人骨髓间充质干细胞与其来源的汗腺样细胞微小RNAs差异表达谱分析  

Differential expression profile of microRNAs between human bone marrow mesenchymal stem cells and sweat gland-like cells

作　　者：谭志军[1] 陈艳[2] 赵洪良[2] 赵换军[1] 赵阿龙[1] 赵智亮[2] 孙梦黎[2] 马奎[2] 张翠萍[2] 付小兵[2] 

机构地区：[1]天津医科大学研究生院,300070 [2]解放军总医院第一附属医院全军创伤修复与组织再生重点实验室

出　　处：《中华医学杂志》2015年第32期2626-2629,共4页National Medical Journal of China

基　　金：国家自然科学基金（81421064,81230041,81171798）

摘　　要：目的 分析入骨髓间充质干细胞（hBM-MSCs）与其来源的汗腺样细胞微小RNAs（miRNAs）差异表达谱.方法 采用成骨、成脂细胞诱导分化液诱导hBM-MSCs分化,油红O和茜素红染色分别进行鉴定.当原代汗腺细胞密度达到70％～ 80％时进行热休克处理,然后将其与hBM-MSCs通过Transwell进行共培养诱导;10 d后分别提取hBM-MSCs和其诱导分化的汗腺样细胞的总RNA,纯化标记后,进行miRNAs杂交和数据分析以及靶基因预测.进一步采用实时荧光定量PCR验证芯片结果的可靠性.结果 差异表达水平绝对值＞2倍的miRNAs有68个,其中上调的19个,下调的49个;其中13个表达水平发生显著变化,差异表达水平＞5倍（miRNA-132-3p、-4467、-4484、-146a-5p、-6126等5个上调;miRNA-708-5p、-138-5p、-6812-5p、-138-1-3p、-1281、-3157-3p、-4298、-4459等8个下调）.靶向汗腺发育相关通路核因子κB、丝裂原活化蛋白激酶/细胞外信号调节激酶、Wnt/β-连环蛋白和外胚叶发育不全基因/外胚叶发育不全基因受体的miRNAs有18个.其中上调的miRNA-146a-5p和下调的miRNA-6812-5p验证结果与芯片结果具有一致性.结论 hBM-MSCs与其来源的汗腺样细胞之间有明显的miRNAs差异性表达,其中13个差异表达＞5倍的miRNAs和18个与汗腺发育通路有关的miRNAs可能在hBM-MSCs向汗腺样细胞分化过程中有重要的调控作用.Objective To explore the differential microRNAs （miRNAs） expression profiles between human bone marrow mesenchymal stem cells （hBM-MSCs） and sweat gland-like cells.Methods The hBM-MSCs were induced to differentiate into osteogenic and adipogenic cells by related osteogenic differentiation basal medium and adipogenic differentiation basal medium respectively.And the results were assayed by oil red O and alizarin red stains respectively.When reaching 70%-80% confluence,primarily cultured sweat gland cells were heat-shocked and co-cultured with hBM-MSCs by a Transwell plate to induce the differentiation of hBM-MSCs.After 10 days,the total RNAs of hBM-MSCs and sweat gland-like cells were extracted,purified,labeled,hybridized and analyzed for predicting miRNAs target genes separately.The microarray results were confirmed by quantitative real-time reverse transcription-polymerase chain reaction （RT-PCR）.Results There were 68 miRNAs of 〉 double differentiation on expression level between hBM-MSCs and sweat gland-like cells.And 19 miRNAs were up-regulated and 49 miRNAs down-regulated.Moreover,there were 13 miRNAs with 〉 5 folds of differential expression level between hBM-MSCs and sweat gland-like cells,including 5 up-regulated miRNAs （miRNA-132-3p,-4467,-4484,-146a-5p and-6126） and 8 down-regulated miRNAs （miRNA-708-5p,-138-5p,-6812-5p,-138-1-3p,-1281,-3157-3 p,-4298 and-4459）.There were 18 miRNAs related to regulation of the signaling pathways of sweat gland development,including nuclear factor-kappa B （NF-κB）,mitogen-activated protein kinase/extracellular regulated protein kinase （MAPK/Erk）,Wnt/beta-Catenin （Wnt/β-Catenin） and ectodysplasin-A1/ectodysplasin-A1 receptor （EDA/EDAR）.The results of RT-PCR on miRNA-146a-5p and miRNA-6812-5p had a high concordance with the results of microarray.Conclusions There are obvious differentiation miRNAs expression profiles between hBM-MSCs and sweat gland-like cells.And 13 miRNAs 〉 5 folds of differential expression and 18 miRNAs related

关 键 词：间质干细胞 汗腺 微RNAS 基因表达谱 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]