發(fā)布時(shí)間：2018-04-03 05:29

  本文選題：microRNA　切入點(diǎn)：電場(chǎng)　出處：《浙江大學(xué)》2016年碩士論文

【摘要】：背景：干細(xì)胞是一類未分化的細(xì)胞,具有自我復(fù)制和多向分化潛能,被廣泛地應(yīng)用到臨床治療中。但是,干細(xì)胞定向遷移能力差制約了其在臨床上的廣泛應(yīng)用。有研究報(bào)道內(nèi)生性電場(chǎng)在胚胎發(fā)育和組織損傷修復(fù)過程中發(fā)揮重要作用,而外生性電場(chǎng)也可以指導(dǎo)一系列細(xì)胞的定向遷移,這些發(fā)現(xiàn)為解決干細(xì)胞定向遷移提供了新的思路。細(xì)胞的趨電性遷移與Ca2+、EGF受體及高爾基體極化等有關(guān),受一系列趨電性相關(guān)基因(如PBKy和PTEN等)的調(diào)控。MicroRNAs是一類由內(nèi)源基因編碼的長(zhǎng)度約為22個(gè)核苷酸的非編碼單鏈小RNA分子,它具有高度保守的特性,廣泛地存在于各種生物體中,對(duì)各種生理、病理過程中均發(fā)揮重要的調(diào)節(jié)作用。但目前關(guān)于細(xì)胞趨電性相關(guān)microRNAs尚無報(bào)道。目的：本研究探索直流電場(chǎng)對(duì)人胚胎干細(xì)胞H9株系(hESC, human embryonic stemcell)遷移的影響,篩選趨電性相關(guān)的microRNAs,并通過生物信息學(xué)進(jìn)行靶基因的預(yù)測(cè)以及功能和通路分析。方法：50mV/mm,150 mV/mm和250 mV/mm直流電場(chǎng)分別刺激hESC 6小時(shí),利用ImageJ軟件追蹤細(xì)胞的遷移情況,選取最適刺激強(qiáng)度進(jìn)行后續(xù)研究。利用microRNA芯片(康成生物)篩選電場(chǎng)刺激后差異表達(dá)的microRNAs,并用qRT-PCR進(jìn)行驗(yàn)證。通過Microcosm、Miranda和Targetsca n進(jìn)行靶基因預(yù)測(cè)。采用GO分析和KEGG分析進(jìn)行功能預(yù)測(cè)和通路預(yù)測(cè)。結(jié)果：在本實(shí)驗(yàn)條件下,直流電場(chǎng)指導(dǎo)hESC向正極遷移且遷移速度增加。在150mV/mm電場(chǎng)刺激6小時(shí)后,立即提取總RNA,芯片分析共檢測(cè)到413個(gè)microRNAs的表達(dá)量,其中有4個(gè)microRNAs的表達(dá)量上調(diào),hsa-miR-4321和hsa-miR-920的表達(dá)量上調(diào)超過3倍,qRT-PCR驗(yàn)證顯示hsa-miR-4321和hsa-miR-920的表達(dá)量上調(diào)超過1.5倍。另外,靶基因預(yù)測(cè)顯示hsa-miR-4321的靶基因有2個(gè),分別為VCL和ITGAM, hsa-miR-920的靶基因有3個(gè),分別為(GIMAP5, STIM1和KCNMB1。功能分析顯示hsa-miR-4321可能與細(xì)胞粘附有關(guān),hsa-miR-920可能與鈣離子通道的調(diào)控有關(guān)。KEGG分析顯示通路adherens junction可能參與細(xì)胞的趨電性。結(jié)論：在本實(shí)驗(yàn)條件下,直流電場(chǎng)(150mV/mm,6小時(shí))可以指導(dǎo)人胚胎干細(xì)胞H9株系向正極遷移,上調(diào)hsa-miR-4321和hsa-miR-920的表達(dá)量。生物信息學(xué)分析表明hsa-miR-4321和hsa-miR-920分別與細(xì)胞粘附和鈣離子通道的調(diào)控有關(guān),電場(chǎng)可能通過hsa-miR-920對(duì)GIMAP5和STIM1的調(diào)控作用和通過hsa-miR-4321涉及的信號(hào)通路Adherens Junction指導(dǎo)人胚胎干細(xì)胞H9株系的定向遷移。
[Abstract]:Background: stem cells are undifferentiated cells with self-replication and multi-differentiation potential and are widely used in clinical therapy.However, the poor ability of directional migration of stem cells restricts its wide application in clinic.It has been reported that endogenous electric field plays an important role in embryonic development and tissue damage repair, but exogenous electric field can also direct the directional migration of a series of cells. These findings provide a new way to solve the directional migration of stem cells.The electrophilic migration of cells is related to the Ca2 receptor and Golgi body polarization. It is regulated by a series of electrophilic related genes (such as PBKy and PTEN, etc.). MicroRNAs are a class of unencoded single-stranded small RNA molecules encoded by endogenous genes with a length of about 22 nucleotides.It has a highly conservative character and widely exists in various organisms and plays an important regulatory role in various physiological and pathological processes.However, there are no reports on microRNAs related to cellular electrotaxis.Aim: to investigate the effect of direct current field on the migration of human embryonic stem cell (human embryonic stem cell) of human embryonic stem cells (H9), and to screen the microRNASs associated with electrophilicity, and to predict the target gene and analyze the function and pathway of the target gene by bioinformatics.Methods the hESC was stimulated by 50 MV / mm 50 mV/mm and 250 mV/mm DC field for 6 hours respectively. The cell migration was tracked by ImageJ software and the optimal stimulation intensity was selected for follow-up study.MicroRNA chip was used to screen differentially expressed microRNas after electric field stimulation, which was verified by qRT-PCR.The target genes were predicted by microcosm Miranda and Targetsca n.Go analysis and KEGG analysis were used to predict function and path.Results: under the experimental conditions, the direct current field guided hESC to positive pole migration and the migration speed increased.鍦，

本文編號(hào)：1703850