本文選題：缺氧損傷 + 低氧誘導(dǎo)因子�。� 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2008年碩士論文

【摘要】： 可變剪接(Alternative splicing)廣泛存在于人類基因組中,是構(gòu)成蛋白質(zhì)多樣性與基因表達(dá)復(fù)雜程度的主要原因。雖然目前有報(bào)道發(fā)現(xiàn)細(xì)胞低氧應(yīng)答通路中的轉(zhuǎn)錄因子——低氧誘導(dǎo)因子HIF-1α(hypoxia inducible factor-1α, HIF-1α)的一種可變剪接可對HIF-1α起負(fù)性調(diào)控作用。但可變剪接機(jī)制作為一種重要的基因表達(dá)調(diào)控手段,在細(xì)胞缺氧、動(dòng)物缺血損傷中發(fā)揮的調(diào)節(jié)作用,目前仍然所知甚少。 為了探討血管內(nèi)皮細(xì)胞缺氧損傷相關(guān)基因的可變剪接變化,我們首先通過NCBI GenBank數(shù)據(jù)庫和ECgene數(shù)據(jù)庫分析獲得低氧通路相關(guān)基因的潛在的可變剪接形式,用RT-PCR鑒定CoCl2誘導(dǎo)的人臍靜脈內(nèi)皮細(xì)胞系(ECV304細(xì)胞)缺氧損傷后HIFs和EGLNs家族各基因的可變剪接異構(gòu)體,發(fā)現(xiàn)HIF-1α的3個(gè)、EGLN1的2個(gè)和EGLN3的1個(gè)可變剪接異構(gòu)體,而且各個(gè)可變剪接異構(gòu)體的表達(dá)水平隨著缺氧時(shí)間的延長而具有不同的變化趨勢,可能與不同的缺氧損傷狀態(tài)有關(guān)。其中新發(fā)現(xiàn)3種可變剪接異構(gòu)體:HIF1α-3,EGLN1-8和EGLN3-5,提交GenBank數(shù)據(jù)庫,獲得的序列接受號(hào)分別為:DQ975378,DQ975380和DQ975379。這些結(jié)果提示可變剪接在細(xì)胞缺氧損傷過程中可能發(fā)揮重要作用。 為了以全基因組水平研究血管內(nèi)皮細(xì)胞缺氧損傷中的可變剪接,我們進(jìn)一步利用Affymetrix GeneChip? Human Exon 1.0 ST Array系統(tǒng)檢測CoCl2誘導(dǎo)的人臍靜脈內(nèi)皮細(xì)胞(HUVEC細(xì)胞)缺氧損傷后基因的可變剪接模式。在HUVEC細(xì)胞缺氧損傷后,顯著差異的基因共1583個(gè),其中表達(dá)上調(diào)的基因300個(gè),表達(dá)下調(diào)的基因1283個(gè);顯著差異的外顯子共342個(gè)(涉及到293個(gè)基因),其中134個(gè)外顯子所屬基因的mRNA表達(dá)水平也顯著差異表達(dá)。通過GO(基因本體論)和KEGG數(shù)據(jù)庫對差異表達(dá)的基因和外顯子所屬基因的編碼產(chǎn)物進(jìn)行生物學(xué)意義注釋、聚類,結(jié)果顯示:“organelle organization and biogenesis”與“nucleobase”屬性相關(guān)基因產(chǎn)物表達(dá)顯著下降,具體表現(xiàn)在以下幾個(gè)表達(dá)顯著下降的通路:focal adhesion,regulation of actin cytoskeleton,cell cycle,pyrimidine metabolism和TGF-Beta signaling pathway;與此相反,“programmed cell death”屬性基因的表達(dá)顯著上升,表現(xiàn)在表達(dá)顯著上調(diào)的通路:MAPK signaling pathway,proteasome,antigen processing and presentation。同時(shí),大量與細(xì)胞低氧反應(yīng)/缺氧損傷相關(guān)的基因表達(dá)發(fā)生了顯著的變化,包括HIF-1α(hypoxia inducible factor-1α, HIF-1α),VEGFC(vascular endothelial growth factor C),ADCY3 ( adenylate cyclase 3), CAD(carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase)基因等。這些結(jié)果提示:在HUVEC細(xì)胞損傷模型中,細(xì)胞外基質(zhì)減少,細(xì)胞間通訊破壞,細(xì)胞的生長受到抑制,細(xì)胞活性下降;同時(shí),一個(gè)新的以熱休克蛋白超家族(Hsp27, Hsp70, Hsp105和DnaJ參與缺氧應(yīng)答)為中心的熱休克蛋白通路被激活起應(yīng)激反應(yīng),通過泛素化降解通路使蛋白降解增加,凋亡通路被啟動(dòng),并發(fā)生細(xì)胞死亡。 UniProt數(shù)據(jù)庫分析表明,差異表達(dá)基因與差異表達(dá)外顯子所屬基因的蛋白功能分類均主要是可變剪接調(diào)控(24.1%, 32.1%)、核蛋白(22.4%, 23.5%)、磷酸化(19.7%, 22.9%)等屬性,說明與可變剪接密切相關(guān)的基因(起調(diào)控作用的剪接因子和被可變剪接調(diào)控的基因)在CoCl2誘導(dǎo)的HUVEC細(xì)胞缺氧損傷過程中發(fā)揮重要的調(diào)控作用,或通過可變剪接調(diào)控對損傷起應(yīng)答反應(yīng)。CoCl2誘導(dǎo)的HUVEC細(xì)胞缺氧損傷后,重要的組成性剪接因子和特異性剪接因子如SF3A2(splicing factor 3a, subunit 2, 66kDa),SFRS7(splicing factor, arginine/serine-rich 7, 35kDa),SFRS1 (splicing factor, arginine/serine-rich 1 (splicing factor 2, alternate splicing factor) ),PTBP1( polypyrimidine tract binding protein 1),RBM14(RNA binding motif protein 14)等的mRNA表達(dá)水平均顯著下降,可改變細(xì)胞內(nèi)眾多基因的剪接事件,這與250個(gè)可變外顯子保留的剪接異構(gòu)體表達(dá)上升或出現(xiàn)、92個(gè)可變外顯子去除的剪接異構(gòu)體表達(dá)水平下降或缺失結(jié)果一致。