發(fā)布時間：2018-05-16 16:15

  本文選題：心房纖顫 + 心臟內(nèi)在自主神經(jīng)重構(gòu)�。� 參考：《山東大學(xué)》2016年博士論文

【摘要】：[研究背景]心房顫動(Atrial Fibrillation, AF,房顫)是臨床上最常見的快速型紊亂性房性心律失常,發(fā)病率約0.4%-1%,且隨年齡增長逐年增加。房顫的主要危害為栓子脫落引起的腦卒中及長期快速心室率誘發(fā)的心力衰竭,房顫患者死亡率是竇性心律人群的2倍。盡管藥物、外科手術(shù)及導(dǎo)管射頻消融等治療房顫的手段在不斷發(fā)展,房顫的治療效果仍不盡人意。究其原因,與房顫發(fā)生機(jī)制不甚明了密切相關(guān)。繼房顫的電重構(gòu)及結(jié)構(gòu)重構(gòu)后,心臟內(nèi)在自主神經(jīng)重構(gòu),即心臟內(nèi)在自主神經(jīng)的再生過度與分布的不均一性,是房顫重要的發(fā)病機(jī)制。神經(jīng)重構(gòu)促進(jìn)房顫發(fā)生與維持的同時,房顫的快速心房率也加劇心臟內(nèi)在自主神經(jīng)重構(gòu)的發(fā)展,兩者互為因果,形成惡性循環(huán)。阻止甚至逆轉(zhuǎn)心臟內(nèi)在自主神經(jīng)重構(gòu)可能是一種新型治療房顫的方法。但目前對房顫神經(jīng)重構(gòu)現(xiàn)象的發(fā)生機(jī)制,仍知之甚少,亟需研究闡明。長鏈非編碼RNAs (long non-coding RNAs,lncRNAs)是一類長度大于200個核苷酸,具有mRNA樣結(jié)構(gòu),但缺少特異完整開放讀碼框架的轉(zhuǎn)錄本。與mRNAs相比,其表達(dá)在不同組織或同一組織的不同生長階段具有明顯的特異性,lncRNAs可從染色質(zhì)重塑、轉(zhuǎn)錄調(diào)控及轉(zhuǎn)錄后加工等多種層面實現(xiàn)對基因表達(dá)的調(diào)控,被證實與蛋白質(zhì)、DNAs和RNAs存在相互作用,因其特異性強和功能多樣化,已成為現(xiàn)階段研究的熱點�，F(xiàn)有研究表明,lncRNAs在心血管系統(tǒng)及神經(jīng)系統(tǒng)的發(fā)育與發(fā)病過程中扮演重要的角色,lncRNAs不僅參與了神經(jīng)元的分化、發(fā)育、突觸可塑性及神經(jīng)退行性疾病的發(fā)生、發(fā)展,且lncRNAs調(diào)控心臟疾病,如心肌梗死、心力衰竭、擴(kuò)張型心肌病、室間隔缺損等的發(fā)病過程。以上均提示lncRNAs在房顫心臟內(nèi)在自主神經(jīng)重構(gòu)的發(fā)生、發(fā)展過程中發(fā)揮重要生物學(xué)功能。但何種lncRNAs有異常表達(dá),發(fā)揮何種作用,與房顫的發(fā)生、發(fā)展又有何種關(guān)聯(lián),尚無系統(tǒng)研究。[研究目的]通過快速右心房起搏建立房顫犬實驗?zāi)Ｐ?應(yīng)用高通量測序檢測房顫及非房顫犬心房前右脂肪墊(anterior right fat pads, ARFPs)中差異lncRNAs的表達(dá),并通過一系列生物信息學(xué)方法,鑒定與心臟內(nèi)在自主神經(jīng)重構(gòu)相關(guān)的lncRNAs;通過體內(nèi)、體外實驗,研究lncRNAs對犬心臟內(nèi)在自主神經(jīng)重構(gòu)及房顫誘發(fā)性的影響；并進(jìn)一步探討lncRNA-032546和lncRNA-026102影響房顫神經(jīng)重構(gòu)的機(jī)理,從lncRNAs的視角研究房顫神經(jīng)重構(gòu)發(fā)生的新機(jī)制,對房顫的防治提供新的干預(yù)靶點。[研究方法]1、房顫犬模型的建立取6只健康成年比格犬,雌雄不拘,隨機(jī)分為對照組和起搏組。兩組均放置起搏電極于右心耳,對照組不起博、喂養(yǎng)4周,起搏組以400次/min起搏4周建立房顫犬模型。為了檢測房顫的發(fā)生,兩組每周均至少行一次體表心電圖。2、驗證神經(jīng)重構(gòu)的發(fā)生取對照組及起搏組犬ARFPs組織,對神經(jīng)細(xì)胞胞漿蛋白9.5(protein gene product 9.5, PGP 9.5)行免疫組化染色,計算神經(jīng)密度,驗證房顫介導(dǎo)神經(jīng)重構(gòu)的發(fā)生。3、高通量lncRNA測序并驗證Triol法提取對照組及起搏組犬ARFPs中總RNAs,通過高通量二代測序檢測lncRNAs及mRNAs的差異表達(dá)。并隨機(jī)選取部分全新的lncRNAs,應(yīng)用實時定量PCR (real-time quantitative PCR, qRT-PCR)的方法,對測序結(jié)果進(jìn)行驗證。