MiR-34a在牙周膜细胞成骨向分化中的作用

Role of Micro-34a in Induced-mineralization of Human Periodontal Ligament Cells

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(P<0.001)。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(P<0.05);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。

Objective： To investigate the effect of miR-34a on the osteogenic differentiation of human periodontal ligament cells （hPDLCs）. Methods： HPDLCs were obtained through in vitro culture combined with enzyme digestion method and tissue pieces culture method. First, the cells were continuously induced by mineralization induction medium for 3,7, and 14 days. The expression of miR-34a was detected by quantitative real-time PCR （qRT-PCR）. Second, hPDLCs were transfected with lentiviral vector containing pre-miR-34a or control to build the miR-34a overexpression model or control model （hPDLCs/34a or hPDLCs/pCDH） and induced by mineralization induction medium for 3, 7, 14, and 21 days. Quantification of osteogenensis-related genes （ALP, Runx2, OCN and BSP） expression, alkaline phosphatase （ALP） activity detection and Alizarin red staining were used to assess the influence of miR-34a on the osteogenic differentiation of hPDLCs. Results： The expression of miR-34a significantly increased in a time-dependent manner （P〈0.001） during the osteogenic differentiation. The mineralization nodule formation and ALP activity were all attenuated by miR-34a. ALP gene expression detected by qRT-PCR was reduced after 7 days＆apos; induction in hPDLCs/34a. Runx2, OCN and BSP gene expression showed no significant differences. Conclusion： MiR-34a may have a negative effect on the osteogenic differentiation of hPDLCs.