摘要
目的:研究miR-34a对人颌骨骨髓间充质干细胞(hOBMSCs)成骨分化的影响。方法:实时定量RT-PCR检测miR-34家族在hOBMSCs成骨诱导过程中的表达情况;采用碱性磷酸酶及茜素红染色检测miR-34a对于hOBMSCs成骨分化能力的影响;Western blot检测hOBMSCs成骨分化过程中miR-34a对于成骨相关蛋白的影响,同时检测hOBMSCs在正常培养条件下miR-34a对于DKK1蛋白表达的影响;双荧光素酶报告基因检测miR-34a与DKK1的靶向关系;通过裸鼠皮下成骨实验检测miR-34a对于hOBMSCs体内成骨的影响。结果:在hOBMSCs成骨诱导过程中,miR-34家族的miRNA表达量均显著上升(P<0.05),其中miR-34a最为明显;过表达miR-34a可显著提高hBMSCs的体外成骨能力;miR-34a可以负向调节DKK1,同时双荧光素酶报告基因实验证实DKK1为miR-34a的靶基因;体内实验同样证实miR-34a可以促进hOBMSCs的成骨分化。结论:miR-34a可能通过抑制DKK1的表达,促进hOBMSCs的成骨分化。
Objective:To investigate the effect of miR-34a on osteogenic differentiation of hOBMSCs.Methods:Real-time quantitative RT-PCR was used to detect the expression of miR-34a/miR-34b/miR-34c in hOBMSCs during osteogenic induction.The effect of miR-34a on osteogenic differentiation of hOBMSCs were detected by alkaline phosphatase staining and alizarin red staining.The regulation role of miR-34a on the expression of osteogenic markers(RUNX2,OSX,OPN,OCN,COL1)during osteogenic induction and the effect of miR-34a on DKK1 protein expression under normal cell culture conditions were detected by westernblot.The target relationship of miR-34a and DKK1 was measured by dual luciferase reporter assay.The effect of miR-34a on osteogenesis of hOBMSCs was examined by subcutaneous osteogenesis in nude mice.Results:MiR-34a was demonstrated to be upregulated during the osteogenic differentiation of hOBMSCs.Overexpression of miR-34a significantly increased alkaline phosphatase activity,mineralization capacity,and the expression of osteogenesis-associated genes in hOBMSCs in vitro.Further investigations revealed that miR-34a inhibited the expression of DKK1,and dual-luciferase reporter gene assay confirmed that DKK1 was the target gene of miR-34a.In vivo experiments also confirmed that miR-34a could promote osteogenic differentiation of hOBMSCs.Conclusions:MiR-34a promoted hOBNSCs osteogenic differentiation via down-regulating the expression of DKK1.
作者
张勃昕
吉爱红
曹正垚
孙秋望月
邓威
郭松松
王晨星
李怀奇
叶金海
ZHANG Boxin;JI Aihong;CAO Zhengyao;SUN Qiuwangyue;DENG Wei;GUO Songsong;WANG Chenxing;LI Huaiqi;YE Jinhai(Jiangsu Key Laboratory of Oral Diseases,Department of Oral and Maxillofacial Surgery,The Affiliated Stomatological Hospital of Nanjing Medical University,Nanjing 210029,China;Zhangqiu Stomatological Hospital,Jinan 250001,China)
出处
《口腔生物医学》
2019年第1期1-5,共5页
Oral Biomedicine
基金
国家自然科学基金(81371123)
"科教强卫工程"医学重点人才项目
江苏高校优势学科建设工程资助项目(2018-87)