4、篩選與房顫神經(jīng)重構(gòu)相關(guān)的lncRNAs對差異表達(dá)的轉(zhuǎn)錄本行Gene Ontology (GO)富集分析及Kyoto Encyclopedia of Genes and Genomes (KEGG)通路分析；通過差異性表達(dá)倍數(shù)、組織特異性、靶基因預(yù)測、文獻(xiàn)檢索等生物信息學(xué)方法,篩選并鑒定與神經(jīng)重構(gòu)相關(guān)的全新lncRNA-032546和lncRNA-026102。5、lncRNA功能缺失實驗1)在體轉(zhuǎn)染體外構(gòu)建lncRNA-032546和lncRNA-026102的沉默慢病毒及對照慢病毒,滴度為1×109TU/ml。根據(jù)轉(zhuǎn)染慢病毒的不同,15只比格犬隨機(jī)分為空白轉(zhuǎn)染組、低表達(dá)lncRNA-032546組和低表達(dá)lncRNA-026102組,開胸后將相應(yīng)慢病毒在體斜行注射至ARFPs中,關(guān)胸,10天后處死,免疫熒光檢測轉(zhuǎn)染效率。2)電生理指標(biāo)的檢測通過程序電刺激分別于開胸后注射慢病毒前、注射慢病毒即刻及注射慢病毒后10天檢測心房有效不應(yīng)期(atrial effective refractory period, AERP)及房顫誘發(fā)率。檢測區(qū)域為ARFPs周圍0.5 cm內(nèi)的心房肌。3)分子生物學(xué)指標(biāo)的檢測分別取轉(zhuǎn)染組及對照組的ARFPs組織,對PGP 9.5行qRT-PCR及免疫組化染色,對神經(jīng)生長因子,即生長蛋白相關(guān)43 (growth-associated protein 43, GAP 43)行qRT-PCR,計算神經(jīng)密度,檢測PGP 9.5及GAP 43 mRNAs的表達(dá)水平。6、lncRNAs潛在的作用機(jī)制1)Cis預(yù)測對lncRNAs進(jìn)行分類,通過cis機(jī)制尋找IncRNAs上下游300 kb內(nèi)的]mRNAs, qRT-PCR檢測1ncRNAs及其上下游InRNAs的表達(dá)水平,明確其表達(dá)的相關(guān)性,結(jié)合文獻(xiàn)進(jìn)行分析。2) Trans預(yù)測根據(jù)皮爾遜相關(guān)系數(shù)及P值,計算并選取前100個與IncRNAs共表達(dá)的mRNAs,進(jìn)行GO富集分析及KEGG通路分析,預(yù)測]lncRNA的trans作用機(jī)制。通過文獻(xiàn)檢索明確與IncRNAs有關(guān)的分子信號通路,進(jìn)一步通過qRT-PCI測通路中關(guān)鍵分子的表達(dá)水平進(jìn)行驗證。[研究結(jié)果]1、通過快速起搏成功建立房顫犬實驗?zāi)Ｐ?起搏組PGP 9.5陽性神經(jīng)纖維的密度較對照組明顯增加。2、通過二代高通量測序,共計獲得61616個推定的IncRNAs,根據(jù)一系列篩選條件,得出1164個候選lncRNAs,其中,差異表達(dá)倍數(shù)2倍的有576個轉(zhuǎn)錄本,410個表達(dá)上調(diào),166個表達(dá)下調(diào),45個轉(zhuǎn)錄本被鑒定為全新的lncRNAs。隨機(jī)選取6個全新的IncRNAs,qRT-PCR證實測序結(jié)果真實、可靠。3、GO富集分析及KEGG通路分析表明差異表達(dá)的基因主要參與了神經(jīng)系統(tǒng)發(fā)育、細(xì)胞遷移及神經(jīng)退行性疾病等生物學(xué)過程；通過篩選差異性表達(dá)倍數(shù)2倍的轉(zhuǎn)錄本、預(yù)測靶基因的生物學(xué)功能并去除在骨骼肌組織中非特異性高表達(dá)的lncRNAs,篩選出2個與神經(jīng)重構(gòu)相關(guān)的全新的表達(dá)下調(diào)的lncRNA-032546和lncRNA-026102。4、免疫熒光檢測轉(zhuǎn)染效率,qRT-PCR驗證沉默效率；心內(nèi)電生理結(jié)果表明,與對照組相比,AERP在轉(zhuǎn)染前及轉(zhuǎn)染即刻均無統(tǒng)計學(xué)差異,而轉(zhuǎn)染后10天,lncRNA-032546表達(dá)下調(diào)可明顯縮短AERP,相應(yīng)的,短陣房速及房顫易誘發(fā)；與之相比,下調(diào)lncRNA-026102明顯延長AERP,短陣房速及房顫不能誘發(fā)。5、PGP 9.5免疫組化結(jié)果表明,相比于對照組,低表達(dá)lncRNA-032546組神經(jīng)密度明顯增加,低表達(dá)lncRNA-026102組神經(jīng)密度明顯減少；qRT-PCR結(jié)果提示,GAP 43表達(dá)趨勢與免疫組化結(jié)果類似,下調(diào)lncRNA-032546表達(dá)后,PGP9.5mRNA表達(dá)水平明顯升高,而下調(diào)lncRNA-026102表達(dá)后,PGP 9.5的mRNA表達(dá)水平無統(tǒng)計學(xué)差異。