摘要
韧带成纤维细胞成骨分化与强直性脊柱炎等多种异位骨化相关性疾病有关,但其机制尚不清楚.本研究目的在于观察韧带成纤维细胞在成骨分化过程中microRNA和mRNA的表达谱变化,以期为揭示成纤维细胞成骨分化机制提供研究基础.原代培养韧带成纤维细胞、地塞米松、抗坏血酸和β-磷酸甘油体外诱导其成骨分化,RT-PCR检测成骨标志物骨钙素和Runx2的表达.采用表达谱基因芯片分析诱导0、7、14 d后韧带成纤维细胞的microRNAs和mRNAs表达,并用实时定量PCR(RT-qPCR)和Western印迹方法验证生物信息学分析结果.结果显示,与未诱导前相比,成骨分化第7 d有66个microRNAs和640个mRNA表达上调,94个microRNAs和744个mRNA表达下调;成骨分化第14 d有58个microRNAs和781个mRNA表达上调,96个microRNAs和603个mRNA表达下调.实时定量PCR和Western印迹验证结果显示,miR-29b在成骨分化过程中表达上调,TGFβ3表达水平降低,与芯片结果一致,miR-29b通过抑制TGFβ3蛋白的翻译来促进Runx2的表达,从而促进韧带成纤维细胞向成骨细胞分化.韧带成纤维细胞成骨分化过程中,microRNA调控基因及mRNA差异表达基因除涉及BMPs、Wnt和Ihh等信号通路外,在成纤维细胞成骨分化过程中可能还存在其它的新机制.
The osteogenic differentiations of ligament fibroblasts are closely associated with ectopic ossification-related diseases,such as ankylosing spondylitis,but the exact mechanism remained unclear.To evaluate the microRNA and mRNA expression profiling of ligament fibroblasts during its osteogenic differentiation,fibroblasts from ligments were isolated and cultured in osteogenic medium containing dexamethasone,ascorbic acid and β-glycerol phosphate.Two osteoblastic markers,Osteocalcin and Runx2,were detected by RT-PCR at day 0,7,and 14.Microarray analyses were performed to profile the expression of microRNAs and mRNAs during the process of osteogenic differentiation.Real-time quantitative PCR（RT-qPCR） and Western blotting were used to for further confirmation following bioinformatic analyses.The results showed that 66 microRNAs and 640 mRNA were up-regulated;94 microRNAs and 744 mRNA down-regulated at day 7.At day 14,58 microRNAs and 781 mRNA were up-regulated,and 96 microRNAs and 603 mRNA down-regulated.The RT-qPCR and Western blot confirmed that miR-29b were up-regulated,while TGFβ3 expression down-regulated.As miR-29b can interact with the 3′-UTR of TGFβ3 mRNA to block its translation,and TGFβ3 suppresses osteoblastic differentiation by either reduce Runx2 cellular protein and activity or induce to undergo Smurf-mediated Runx2 degradation,thus lead to the promotion of osteoblastic differentiation.The microRNAs and genes changed during fibroblast osteogenic differentiation were mainly involved in BMPs,Wnt and Ihh signal pathways,suggesting that besides of some unique characteristics in the gene expression profile,ligment fibroblasts might share a common pathway with osteoblasts during osteogenic differentiation.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2011年第6期540-547,共8页
Chinese Journal of Biochemistry and Molecular Biology
