化處理后,將10個(gè)樣本中至少有5個(gè)樣本出現(xiàn)lncrna-mrna表達(dá)(包括模糊表達(dá))定義為有意義的靶基因。結(jié)果發(fā)現(xiàn),10例樣本中,共2116個(gè)lncrnas、540個(gè)mrnas滿足以上標(biāo)準(zhǔn)。質(zhì)量評估:火山圖法、聚類分析熱圖法處理上述數(shù)據(jù),結(jié)果提示:兩組樣本的基因表達(dá)存在了顯著差異性,ais樣本內(nèi)某些基因的表達(dá)水平與正常樣本呈現(xiàn)出顯著的差異。以表達(dá)水平差異倍數(shù)超過2倍且studentt檢驗(yàn)后p0.05,我們以此為差異性的評定標(biāo)準(zhǔn),之后對基因芯片數(shù)據(jù)進(jìn)行篩選。發(fā)現(xiàn),ais組中有2116個(gè)lncrnas和540個(gè)mrnas發(fā)生差異表達(dá)現(xiàn)象,其中l(wèi)ncrnas表達(dá)上調(diào)為1015個(gè),下調(diào)為1101個(gè)。篩選的差異表達(dá)的lncrnas、mrnas中,差異超過5倍的分別為64個(gè)(其中1個(gè)上調(diào),63個(gè)下調(diào))和26個(gè)(0個(gè)上調(diào),26個(gè)下調(diào))。與正常組相對比,ais患者樣本基因中有346個(gè)一致性表現(xiàn)為上調(diào)或下調(diào)(≥2個(gè)差異倍數(shù))。nonhsat137367是最顯著的下調(diào)lncrna基因(差異倍數(shù)=11.27617),而nonhsat103134是最顯著的上調(diào)lncrna(差異倍數(shù)=5.174644)。本研究發(fā)現(xiàn),與正常組相比,arc,set及tpm3基因在ais患者樣本中表現(xiàn)出顯著的差異。第二部分:選取5條lncrnas,在15例ais外周血樣本和15例正常外周血樣本中驗(yàn)證上述結(jié)果,在ais外周血樣本和正常外周血樣本中對其進(jìn)行了qrt-pcr檢測,驗(yàn)證結(jié)果與前述結(jié)果高度一致。由此可見,高通量的基因芯片結(jié)果具有較高的可信度。第三部分:生物信息量預(yù)測:kegg生物學(xué)通路、go分析及david功能注釋簇集法的應(yīng)用對差異表達(dá)的lncrna、mrna進(jìn)行功能注釋和預(yù)測。通過kegg法,我們發(fā)現(xiàn)focaladhesion、amoebiasis、adherensjunction、regulationofactincytoskeleton、leukocytetransendothelialmigration、salivarysecretion、arginineandprolinemetabolism和thyroidcancer八個(gè)生物學(xué)通路具體潛在的研究價(jià)值。對差異表達(dá)的mrna進(jìn)行的david功能注釋簇集分析的結(jié)果中出現(xiàn)了中央神經(jīng)系統(tǒng)的發(fā)育、表皮發(fā)育、跨膜蛋白受體色氨酸激酶信號(hào)通路調(diào)節(jié)、肺部發(fā)育、細(xì)胞轉(zhuǎn)移及生長因子等,提示在ais患者中,上述病理變化廣泛存在。pearson分析表明,差異顯著的10個(gè)lncrnas,與相關(guān)共表達(dá)的mrna構(gòu)建成cnc網(wǎng)絡(luò)關(guān)系圖。go分析提示,上述lncrnas在蛋白轉(zhuǎn)移、增殖或凋亡、基因表達(dá)的轉(zhuǎn)錄后調(diào)控、細(xì)胞分化、凋亡調(diào)控以及蛋白的代謝等多個(gè)與ais相關(guān)的病理進(jìn)化過程發(fā)揮著重要的作用�；诖�,這10個(gè)差異表達(dá)最為顯著的lncrnas,究竟在ais發(fā)病及加重過程中發(fā)揮何種作用值得進(jìn)行下一步深入的研究。第四部分:我們招募了ais患者男女各四名;正常的青少年男女各四名,研究AIS患者性別之間的遺傳差異。Venn、GO分析和KEGG分析是用來篩選與AIS女性多發(fā)相關(guān)的lncRNAs。Venn分析挑選出134條與AIS女性多發(fā)相關(guān)的lncRNAs。其中NONHSAG015771基因下調(diào)4.4倍,CUST_8497_PI429545388基因上調(diào)5.5倍。在這項(xiàng)研究中用微陣列篩選與女性多發(fā)相關(guān)的lncRNA。共有134 lncRNAs作為候選靶基因,通過GO分析和KEGG分析得出:他們參與吞噬體,溶酶體和HTLV-1感染生物學(xué)過程,可能參與RNA聚合酶II啟動(dòng)子轉(zhuǎn)錄的負(fù)調(diào)控。這些生物過程均可能是女性AIS多發(fā)的病理機(jī)制。在本實(shí)驗(yàn)中,我們首次對AIS發(fā)病LncRNA的相關(guān)性進(jìn)行研究,進(jìn)一步發(fā)現(xiàn)其潛在致病因素。在未來的研究中,深入了解特定LncRNA在AIS病理機(jī)制中發(fā)的明確作用。
[Abstract]:Background: adolescent idiopathic scoliosis (Adolescent Idiopathic Scoliosis, AIS) is the most common type of idiopathic scoliosis (Idiopathic Scoliosis, IS), most of which are female patients. The age of onset is usually 10-18 years old and increases with the growth of the body. It will not only affect the appearance of the patient, but also affect the patient's appearance. The heart and lung function of the patient. Although there are many clinical, epidemiological and molecular biological analysis of the pathogenesis of AIS, the pathogenesis is still unclear. Related reports on the study of Harrington have occurred among patients with more than 15 degrees of Cobb, and 27% of their descendants may have scoliosis. In the AIS related basic and clinical study of twin children, the common occurrence rate of monozygotic twins is about 73%, and the possibility rate of double egg twins is only 27%.Wynne through follow-up study of AIS patients. It has obvious familial tendency, but the mechanism