發(fā)布時間：2018-05-29 23:24

  本文選題：表皮干細胞 + 熱損傷��； 參考：《南昌大學》2017年碩士論文

【摘要】：目的:探討離體人表皮干細胞熱損傷后的微小RNA(miRNA)的差異表達;并預測差異表達miRNA的靶基因及其參與的信號通路,為進一步研究創(chuàng)面愈合的機制及相關靶向治療提供新的線索。方法:(1)應用胰酶消化法分離并培養(yǎng)離體人原代表皮干細胞。(2)將細胞隨機分為2組,將實驗組細胞置于51.5℃水箱中孵育35s,對照組細胞置于37℃恒溫水箱中孵育35s,放置于倒置相差顯微鏡下,觀察兩組細胞的形態(tài)及生長情況。37℃培養(yǎng)箱中再培養(yǎng)6h后收集細胞樣本,用于提取總RNA。(3)應用TRIzol法提取兩組樣本細胞的總RNA,隨后采用RNeasy Mini Kit對RNA進行純化及質檢,以1ug Total RNA≥50ng small RNA起始量進行文庫構建,并用Agilent 2200TapeStation進行文庫質檢;質檢后文庫,按照HiSeq 2500操作指南中要求制備上機樣本;使用Single Read Flow Cell,按照Hi Seq 2500 User Guide的指示進行Hi Seq 2500上機測序;得到測序結果;(4)對差異表達mi RNAs進行miRNA家族分析及相關生物信息學分析。。結果:(1)倒置相差顯微鏡下觀察可見,表皮干細胞剛接種時貼壁牢固,形狀小而圓,折光性好。培養(yǎng)2d后,細胞克隆生長較快,貼壁牢固,對照組37℃水浴后細胞數(shù)量未見明顯減少,細胞外形呈類圓形,貼壁牢固。實驗組53.5℃水浴后細胞數(shù)量明顯減少,細胞呈不規(guī)則外形,貼壁欠牢固;(2)測序結果顯示,兩組細胞差異表達的miRNA共54個,其中表達上調(diào)的33個,表達下調(diào)的21個。其中上調(diào)最明顯的為hsa-mi R-1973,下調(diào)最明顯的為hsa-miR-4520-5p。差異表達明顯上調(diào)的miRNA還有hsa-miR-4485-3p,hsa-miR-548j-5p,hsa-miR-212-3p,hsa-miR-4461;下調(diào)的miRNA有hsa-miR-4661-5p,hsa-miR-191-3p,hsa-miR-129-5p,hsa-miR-147b,hsa-mi R-6868-3p;(3)通過對顯著差異表達的miRNA家族分析,探索其所屬的miRNA家族的分布情況,結果顯示差異miRNA家族為: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靶基因預測及相關生物信息學分析結果提示,差異表達的miRNAs參與細胞增殖分化、生長凋亡、粘附遷移等生物學過程。結論:1、離體人表皮干細胞熱損傷后miRNAs表達譜存在明顯差異;2、差異表達的miRNAs可能參與調(diào)控創(chuàng)面愈合過程。
[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，

本文編號：1952882