基于高通量测序的离体人表皮干细胞热损伤后microRNAs表达谱分析

发布时间：2018-05-29 23:24

  本文选题：表皮干细胞 + 热损伤　； 参考：《南昌大学》2017年硕士论文

【摘要】：目的:探讨离体人表皮干细胞热损伤后的微小RNA(miRNA)的差异表达;并预测差异表达miRNA的靶基因及其参与的信号通路,为进一步研究创面愈合的机制及相关靶向治疗提供新的线索。方法:(1)应用胰酶消化法分离并培养离体人原代表皮干细胞。(2)将细胞随机分为2组,将实验组细胞置于51.5℃水箱中孵育35s,对照组细胞置于37℃恒温水箱中孵育35s,放置于倒置相差显微镜下,观察两组细胞的形态及生长情况。37℃培养箱中再培养6h后收集细胞样本,用于提取总RNA。(3)应用TRIzol法提取两组样本细胞的总RNA,随后采用RNeasy Mini Kit对RNA进行纯化及质检,以1ug Total RNA≥50ng small RNA起始量进行文库构建,并用Agilent 2200TapeStation进行文库质检;质检后文库,按照HiSeq 2500操作指南中要求制备上机样本;使用Single Read Flow Cell,按照Hi Seq 2500 User Guide的指示进行Hi Seq 2500上机测序;得到测序结果;(4)对差异表达mi RNAs进行miRNA家族分析及相关生物信息学分析。。结果:(1)倒置相差显微镜下观察可见,表皮干细胞刚接种时贴壁牢固,形状小而圆,折光性好。培养2d后,细胞克隆生长较快,贴壁牢固,对照组37℃水浴后细胞数量未见明显减少,细胞外形呈类圆形,贴壁牢固。实验组53.5℃水浴后细胞数量明显减少,细胞呈不规则外形,贴壁欠牢固;(2)测序结果显示,两组细胞差异表达的miRNA共54个,其中表达上调的33个,表达下调的21个。其中上调最明显的为hsa-mi R-1973,下调最明显的为hsa-miR-4520-5p。差异表达明显上调的miRNA还有hsa-miR-4485-3p,hsa-miR-548j-5p,hsa-miR-212-3p,hsa-miR-4461;下调的miRNA有hsa-miR-4661-5p,hsa-miR-191-3p,hsa-miR-129-5p,hsa-miR-147b,hsa-mi R-6868-3p;(3)通过对显著差异表达的miRNA家族分析,探索其所属的miRNA家族的分布情况,结果显示差异miRNA家族为:mir-4520,mir-548,mir-132,mir-4510,mir-191,mir-129,mir-549,mir-154,mir-7641,mir-1976,mir-147,mir-6511,mir-154,mir-744,mir-1287,mir-3064,mir-1295,mir-1248,mir-181,mir-193,mir-1294,mir-149,mir-515,mir-506,mir-338,mir-17,mir-486,mir-431,mir-4504,mir-29,mir-134,mir-221,mir-194,mir-6511;miRNAs靶基因预测及相关生物信息学分析结果提示,差异表达的miRNAs参与细胞增殖分化、生长凋亡、粘附迁移等生物学过程。结论:1、离体人表皮干细胞热损伤后miRNAs表达谱存在明显差异;2、差异表达的miRNAs可能参与调控创面愈合过程。
[Abstract]:Objective: to investigate the differential expression of minimal RNA-miRNAs after heat injury of human epidermal stem cells in vitro and to predict the target genes and signal pathways involved in the differential expression of miRNA in order to provide a new clue for further study on the mechanism of wound healing and related targeted therapy. Methods the cells were isolated by trypsin digestion and cultured in vitro. The cells were randomly divided into two groups. The cells of the experimental group were incubated in water tank at 51.5 鈩，

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