is still not clear. Therefore, the genetic factors are found to be in the pathogenesis. The development of gene technology, the perfection of the genome project, the development of gene function will be the focus of attention. The AIS molecular typing based on Transcriptome has gradually become the focus of research. The research on the pathogenesis of AIS and how to treat it is still still more than yet. At the initial stage, the clinical application has a broad prospect. With the maturation and development of gene technology, the transcriptional analysis of the two generation gene sequencing technology (RNA-seq) and the whole genome Gene Expression Microarray plays a very important role in the study of functional genomics, not only for use in the study of functional genomics. A variety of different regulatory noncoding RNA (microRNA and lncRNA, etc.) can be detected in the detection of mRNA encoded proteins. The study of transcriptional studies has important application prospects and clinical value on how to further screen the possible molecules associated with AIS pathogenesis, reveal its development mechanism and seek new molecular targets for AIS treatment. Chain non coding RNA (Long noncoding RNA (lncRNA)) is a hot spot of current research. It can regulate gene expression through epigenetic, transcriptional and post transcriptional levels. In many complex diseases such as tumor, neurodegenerative disease, cardiovascular disease, the expression of lncRNA is abnormal, and part of lncRNA is promoted. But so far, there are few reports on the expression of AIS related lncRNA. This study has obtained the lncRNA expression profiles of AIS patients and normal people by using RNA-Seq sequencing technology and obtained the mRNA expression profiles, and further explored the differences in the expression of lncRNA in AIS patients, providing a further study for further research. The main contents of this study are summarized as follows: the first part: Study on the expression of lncRNA and mRNA in the peripheral blood samples of normal adolescents and AIS patients. In this part of the study, we selected 10 cases of peripheral blood (5 normal samples, 5 AIS samples). After standardization, at least 5 samples of the 10 samples appeared lncrna-mrna The expression (including fuzzy expression) was defined as a meaningful target gene. The results showed that in 10 samples, 2116 lncrnas and 540 mRNAs met the above criteria. Quality assessment: Volcano mapping and cluster analysis thermograph method to deal with the above data. The results showed that there were significant differences in gene tables in the two groups, and the expression of some genes in AIS samples. There was a significant difference between the level and the normal sample. In order to express the horizontal difference multiplier more than 2 times and P0.05 after studentt test, we used this as the standard of differential evaluation and then screened the gene chip data. It was found that there were 2116 lncrnas and 540 mRNAs expressions in the AIS group, and the lncrnas expression was up to 1015, The down regulation was 1101. The differential expression of lncrnas and mRNAs were 64 (1 up, 63 down-regulation) and 26 (0 up-regulated, 26 down-regulation). Compared with the normal group, there were 346 congruence in the AIS patient's