6、lncRNA-032546和lncRNA-026102均為基因間IncRNAs,易通過cis機(jī)制調(diào)控鄰近靶基因的表達(dá)。基于cis預(yù)測,找出lncRNA-032546和lncRNA-026102上下游300 kb內(nèi)的mRNAs,qRT-PCR結(jié)果示下調(diào)lncRNA-032546和lncRNA-026102的表達(dá)分別上調(diào)CCND1-FGF19-FGF4-FGF3基因簇和SLC25A4的表達(dá)；通過文獻(xiàn)檢索明確差異表達(dá)的CCND 1-FGF 19-FGF4-FGF3基因簇和SLC25A4與神經(jīng)生長、發(fā)育密切相關(guān)；進(jìn)一步通過生物信息學(xué)計算lncRNA與mRNA間的共表達(dá)系數(shù),推測出lncRNA-032546和lncRNA-026102潛在的靶基因分別為CCND1-FGF19-FGF4-FGF3基因簇和SLC25A4。7、根據(jù)trans預(yù)測,分別找出與lncRNA-032546、lncRNA-026102顯著共表達(dá)的前100個mRNAs,行GO富集分析及KEGG通路分析。文獻(xiàn)表明絲裂原活化蛋白激酶(MAPK)通路與CCND 1-FGF 19-FGF4-FGF3基因簇在促神經(jīng)發(fā)生上密切相關(guān),qRT-PCR結(jié)果表明ERK1、ERK2、JN及p38的mRNA表達(dá)水平均明顯上調(diào),提示下調(diào)lncRNA-032546的表達(dá)可激活MAPK通路發(fā)揮功能。同時,細(xì)胞核因子酉乙蛋白(NF-kappa B)通路在神經(jīng)系統(tǒng)生長、發(fā)育中發(fā)揮重要作用,過表達(dá)SLC25A4通過招NF-kappa B誘導(dǎo)細(xì)胞凋亡,低表達(dá)lncRNA-026102組SLC25A4表達(dá)上調(diào)、神經(jīng)細(xì)胞密度下調(diào),提示下調(diào)lncRNA-026102介導(dǎo)神經(jīng)重構(gòu)的發(fā)生可能通過NF-kappa B通路上調(diào)SLC25A4的表達(dá)實現(xiàn)。[研究結(jié)論]1、在房顫／非房顫犬ARFP內(nèi)一系列l(wèi)ncRNAs存在顯著差異性表達(dá),這些差異表達(dá)的lncRNAs參與了房顫心臟內(nèi)在自主神經(jīng)重構(gòu)的發(fā)生；2、我們發(fā)現(xiàn)了2個全新的lncRNA-032546和lncRNA-026102與房顫神經(jīng)重構(gòu)密切相關(guān),并通過功能缺失實驗明確lncRNA-032546通過促進(jìn)心臟內(nèi)在自主神經(jīng)的重構(gòu)易化房顫的發(fā)生,而lncRNA-026102則抑制心臟內(nèi)在自主神經(jīng)重構(gòu)進(jìn)而阻止房顫的發(fā)生；3、我們預(yù)測了這2個lncRNAs的作用機(jī)制,即]lncRNA-032546通過cis調(diào)控鄰近CCND1-FGF19-FGF4-FGF3基因簇并通過trans調(diào)控下游MAPK信號通路發(fā)揮作用；lncRNA-026102則通過c西調(diào)控鄰近靶基因SLC25A4, trans調(diào)控TF-kappa B通路發(fā)揮生物學(xué)功能。[創(chuàng)新性]1、從lncRNAs的視角,觀察其表達(dá)水平變化對房顫心臟內(nèi)在自主神經(jīng)重構(gòu)的影響；2、探索lncRNAs通過cis調(diào)控鄰近靶基因及trans調(diào)控下游分子信號通路影響房顫心臟內(nèi)在自主神經(jīng)重構(gòu)的分子機(jī)制；3、應(yīng)用犬心臟脂肪墊在體轉(zhuǎn)染慢病毒介導(dǎo)的lncRNA沉默載體技術(shù),研究lncRNAs對房顫心臟內(nèi)在自主神經(jīng)重構(gòu)的影響及作用機(jī)制。
[Abstract]:Atrial fibrillation ( AF ) is one of the most common types of atrial fibrillation in patients with atrial fibrillation . AIM : To establish an experimental model of atrial fibrillation by rapid and right atrial pacing , and to identify the expression of different lncRNAs in anterior right fat pad ( ARFPs ) of atrial fibrillation and non - atrial fibrillation dogs by rapid and right atrial pacing .
