本文選題：miR-150 + 診斷指標(biāo)��； 參考：《華中科技大學(xué)》2014年博士論文

【摘要】：近幾年,microRNA (miRNA)在心血管生理及疾病機(jī)制中的研究獲得了重大的進(jìn)步,非編碼RNA在生物學(xué)機(jī)理過程中的重要作用目前已經(jīng)取得共識,而miRNAs就是其中的一類,其大約只有22個堿基的RNA序列,通過部分互補(bǔ)結(jié)合信使RNA上的靶位點(diǎn),調(diào)控蛋白的合成。在心血管系統(tǒng)中,miRNAs已經(jīng)被證實(shí)起著重要的作用,它幾乎參與所有和心血管系統(tǒng)相關(guān)細(xì)胞的功能調(diào)控,例如內(nèi)皮細(xì)胞、心肌細(xì)胞、平滑肌細(xì)胞,炎癥細(xì)胞和成纖維細(xì)胞,因此,miRNAs直接參與了很多心血管疾病的病理發(fā)展�；谒鼈冊诩膊≈械淖饔�,研究它們對于疾病的診斷、預(yù)防和治療都成為了目前的熱點(diǎn),然而要使它們進(jìn)入臨床現(xiàn)在還需要克服許多的困難,本課題主要探討miRNAs在心血管疾病中的診斷以及作用機(jī)制。 一、血漿中降低的miRNA-150作為潛在診斷房顫的新指標(biāo) 1、基因篩查并驗證miRNA-150在房顫病人血漿中表達(dá)降低 本課題分別收集5例孤立性陣發(fā)性房顫(PAF)病人血漿、5例孤立性永久性房顫(PersAF)病人血漿和5例正常對照組,運(yùn)用深度測序技術(shù)篩查出22個miRNAs表達(dá)異常,其中miRNA-146a、miRNA-150、miRNA-19a和miRNA-375符合標(biāo)準(zhǔn)進(jìn)一步驗證。同時收集90例獨(dú)立血漿標(biāo)本運(yùn)用探針PCR技術(shù)驗證這4個miRNAs,發(fā)現(xiàn)只有miRNA-150的表達(dá)趨勢符合篩查結(jié)果,其表達(dá)分別在PersAF病人血漿中及PAF病人血漿中相對于正常對照組下降約17倍和20倍。 2、下調(diào)的miRNA-150表達(dá)與房顫相關(guān)聯(lián) 結(jié)合病人資料和測得的miRNA-150表達(dá)值,邏輯回歸分析顯示,降低的miRNA-150表達(dá)(優(yōu)勢比OR1.96,95%可信區(qū)間CI1.5～3.57,P0.001)、年齡(OR1.1,95%CI1.36-2.73, P0.001)和左房直徑(LAD)(OR1.5,95%CI1.36～1.8, P0.001)分別為房顫的獨(dú)立相關(guān)因素。數(shù)據(jù)庫分析顯示miRNA-150的靶基因中有18個基因與AF相關(guān),其中11個與炎癥系統(tǒng)有聯(lián)系,炎癥因子不僅可以作為診斷AF的標(biāo)準(zhǔn),也參與了AF的發(fā)病機(jī)理,為此我們探測了病人血漿中C-反應(yīng)蛋白(CPR)的表達(dá)水平,發(fā)現(xiàn)其與miRNA-150的表達(dá)呈負(fù)相關(guān)(相關(guān)系數(shù)0.77)。 二、miRNA-150在急性心肌梗死(AMI)中調(diào)控單核細(xì)胞功能保護(hù)心肌功能 1. miRNA-150在AMI后的外周單核細(xì)胞中表達(dá)下調(diào) 收集20例AMI病人外周血和10例正常對照組,磁珠分選出外周血CD14+單核細(xì)胞,qRT-PCR測試發(fā)現(xiàn)miRNA-150表達(dá)量下調(diào),而在CD14的外周單核細(xì)胞(PBMCs)中表達(dá)無顯著差異。同時建立小鼠心梗模型,從心梗小鼠和對照組中的PBMCs中磁珠分選出CD115+單核細(xì)胞,相對于對照組,心梗后單核細(xì)胞中的miRNA-150表達(dá)顯著下調(diào),而在CD115-PBMCs中表達(dá)無差異。 2、miRNA-150調(diào)控人類單核細(xì)胞(THP-1)的遷移和炎癥因子的產(chǎn)生 體外實(shí)驗在THP-1細(xì)胞系中轉(zhuǎn)染miRNA-150的擬合物(agomiRNA150)、抑制劑(antagomiRNA-150)和對照物,遷移實(shí)驗發(fā)現(xiàn)高表達(dá)組相對對照組阻止了THP-1的遷移(3.7±0.5倍),而抑制miRNA-150組促進(jìn)了遷移(6.3±0.8倍)。另外,轉(zhuǎn)然后的THP-1給予脂多糖(LPS)刺激,ELLSA測試發(fā)現(xiàn),相對于對照組高表達(dá)miRNA-150組減少了促炎因子的產(chǎn)生,增加了白介素-10(IL-10)的表達(dá)；抑制miRNA-150組得到了相反的結(jié)果。 3、上調(diào)單核細(xì)胞中miRNA-150的表達(dá)保護(hù)AMI后的心肌功能 動物實(shí)驗顯示在外周細(xì)胞高表達(dá)miRNA-150的心梗小鼠模型中,相對于對照組小鼠改善了心臟功能,減少了心梗的面積,阻止了心肌細(xì)胞的凋亡,并且降低了炎癥單核細(xì)胞Ly-6Chigh的聚集；而在移植了miRNA-150敲除鼠骨髓的小鼠心梗模型中,這些保護(hù)效應(yīng)則被消除。 4、miRNA-150調(diào)控趨化因子受體4(CXCR4)的表達(dá) 數(shù)據(jù)庫分析得出CXCR4為miRNA-150的靶基因,體外實(shí)驗在THP-1細(xì)胞中分別轉(zhuǎn)染agomiRNA-150和對照擬合物,WESTERN實(shí)驗發(fā)現(xiàn)CXCR4表達(dá)相對于對照組下調(diào)；在高表達(dá)miRNA-150的小鼠中,分離CD115+單核細(xì)胞,同樣發(fā)現(xiàn)CXCR4表達(dá)相對于對照組下調(diào)。 結(jié)論 1、血漿中降低的miRNA-150可以作為潛在診斷房顫的新指標(biāo)； 2、miRNA-150調(diào)控單核細(xì)胞功能,對AMI造成的心肌損傷起著保護(hù)作用,為治療急性心梗提供了新的研究靶點(diǎn)。
[Abstract]:In recent years , microRNA ( miRNA ) has made great progress in the research of cardiovascular physiology and disease mechanism , and the important role of non - coding RNA in the process of biological mechanism has now reached consensus , and the miRNA is one of them , which has been proved to play an important role in the pathogenesis of cardiovascular diseases , such as endothelial cells , myocardial cells , smooth muscle cells , inflammatory cells and fibroblasts .