在342個(gè)可變剪接事件中,基因可變剪接類型的發(fā)生率:47.95%為cassette exon,16.96%為alternative promoter,19.01%為alternative polyadenylation,其它的為內(nèi)含子保留、互斥外顯子等剪接類型。這與文獻(xiàn)報(bào)道的cassette exon是可變剪接的主要類型一致�？勺兗艚油ㄟ^改變蛋白質(zhì)的結(jié)構(gòu)域而改變基因的功能,改變啟動(dòng)子結(jié)構(gòu)而調(diào)控基因的表達(dá)。在HUVEC細(xì)胞缺氧損傷后,我們通過RT-PCR驗(yàn)證的具有可變剪接異構(gòu)體的14個(gè)基因中,僅有HNRPD, TNFRSF10B, PHF14, ALAS1, BBS9基因的可變剪接異構(gòu)體具有RefSeq注釋。 基因的可變剪接調(diào)節(jié)機(jī)制十分復(fù)雜。在HUVEC細(xì)胞缺氧損傷中,可變剪接事件不僅發(fā)生在與缺氧相關(guān)的基因上,在剪接因子中也普遍存在。這提示:可變剪接在細(xì)胞缺氧損傷中通過剪接因子來發(fā)揮重要的調(diào)控作用;剪接因子可能通過可變剪接調(diào)節(jié)自身的表達(dá),然后調(diào)控一系列與之相關(guān)的基因發(fā)生剪接改變以適應(yīng)環(huán)境的改變。然而這些剪接因子在缺氧損傷中究竟怎樣調(diào)控基因的表達(dá)以及調(diào)節(jié)哪些基因的可變剪接事件,目前還不清楚,尚需深入對剪接因子的調(diào)控進(jìn)行系統(tǒng)的功能研究。目前,我們的研究表明:CoCl2誘導(dǎo)的HUVEC細(xì)胞缺氧損傷模型中,基因轉(zhuǎn)錄表達(dá)水平與基因的可變剪接調(diào)控機(jī)制共同調(diào)節(jié)細(xì)胞對缺氧損傷起應(yīng)答反應(yīng)。 另外,我們已成功制備C57BL/6J小鼠大腦中動(dòng)脈阻塞模型,并利用該模型研究可變剪接在大腦缺血及缺血再灌注損傷中的調(diào)控機(jī)制,目前已獲得初步結(jié)果。 綜上所述,本研究初步揭示細(xì)胞缺氧損傷中,可變剪接起到重要的調(diào)控作用。為了解細(xì)胞缺氧損傷機(jī)制提供了重要數(shù)據(jù),有助于從基因轉(zhuǎn)錄表達(dá)水平和可變剪接調(diào)控水平理解腦血管病的發(fā)病機(jī)制。
[Abstract]:Variable splicing (Alternative splicing) is widely found in the human genome, which is the main cause of the complexity of protein diversity and gene expression. Although there are reports of a variable splicing of the transcription factor of hypoxia inducible factor HIF-1 a (hypoxia inducible factor-1 alpha, HIF-1 a) in the cellular hypoxia response pathway However, as an important means of gene expression regulation, variable splicing mechanism, as an important means of gene expression and regulation, is still poorly understood in cell hypoxia and animal ischemic injury.
In order to investigate the variable splicing changes of hypoxia injury related genes in vascular endothelial cells, we first analyzed the potential variable splicing form of hypoxia pathway related genes through NCBI GenBank database and ECgene database, and identified HIFs and EGLNs in CoCl2 induced human umbilical vein endothelial cell line (ECV304 cells) induced by CoCl2 after hypoxia injury. The variable splicing isomers of the family genes found 3 HIF-1 alpha, 2 EGLN1 and 1 variable splicing isomers of EGLN3, and the expression level of the variable splice isomers varied with the prolongation of the anoxic time, which may be related to the different state of hypoxia injury. Among them, 3 alterable splicing isomers were discovered. HIF1 alpha -3, EGLN1-8 and EGLN3-5 were submitted to the GenBank database. The sequence numbers obtained were DQ975378, DQ975380, and DQ975379., which suggested that the variable splicing might play an important role in the process of cell hypoxia injury.