In order to detect the occurrence of atrial fibrillation , two groups were randomly divided into two groups : control group and pacing group . In order to detect the occurrence of atrial fibrillation , two groups were randomly divided into control group and pacing group .
The expression levels of lncRNA - 032546 and lncRNA - 26102.5 and lncRNA - 26102.5 and lncRNA - 26102.5 and lncRNA - 26102.5 and lncRNA - 26102.5 , lncRNA - 032546 and GAP - 43 mRNAs were randomly divided into blank transfection group , low - expression lncRNA - 032546 group and low - expression lncRNA - 26102 group . Based on a series of screening conditions , 1,164 candidate lncRNAs were obtained , including 576 transcripts , 410 expression up - regulation , 166 expression downregulation , 45 transcripts were identified as completely new lncRNAs .
by screening transcripts with multiple times of differential expression , predicting the biological function of the target gene and removing the lncRNAs expressed in the non - specific high expression in the skeletal muscle tissue , two novel expression downregulated lncRNA - 032546 and lncRNA - 26102.4 related to nerve reconstruction are screened , the transfection efficiency of immunofluorescence detection is detected , and the silencing efficiency is verified by qRT - PCR ;
The results showed that the expression of lncRNA - 032546 could significantly shorten AERP after transfection .
Compared with the control group , the lower expression of lncRNA - 032546 group significantly increased the nerve density and the low expression of lncRNA - 032546 group was significantly decreased compared with the control group .
The results of qRT - PCR showed that the expression of GAP 43 was similar to that of immunohistochemistry . After downregulating the expression of lncRNA - 032546 , the mRNA expression level of PGP9.5 mRNA was significantly increased , but the expression of mRNA was regulated by cis mechanism . On the basis of cis prediction , the expression of lncRNA - 032546 and lncRNA - 26102 was found . The expression of lncRNA - 032546 and lncRNA - 26102 was downregulated and the expression of CCND1 - FGF19 - FGF4 - FGF3 gene cluster and SLC25A4 were regulated respectively .
The CCN1 - FGF - 19 - FGF4 - FGF3 gene cluster and SLC25A4 expressed by literature search were closely related to the growth and development of nerve ;
In this paper , we found that the potential target genes of lncRNA - 032546 and lncRNA - 26102 were significantly higher than that of CCND1 - FGF19 - FGF4 - FGF3 gene cluster and SLC25A4.7 .
2 . We have found that two new lncRNA - 032546 and lncRNA - 26102 are closely related to the remodeling of atrial fibrillation nerve , and through functional deletion experiments , lncRNA - 032546 is established to promote the reconstruction of the intrinsic autonomic nerve in the heart .
3 . We predict the mechanism of action of these two lncRNAs , that is , by cis - regulating the adjacent CCND1 - FGF19 - FGF4 - FGF3 gene cluster and regulating the MAPK signal pathway downstream through trans .
The effects of changes of expression level on the internal autonomic nerve remodeling in AF heart were observed from the perspective of lncRNAs from the perspective of lncRNAs .
2 . To explore the molecular mechanism of lncRNAs regulating the intrinsic autonomic nerve remodeling in patients with AF by regulating the molecular signaling pathway adjacent to the target gene and trans - regulating the downstream molecular signal pathway ;
3 . The effect and mechanism of lncRNAs on the internal autonomic nerve remodeling in patients with atrial fibrillation were studied by using canine cardiac fat pad in vivo .

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R541.75
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本文編號：1897546