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

文题释义： 羟基磷灰石：是目前最为理想的生物活性材料，具有生物相容性、骨传导性和骨诱导性，植入人体后对组织无刺激和排斥作用，能与骨形成很强的化学结合，用作骨缺损的充填材料，能为新骨的形成提供支架，发挥骨传导作用，是理想的硬组织替代材料。 MicroRNA(miRNA)：是一类内生的、长度为20-24个核苷酸的单链小分子RNA，由具有发夹结构的70-90个碱基大小的单链RNA前体经过Dicer酶加工后生成，其可以通过几个miRNAs的组合在转录后水平精细调控基因的表达。 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miRNAs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miRNA分子并进行qRT-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)；②microRNA高通量测序结果显示miR-210-3p、miR-146a-5p等13个miRNAs明显上调；let-7c-3p、let-3615等17个miRNAs明显下调；③GO分析上调的miRNA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qPCR验证结果显示miRNA-210在实验组上调15倍，miR-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miRNA-210-3p和miR-146a表达，促进骨髓间充质干细胞的成骨分化。 ORCID: 0000-0002-8722-1548(郑佳俊) 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

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

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 bone marrow mesenchymal stem cells by up-regulating miRNA-210-3 p and miR-146a.