微渠多孔羟基磷灰石支架诱导骨髓间充质干细胞成骨分化的miRNA表达谱分析  

miRNA expression profiling of osteogenic differentiation of bone marrow mesenchymal stem cells induced by microchannel porous hydroxyapatite scaffold

作　　者：郑佳俊 青薇 黄丽娟 任静 刘春晖 彭湃然 黄杰 牟雁东 Zheng Jiajun;Qing Wei;Huang Lijuan;Ren Jing;Liu Chunhui;Peng Pairan;Huang Jie;Mu Yandong(School of Stomatology,Southwest Medical University,Luzhou 646000,Sichuan Province,China;School of Medicine,University of Electronic Science and Technology,Chengdu 610000,Sichuan Province,China)

机构地区：[1]西南医科大学口腔医学院,四川省泸州市646000 [2]电子科技大学医学院,四川省成都市610000

出　　处：《中国组织工程研究》2020年第13期1989-1995,共7页Chinese Journal of Tissue Engineering Research

基　　金：四川省科技厅项目(2016TD0008)，项目负责人：牟雁东~~

摘　　要：背景:多孔羟基磷灰石支架具有良好的体内外成骨效能,但其所涉及的mi RNAs复杂调控机制相关研究较少。目的:探讨多孔羟基磷灰石支架材料介导大鼠骨髓间充质干细胞成骨矿化过程中相关miRNA表达谱的变化。方法:体外分离、培养和鉴定大鼠骨髓间充质干细胞,将骨髓间充质干细胞与多孔羟基磷灰石支架共培养为实验组,骨髓间充质干细胞单独培养为空白对照组,分别进行成骨诱导7 d,运用miRNA高通量测序技术分析两组骨髓间充质干细胞成骨矿化过程中相关miRNA表达谱的变化并进行GO分析,筛选出两组中表达差异明显的mi RNA分子并进行q RT-PCR验证。结果与结论:①与空白对照组比较,成骨诱导7 d时实验组BMP2、ALP、Runx2 mRNA表达上调,其中BMP2上调明显(P <0.05);②micro RNA高通量测序结果显示mi R-210-3p、mi R-146a-5p等13个mi RNAs明显上调;let-7c-3p、let-3615等17个mi RNAs明显下调;③GO分析上调的mi RNA靶基因主要参与生物学调节、细胞基因表达、基因表达调节等,包括NF-κB、Toll样受体9、细胞间黏附、白细胞介素1调节、血管生成、Hippo等信号通路;④实时荧光定量q PCR验证结果显示mi RNA-210在实验组上调15倍,mi R-146a-5p在实验组上调10倍(P <0.05);⑤结果表明,新型微渠多孔羟基磷灰石支架可以通过上调骨髓间充质干细胞mi RNA-210-3p和mi R-146a表达,促进骨髓间充质干细胞的成骨分化。BACKGROUND: Porous hydroxyapatite scaffolds have good osteogenesis in vivo and in vitro. However, little research has been done on the complex regulation mechanisms of miRNAs involved. OBJECTIVE: To investigate the changes of related miRNA expression in rat bone marrow mesenchymal stem cells during osteogenic mineralization by porous hydroxyapatite scaffolds. METHODS: Rat bone marrow mesenchymal stem cells were isolated, cultured and identified in vitro. Bone marrow mesenchymal stem cells co-cultured with porous hydroxyapatite scaffold were as experimental group, and bone marrow mesenchymal stem cells cultured alone served as blank control group, both of which underwent osteogenic induction for 7 days. During the osteogenic mineralization, miRNA high-throughput sequencing technology was used to analyze the changes of miRNA expression profiles followed by GO analysis. The miRNA molecules with obvious expression differences were screened and verified by qRT-PCR. RESULTS AND CONCLUSION:(1) Compared with the blank control group, in the experimental group, the expression levels of BMP2, ALP and Runx2 mRNA were up-regulated, and the expression level of BMP2 was up-regulated significantly(P < 0.05).(2) Results of miRNA high-throughput sequencing showed that 13 miRNAs such as miR-210-3 p and miR-146 a-5 p were up-regulated, and 17 miRNAs such as let-7 c-3 p and let-3615 were down-regulated significantly.(3) GO analysis revealed that up-regulated miRNA target genes were mainly involved in biological regulation, cellular gene expression, and gene expression regulation, mainly including nuclear factor-κB, Toll-like receptor 9, intercellular adhesion, interleukin-1 regulation, and signaling pathways such as angiogenesis and Hippo.(4) Real-time fluorescence quantitative qPCR results showed that miRNA-210 was up-regulated 15 times and miR-146 a-5 p was up-regulated 10 times in the experimental group(P < 0.05). These results indicate that the new microchannel porous hydroxyapatite scaffold can promote the differentiation of bo

关 键 词：羟基磷灰石 骨髓间充质干细胞 MIRNA 功能分析 转录组测序 成骨诱导分化 MIR-210 MIR-146A 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]