In order to study the variable splicing in the anoxic damage of vascular endothelial cells at the whole genome level, we further used the Affymetrix GeneChip? Human Exon 1 ST Array system to detect the variable splicing pattern after hypoxia injury induced by CoCl2 induced human umbilical vein endothelial cells (HUVEC cells). There were 1583 genes, including 300 up-regulated genes and 1283 down-regulated genes, and 342 exons of significant differences (involving 293 genes), of which 134 exons were also significantly expressed in the mRNA expression level. The genes and exons expressed by the GO (gene ontology) and KEGG database The results showed that the expression of "organelle organization and biogenesis" and "nucleobase" attribute related gene products decreased significantly, which was manifested in the following pathways: focal adhesion, regulation of actin cytoskeleton, cell cell. Abolism and TGF-Beta signaling pathway, on the contrary, the expression of the "programmed cell death" attribute gene is significantly increased, manifested in a significant up-regulated pathway: MAPK signaling pathway, proteasome, antigen, and a large number of gene expressions associated with cellular hypoxia response / hypoxia injury. The changes include HIF-1 alpha (hypoxia inducible factor-1 alpha, HIF-1 alpha), VEGFC (vascular endothelial growth factor C), ADCY3 (2) genes, etc. These results suggest that the extracellular matrix is in the cell damage model. Decreased cell communication was destroyed, cell growth was inhibited and cell activity decreased; at the same time, a new heat shock protein pathway centered on the heat shock protein superfamily (Hsp27, Hsp70, Hsp105 and DnaJ) was activated to activate the stress response, and the protein degradation increased through the ubiquitination degradation pathway and the apoptotic pathway was activated. And cell death occurred.
UniProt database analysis showed that the protein functional classification of genes of differentially expressed and differentially expressed exons were mainly variable splicing regulation (24.1%, 32.1%), nuclear protein (22.4%, 23.5%), phosphorylation (19.7%, 22.9%), indicating genes closely related to variable splicing (the splicing factors regulating the splicing and variable splicing). Regulatory genes play an important regulatory role in the process of CoCl2 induced HUVEC cell hypoxia injury, or through variable splicing to regulate the hypoxia injury induced by.CoCl2 induced HUVEC cells induced by the damage response, important splicing factors and specific splicing factors such as SF3A2 (splicing factor 3a, subunit 2, 66kDa), SFRS7 (splici) (splici) Ng factor, arginine/serine-rich 7, 35kDa), SFRS1 (splicing factor, arginine/serine-rich 1 (splicing factor 2, alternate splicing)), which are significantly reduced, can change the splicing of numerous genes in the cells. Events, the expression of splice isomers retained with 250 variable exons rose or appeared, and the expression level of splicing isomers removed by 92 exons was reduced or the results were consistent. In the 342 variable splicing events, the incidence of variable splicing types was: 47.95% cassette exon, 16.96% alternative promoter, 19.01% alter Native polyadenylation, other splicing types such as introns, exons and exons. This is the same as the main type of variable splicing that was reported by cassette exon. Variable splicing changes the function of the gene by changing the domain of protein, changes the promoter structure and regulates the expression of genes. After the hypoxia injury of HUVEC cells, Of the 14 genes with variable splicing isomers verified by RT-PCR, the variable splicing isomers with only HNRPD, TNFRSF10B, PHF14, ALAS1, and BBS9 genes have RefSeq annotations.
Variable splicing mechanism is very complex. In HUVEC cell hypoxia damage, variable splicing events occur not only in anoxia related genes, but also in splicing factors. This suggests that variable splicing can play a critical role in cell hypoxia injury through splicing factors, and splicing factors may pass by variable shear. It is not clear how these splicing factors regulate the expression of genes and regulate which genes are variable splicing, but it is still unclear how to regulate the regulation of splicing factors. At present, our study shows that in the CoCl2 induced HUVEC cell hypoxia damage model, the transcriptional expression level of the gene and the variable splicing regulation mechanism of the gene regulate the response of the cell to hypoxia injury.
In addition, we have successfully prepared the C57BL/6J mouse model of middle cerebral artery occlusion and used this model to study the mechanism of variable splicing in cerebral ischemia and ischemia-reperfusion injury, and the preliminary results have been obtained.
To sum up, this study has preliminarily revealed that the variable splicing plays an important regulatory role in the cell hypoxia injury. It provides important data for understanding the mechanism of cell hypoxia injury, and is helpful to understand the pathogenesis of cerebrovascular disease from the level of gene transcription and the regulation of variable splicing.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R346

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 王穩(wěn);利用RT-PCR驗(yàn)證基于小鼠外顯子芯片發(fā)現(xiàn)的腦缺血相關(guān)基因表達(dá)的研究[D];西北農(nóng)林科技大學(xué);2010年

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本文編號(hào)：2084166