sample gene, up or down (more than 2 differential multiple).Nonhsat137367 was the most significant down-regulation of L The ncRNA gene (differential multiple =11.27617) was the most significant up-regulated lncrna (differential multiplier =5.174644). This study found that the arc, set, and TPM3 genes were significantly different in the AIS patient samples compared with the normal group. Second part: selected 5 lncrnas, in 15 AIS peripheral blood samples and 15 normal peripheral blood samples. The above results were detected by qRT-PCR in AIS peripheral blood samples and normal peripheral blood samples. The results were highly consistent with the previous results. Thus, high throughput gene chip results have high reliability. Third parts: bioinformatics prediction: KEGG biologic pathway, go analysis and the application of David function annotation cluster method Functional annotation and prediction for differentially expressed lncrna, mRNA. Through KEGG, we found specific potential research prices for eight biological pathways: focaladhesion, amoebiasis, adherensjunction, regulationofactincytoskeleton, leukocytetransendothelialmigration, salivarysecretion, arginineandprolinemetabolism and thyroidcancer. The results of the David functional annotation cluster analysis of differentially expressed mRNA showed the development of the central nervous system, the development of the epidermis, the regulation of tryptophan kinase signaling pathway, lung development, cell metastasis and growth factor, suggesting that in the AIS patients, the above pathological changes were widely found by.Pearson analysis. 10 different significant lncrnas, and related co expressed mRNA constructed into CNC network diagram.Go analysis, it is suggested that the lncrnas plays an important role in the process of AIS related pathogenesis, such as protein transfer, proliferation or apoptosis, post transcriptional regulation of gene expression, cell differentiation, apoptosis regulation and protein metabolism. Lncrnas, the most significant difference expression, is worthy of further research in the course of the pathogenesis and aggravation of AIS. The fourth part: We recruited four men and women in AIS patients; four normal young men and women, studied the genetic difference between sex of AIS patients.Venn, GO analysis and KEGG analysis were used to screen. The lncRNAs.Venn analysis of multiple hair related to AIS women selected 134 lncRNAs. associated with AIS women, in which the NONHSAG015771 gene was down 4.4 times, and the CUST_8497_PI429545388 gene was up 5.5 times. In this study, microarrays were used to screen a total of 134 lncRNAs as a candidate target gene for lncRNA.. KEGG analysis shows that they participate in the phagocytic, lysosome and HTLV-1 infection biological processes, and may be involved in the negative regulation of RNA polymerase II promoter transcription. These biological processes may be the pathological mechanism of the multiple AIS in female AIS. In this experiment, we first studied the phase of LncRNA in the pathogenesis of AIS and further found its underlying cause. In future research, we will further understand the specific role of specific LncRNA in the pathogenesis of AIS.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R726.8

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