人脂肪来源干细胞成软骨分化过程中成软骨相关微小RNA表达的初步研究  

CHONDROGENESIS-SPECIFIC MICRORNA EXPRESSION PATTERN ANALYSIS IN CHONDROGENIC DIFFERENTIATION OF HUMAN ADIPOSE-DERIVED STEM CELLS

作　　者：张紫机[1] 康焱[1] 张志奇[1] 杨子波[1] 方淑莺[1] 盛璞义[1] 何爱珊[1] 傅明[1] 廖威明[1] 

机构地区：[1]中山大学附属第一医院关节外科,广州510080

出　　处：《中国修复重建外科杂志》2015年第1期74-80,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81171709;81301558;81371941;81201388);教育部高等学校博士点基金新教师类资助项目(20130171120074);广东省自然科学基金博士启动项目(S2013040016269)~~

摘　　要：目的探讨微小RNA(micro RNA,mi RNA)在人脂肪来源干细胞(human adipose-derived stem cells,h ADSCs)诱导成软骨分化过程中的表达规律及其影响软骨分化的可能机制。方法取行抽脂术或其他腹部手术患者自愿捐赠的脂肪组织,分离、培养h ADSCs并鉴定。取第3代细胞成软骨分化,倒置相差显微镜下观察细胞形态,于诱导21 d行阿尔新蓝染色观察软骨形成情况,诱导0、7、14、21 d行ELISA检测成软骨相关蛋白Ⅱ型胶原蛋白(collagen typeⅡ,Col2a1)、蛋白聚糖(Aggrecan)、Col10a1以及硫酸软骨素表达。采用基因芯片技术筛选h ADSCs成软骨诱导前及诱导后21 d差异性表达mi RNA,并预测筛选出的mi RNA靶基因。结果实验成功培养h ADSCs,经成软骨诱导培养后,随时间延长可形成软骨球;21 d阿尔新蓝染色呈阳性;h ADSCs成软骨诱导后7、14、21 d,Col2a1、Aggrecan、Col10a1及硫酸软骨素表达水平均较成软骨诱导前h ADSCs显著增高,差异均有统计学意义(P<0.05)。基因芯片技术共筛选出11个差异性表达mi RNA,其中7个mi RNA表达上调,4个mi RNA表达下调。筛选出的成软骨相关mi RNAs预测靶基因可能参与了干细胞成软骨分化、增殖、凋亡、细胞周期调控,以及介导细胞内级联反应和自我更新等。结论实验筛选出11个成软骨分化差异性表达超过2倍的mi RNAs,并对其靶基因进行预测,加深了对h ADSCs成软骨分化机制的理解,为定向控制h ADSCs成软骨分化及筛选组织工程改良种子细胞提供了理论依据。Objective To investigate the microRNA（miRNA） expression profile during chondrogenic differentiation of human adipose-derived stem cells（hADSCs）,and assess the roles of involved miRNAs during chondrogenesis. Methods h ADSCs were harvested and cultured from donors who underwent elective liposuction or other abdominal surgery. When the cells were passaged to P3,chondrogenic induction medium was used for chondrogenic differentiation. The morphology of the cells was observed by inverted phase contrast microscopy. Alcian blue staining was carried out at 21 days after induction to access the chondrogenic status. The expressions of chondrogenic proteins were detected by ELISA at 0,7,14,and 21 days. The miRNA expression profiles at pre- and post-chondrogenic induction were obtained by microarray assay,and differentially expressed miRNAs were verified by real-time quantitative PCR（q RT-PCR）. The targets of the miRNAs were predicted by online software programs. Results hADSCs were cultured successfully and induced with chondrogenic medium. At 21 days after chondrogenic induction,the cells were stained positively for alcian blue staining. At 7,14,and 21 days after chondrogenic induction,the levels of collogen type II,Col2a1,aggrecan,Col10a1,and chondroitin sulfate in induced h ADSCs were significantly higher than those in noninduced h ADSCs（P〈0.05）. Eleven differentially expressed miRNAs were found,including seven up-regulated and four down-regulated. Predicted target genes of the differentially expressed miRNAs were based on the overlap from three public prediction algorithms,with the known functions of regulating chondrogenic differentiation of stem cells,selfrenewal,signal transduction,intracellular signaling cascade,and cell cycle control. Conclusion A group of mi RNAs and their target genes are identified,which may play important roles in regulating chondrogenic differentiation of hADSCs. These results will facilitate the initial understanding of the molecular mechanism of chondrogenic differentiation

关 键 词：微小RNA 人脂肪来源干细胞 成软骨分化 基因芯片 靶基因 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]