中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2010年
10期
1711-1715
,共5页
王世华%边春景%黄珊%赵春华
王世華%邊春景%黃珊%趙春華
왕세화%변춘경%황산%조춘화
microRNA-125b%脂肪源间充质干细胞%神经分化%miRNA%miRNA芯片
microRNA-125b%脂肪源間充質榦細胞%神經分化%miRNA%miRNA芯片
microRNA-125b%지방원간충질간세포%신경분화%miRNA%miRNA심편
背景:间充质干细胞的分化受遗传和表观遗传因素的严格调控,越来越多的研究表明microRNA也在这一过程中发挥重要的调控作用.目的:观察microRNA-125b在Flk1~+ 1间充质干细胞向神经细胞分化过程中的表达变化.方法:成人脂肪样品取自15~35岁健康志愿者,贴壁培养法获得间充质干细胞,采用神经诱导培养基对第3代间充质干细胞进行诱导培养.在诱导第0,4,8,12天检测microRNA-125b的表达情况.应用microRNA芯片技术检测间充质干细胞在神经诱导前后microRNA-125b的表达差异并采用反转录-聚合酶链反应和Taqman real-time PCR的方法进行验证.检测inhibitor抑制microRNA-125b表达的有效性.结果与结论:①实验成功诱导间充质干细胞向神经分化,诱导培养后12 d观察到细胞形态发生变化;大部分细胞胞体回缩,呈球形,并有轴突形成,免疫荧光检测结果显示,分化后的细胞表达神经元、星形胶质细胞特征性表面标志.②RT-PCR和Taqman real-time PCR显示神经诱导后microRNA-125b表达明显升高,与microRNA芯片结果一致.③inhibitor可以有效抑制microRNA-125b的表达.提示microRNA-125b可能在间充质干细胞成神经分化过程中起调控作用.
揹景:間充質榦細胞的分化受遺傳和錶觀遺傳因素的嚴格調控,越來越多的研究錶明microRNA也在這一過程中髮揮重要的調控作用.目的:觀察microRNA-125b在Flk1~+ 1間充質榦細胞嚮神經細胞分化過程中的錶達變化.方法:成人脂肪樣品取自15~35歲健康誌願者,貼壁培養法穫得間充質榦細胞,採用神經誘導培養基對第3代間充質榦細胞進行誘導培養.在誘導第0,4,8,12天檢測microRNA-125b的錶達情況.應用microRNA芯片技術檢測間充質榦細胞在神經誘導前後microRNA-125b的錶達差異併採用反轉錄-聚閤酶鏈反應和Taqman real-time PCR的方法進行驗證.檢測inhibitor抑製microRNA-125b錶達的有效性.結果與結論:①實驗成功誘導間充質榦細胞嚮神經分化,誘導培養後12 d觀察到細胞形態髮生變化;大部分細胞胞體迴縮,呈毬形,併有軸突形成,免疫熒光檢測結果顯示,分化後的細胞錶達神經元、星形膠質細胞特徵性錶麵標誌.②RT-PCR和Taqman real-time PCR顯示神經誘導後microRNA-125b錶達明顯升高,與microRNA芯片結果一緻.③inhibitor可以有效抑製microRNA-125b的錶達.提示microRNA-125b可能在間充質榦細胞成神經分化過程中起調控作用.
배경:간충질간세포적분화수유전화표관유전인소적엄격조공,월래월다적연구표명microRNA야재저일과정중발휘중요적조공작용.목적:관찰microRNA-125b재Flk1~+ 1간충질간세포향신경세포분화과정중적표체변화.방법:성인지방양품취자15~35세건강지원자,첩벽배양법획득간충질간세포,채용신경유도배양기대제3대간충질간세포진행유도배양.재유도제0,4,8,12천검측microRNA-125b적표체정황.응용microRNA심편기술검측간충질간세포재신경유도전후microRNA-125b적표체차이병채용반전록-취합매련반응화Taqman real-time PCR적방법진행험증.검측inhibitor억제microRNA-125b표체적유효성.결과여결론:①실험성공유도간충질간세포향신경분화,유도배양후12 d관찰도세포형태발생변화;대부분세포포체회축,정구형,병유축돌형성,면역형광검측결과현시,분화후적세포표체신경원、성형효질세포특정성표면표지.②RT-PCR화Taqman real-time PCR현시신경유도후microRNA-125b표체명현승고,여microRNA심편결과일치.③inhibitor가이유효억제microRNA-125b적표체.제시microRNA-125b가능재간충질간세포성신경분화과정중기조공작용.
BACKGROUND:The differentiation of mesenchymal stem cells(MSCs)is strictly regulated by both genetic and epigenetic Factors .Emerging evidences have demonstrated that microRNA also plays an important role in this process.OBJECTIVE:To explore the differential expression of microRNA-125b during neuronal dliferentiation of Flk1~+ adipose-derived MSCs(AD-MSCs).MATERIALS:The fat samples were provided bv healthy female volunteers aged 15-35 years and recruited from the Plastic Surgery Hospital affiliated to the Chinese Academy of Medical Sciences.METHODS:Adult adipose tissues were obtained from healthy voluntears with age of 15-35 years .Using adherence method,Flk1~+ MSCs were obtained and the 3~(rd) passage cells were taken in the experiment.Cultured in neuronalinduction medium.these MSC were induced to differentiate towards neuronal lineage.The expression of microRNA-125b was examined at days 0,4,8 and 12.To explore its role in neuronal differentiation,we need to change its expression.RT-PCR and Taqman real-time PCR were carried out to explore the difierentially expression of microRNA-125b during neuronal differentiation of AD-MSCs.The effect of inhibitor on the expression of microRNA-125b was detected.RESULTS AND CONCLUTION:①The Flk-1~+ MSCs were successfully induced into neuronal differentiation and displayed typical morphological changes 12 days after induction:Most cells retracted their cytoplasm ,fomling spherical cell body and emitted cellular protrusions.RT-PCR and immunocytochemistry studies confirmed their phenotype with expression of known neuronal cell markers including neurofilament,glial fibrillary acidic protein.②The expression of microRNA-125b was significant up-regulated during neuronal differentiation .Results of RT-PCR and Taqman real-time PCR were concordarlce with that of microRNA chip technology.③Inhibitor could down-regulate microRNA-125b.The results implied that microRNA-125b may play an important role in neuronal differentiation.