I . miRNA - 150 lowered in plasma as a new indicator for potential diagnosis of atrial fibrillation

1 . Gene screening and verification of reduced expression of miRNA - 150 in patients with atrial fibrillation

In this study , five patients with isolated paroxysmal atrial fibrillation ( PAF ) were collected from plasma and 5 normal controls . Twenty - five normal controls were screened by depth - sequencing .

2 . Down - regulated miRNA - 150 expression is associated with AF

The results showed that 18 of the target genes of miRNA - 150 were associated with AF , 11 of them were associated with AF , 11 of them were associated with the inflammatory system , and inflammatory factors could not only be used as criteria for diagnosis of AF , but also involved in the pathogenesis of AF , and we detected the expression level of C - reactive protein ( CPR ) in plasma of patients , and found that it had negative correlation with the expression of miRNA - 150 ( correlation coefficient 0.77 ) .

II . Function of miRNA - 150 in regulating the function of monocytes in acute myocardial infarction ( AMI )

1 . Down regulation of miRNA - 150 in peripheral blood mononuclear cells after AMI

The expression of miRNA - 150 was downregulated in peripheral blood mononuclear cells ( PBMCs ) of 20 AMI patients and 10 normal controls , and no significant difference was found in peripheral blood mononuclear cells ( PBMCs ) of CD14 + monocytes .

2 . miRNA - 150 regulates the migration of human monocytes ( THP - 1 ) and the production of inflammatory factors

In vitro experiments were carried out in THP - 1 cell line transfected miRNA - 150 ( agomiRNA150 ) , inhibitor ( miRNA - 150 ) and control . The migration of THP - 1 was inhibited ( 3.7 鹵 0.5 times ) in high expression group compared with control group . In addition , THP - 1 inhibited migration ( 6.3 鹵 0.8 times ) . In addition , THP - 1 inhibited the production of LPS - 150 group and increased the expression of IL - 10 ( IL - 10 ) .
The suppression of miRNA - 150 groups yielded the opposite results .

3 . Up - regulate the expression of miRNA - 150 in monocytes and protect the myocardial function after AMI

Animal experiments show that in the stem mouse model of the high expression miRNA - 150 of the peripheral cells , the heart function is improved relative to the control group , the area of the myocardial infarction is reduced , the apoptosis of the myocardial cells is prevented , and the aggregation of the inflammatory monocytes is reduced ;
These protective effects were eliminated in mice transplanted with miRNA - 150 knockout mice bone marrow .

4 . miRNA - 150 regulates the expression of chemokine receptor 4 ( CXCR4 )

The results showed that CXCR4 was the target gene of miRNA - 150 , and in vitro experiments were carried out in THP - 1 cells transfected with agomiRNA - 150 and control fitting respectively . Western experiments showed that CXCR4 expression was down - regulated with respect to the control group ;
In mice with high expression of miRNA - 150 , CD115 + monocytes were isolated and CXCR4 expression was also found to be down - regulated with respect to the control group .

Conclusion

1 . miRNA - 150 reduced in plasma can be used as a new index for diagnosis of atrial fibrillation .


2 . miRNA - 150 regulates the function of monocytes , plays a protective role in myocardial injury caused by AMI , and provides a new research target for the treatment of acute myocardial infarction .
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R541.75;R542.22

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Mustafa Abdo Saif Dehwah;;MicroRNAs and Type 2 Diabetes/Obesity[J];遺傳學(xué)報;2012年01期

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本文編號：2094290