干细胞趋电性相关的microRNAs的表达量变化

发布时间：2018-04-03 05:29

  本文选题：microRNA　切入点：电场　出处：《浙江大学》2016年硕士论文

【摘要】：背景：干细胞是一类未分化的细胞,具有自我复制和多向分化潜能,被广泛地应用到临床治疗中。但是,干细胞定向迁移能力差制约了其在临床上的广泛应用。有研究报道内生性电场在胚胎发育和组织损伤修复过程中发挥重要作用,而外生性电场也可以指导一系列细胞的定向迁移,这些发现为解决干细胞定向迁移提供了新的思路。细胞的趋电性迁移与Ca2+、EGF受体及高尔基体极化等有关,受一系列趋电性相关基因(如PBKy和PTEN等)的调控。MicroRNAs是一类由内源基因编码的长度约为22个核苷酸的非编码单链小RNA分子,它具有高度保守的特性,广泛地存在于各种生物体中,对各种生理、病理过程中均发挥重要的调节作用。但目前关于细胞趋电性相关microRNAs尚无报道。目的：本研究探索直流电场对人胚胎干细胞H9株系(hESC, human embryonic stemcell)迁移的影响,筛选趋电性相关的microRNAs,并通过生物信息学进行靶基因的预测以及功能和通路分析。方法：50mV/mm,150 mV/mm和250 mV/mm直流电场分别刺激hESC 6小时,利用ImageJ软件追踪细胞的迁移情况,选取最适刺激强度进行后续研究。利用microRNA芯片(康成生物)筛选电场刺激后差异表达的microRNAs,并用qRT-PCR进行验证。通过Microcosm、Miranda和Targetsca n进行靶基因预测。采用GO分析和KEGG分析进行功能预测和通路预测。结果：在本实验条件下,直流电场指导hESC向正极迁移且迁移速度增加。在150mV/mm电场刺激6小时后,立即提取总RNA,芯片分析共检测到413个microRNAs的表达量,其中有4个microRNAs的表达量上调,hsa-miR-4321和hsa-miR-920的表达量上调超过3倍,qRT-PCR验证显示hsa-miR-4321和hsa-miR-920的表达量上调超过1.5倍。另外,靶基因预测显示hsa-miR-4321的靶基因有2个,分别为VCL和ITGAM, hsa-miR-920的靶基因有3个,分别为(GIMAP5, STIM1和KCNMB1。功能分析显示hsa-miR-4321可能与细胞粘附有关,hsa-miR-920可能与钙离子通道的调控有关。KEGG分析显示通路adherens junction可能参与细胞的趋电性。结论：在本实验条件下,直流电场(150mV/mm,6小时)可以指导人胚胎干细胞H9株系向正极迁移,上调hsa-miR-4321和hsa-miR-920的表达量。生物信息学分析表明hsa-miR-4321和hsa-miR-920分别与细胞粘附和钙离子通道的调控有关,电场可能通过hsa-miR-920对GIMAP5和STIM1的调控作用和通过hsa-miR-4321涉及的信号通路Adherens Junction指导人胚胎干细胞H9株系的定向迁移。
[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，

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