CAJ | 학술논문

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.
목적 분석입골수간충질간세포(hBM-MSCs)여기래원적한선양세포미소RNAs(miRNAs)차이표체보.방법 채용성골、성지세포유도분화액유도hBM-MSCs분화,유홍O화천소홍염색분별진행감정.당원대한선세포밀도체도70％～ 80％시진행열휴극처리,연후장기여hBM-MSCs통과Transwell진행공배양유도;10 d후분별제취hBM-MSCs화기유도분화적한선양세포적총RNA,순화표기후,진행miRNAs잡교화수거분석이급파기인예측.진일보채용실시형광정량PCR험증심편결과적가고성.결과 차이표체수평절대치＞2배적miRNAs유68개,기중상조적19개,하조적49개;기중13개표체수평발생현저변화,차이표체수평＞5배(miRNA-132-3p、-4467、-4484、-146a-5p、-6126등5개상조;miRNA-708-5p、-138-5p、-6812-5p、-138-1-3p、-1281、-3157-3p、-4298、-4459등8개하조).파향한선발육상관통로핵인자κB、사렬원활화단백격매/세포외신호조절격매、Wnt/β-련배단백화외배협발육불전기인/외배협발육불전기인수체적miRNAs유18개.기중상조적miRNA-146a-5p화하조적miRNA-6812-5p험증결과여심편결과구유일치성.결론 hBM-MSCs여기래원적한선양세포지간유명현적miRNAs차이성표체,기중13개차이표체＞5배적miRNAs화18개여한선발육통로유관적miRNAs가능재hBM-MSCs향한선양세포분화과정중유중요적조공작용.
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 to sweat gland development may play essential roles in regulated differentiation of hBM-MSCs into sweat gland-like cells.