MiR-34a在牙周膜细胞成骨向分化中的作用

发布时间：2018-05-19 03:13

本文选题：人牙周膜细胞 + 成骨分化　；参考：《口腔医学研究》2017年09期

【摘要】：目的:探讨微小RNA-34a在牙周膜细胞成骨向分化中的作用。方法:体外分离培养人牙周膜细胞(hPDLCs),取第3~6代用于实验。首先,对hPDLCs进行成骨诱导液处理,在3、7、14d后利用实时荧光定量PCR(qRT-PCR)检测miR-34a基因的表达变化。然后,构建慢病毒载体pCDH-pre-miR-34a和pCDH,将慢病毒载体感染hPDLCs,构建pre-miR-34a过表达细胞模型(hPDLCs/34a)和空载体对照细胞模型(hPDLCs/pCDH),并诱导hPDLCs成骨分化3、7、14和21d。分析成骨标志基因碱性磷酸酶(ALP)、Runt相关转录因子2(Runx2)、骨钙素(OCN)及骨涎蛋白(BSP)表达变化,ALP活性及钙化结节形成的茜素红染色情况。结果:hPDLCs经成骨分化诱导后,miR-34a基因表达在第3天开始明显升高,并呈逐渐增高趋势,差异均有统计学意义(P0.001)。与hPDLCs/pCDH组相比,hPDLCs/34a组的ALP活性和茜素红染色均有明显减弱。qRT-PCR结果显示:hPDLCs/34a组的ALP表达量在成骨诱导7d时较hPDLCs/pCDH组降低(P0.05);Runx2、OCN及BSP表达量的差异在各时间点基本无统计学意义。结论:在体外条件下,miR-34a抑制人牙周膜细胞成骨向分化。
[Abstract]:Objective: to investigate the role of small RNA-34a in osteogenic differentiation of periodontal ligament cells. Methods: human periodontal ligament cells were isolated and cultured in vitro. Firstly, hPDLCs was treated with osteoblast inducer and the expression of miR-34a gene was detected by real-time fluorescence quantitative PCR qRT-PCRat 14 days later. Then, lentivirus vectors pCDH-pre-miR-34a and pCDH were constructed, lentivirus vectors were infected with hPDLCs, and pre-miR-34a overexpression cell models were constructed. The changes of ALP activity and alizarin red staining in calcified nodules were analyzed by analyzing the expression of osteoblastic marker gene ALPT-Runt related transcription factor 2runx2, osteocalcin OCNand bone sialoprotein (BSPP). Results the expression of miR-34a gene began to increase significantly on the 3rd day after osteogenic differentiation induced by% hPDLCs, and the expression of miR-34a gene increased gradually. The difference was statistically significant (P 0.001). Compared with hPDLCs/pCDH group, ALP activity and alizarin red staining in hPDLC / 34a group were significantly decreased. The results of qRT-PCR showed that the expression of ALP in 10 hPDLC / 34a group was significantly lower than that in hPDLCs/pCDH group on the 7th day after osteogenesis. There was no significant difference in the expression of ALP and BSP between the two groups at all time points. Conclusion: in vitro, miR-34a inhibits osteogenic differentiation of human periodontal ligament cells.
【作者单位】：武汉大学口腔医学院口腔基础医学省部共建国家重点实验室培训基地和口腔生物医学教育部重点实验室;武汉大学口腔医院修复科;
【基金】：国家自然科学基金(编号:81200812) 武汉市应用基础研究计划项目(编号:2016060101010043)
【分类号】：R781.4

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