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目的观察重组腺病毒载体Ad5-h OPG-EGFP转染的Beagle犬牙周膜细胞(PDLCs)体内和体外的骨化能力。方法将体外构建的携带人骨保护素(human osteoprotegerin,h OPG)基因的腺病毒载体Ad5-h OPG-EGFP转染Beagle犬PDLCs,通过体外Von Kossa染色实验、实时荧光定量PCR检测成骨指标的表达情况、酶联免疫检测RANKL/OPG的值及裸鼠体内胶原膜成骨实验比较转染组和对照组的成骨能力。结果组织学和形态学结果显示,转染了重组腺病毒Ad5-h OPG-EGFP的Beagle犬PDLCs形成的矿化结节数目多且体积大、深染。转染组中犬碱性磷酸酶(alkaline phosphates,ALP)、骨钙素(osteocalcin,OC)、骨涎蛋白(bone sialoprotein,BSP)基因相对表达水平均高于空载体组和空白对照组(P<0.05)。转染组中犬RANKL/OPG的值下降趋势最为明显。裸鼠体内实验中转染组胶原膜也体现出较好的成骨能力。结论重组腺病毒介导的h OPG基因可以促进PDLCs成骨。
Objective To observe the in vivo and in vitro ossification of human periodontal ligament cells (PDLCs) transfected with recombinant adenovirus vector Ad5-h OPG-EGFP. Methods Ad5-h OPG-EGFP carrying human osteoprotegerin (h OPG) gene was transfected into PDLCs of Beagle dogs in vitro. The expression of osteogenic markers was detected by Von Kossa staining in vitro and by real-time fluorescence quantitative PCR , The value of RANKL / OPG by enzyme-linked immunosorbent assay and collagen membrane osteogenesis in nude mice were compared between the transfected group and the control group. Results Histological and morphological results showed that Beagle canine PDLCs transfected with recombinant adenovirus Ad5-h OPG-EGFP formed a large number of mineralized nodules and were bulky and deeply stained. The relative expression levels of alkaline phosphatase (ALP), osteocalcin (OC) and bone sialoprotein (BSP) in transfected group were higher than those in blank vector group and blank control group <0.05). The decreasing tendency of RANKL / OPG in the transfection group was the most obvious. In vivo experiments in nude mice transfection group collagen membrane also showed good osteogenic ability. Conclusion Recombinant adenovirus mediated h OPG gene can promote PDLCs osteogenic.