hOPG转染对大鼠BMSCs及种植体周围成骨的影响

Effect of hOPG Gene Transfection on Osteogenesis of Rat BMSCs and around Implant

目的:研究体内、体外人骨保护素(hOPG)基因对大鼠骨髓间充质干细胞(BMSCs)成骨功能和种植体周围成骨的影响。方法:构建含hOPG的重组腺病毒,蛋白印迹杂交法Western Blot检测转染细胞OPG蛋白功能表达。倒置显微镜观察转染细胞形态差别,茜素红S染色鉴定转染细胞的体外成骨能力。建立大鼠股骨干骺端羟基磷灰石涂层种植体植入动物模型。植入种植体前,实验组于植入体窝注入10μL的病毒悬浮液;对照组注入10μL的PBS。4周后取材,HE染色,光镜下观察,定量分析。结果:Western blot结果显示OPG在蛋白水平高表达;茜素红S染色转染组和未转染组都出现了较多散在的致密圆形矿化结节,萃取染色后分光光度检测两者无明显差异;体内实验显示实验组种植体周围成骨明显高于对照组。结论:体外pDC316-hOPG-EGFP成功转染大鼠BMSCs,转染hOPG基因的BMSCs能持续稳定的高表达hOPG。转染不影响BMSCs的成骨活性。种植体周围注射hOPG具有促进种植体周围成骨的作用。

Objective： To study effect of hOPG gene transfection on osteogenic differentiation of rat BMSCs and osteogenesis around implant in vitro and in vivo. Methods： Recombinant adenovirus containing hOPG was constructed. PCR and western blot were used to detect the transcription of hOPG DNA and protein. We used inverted microscope to observe the transfection cell morphological differences and Alizarin red S staining to evaluate transfection cells in vitro osteogenesis ability. 10ul hOPG adenovirus was injected into defect around HA coating implant in experimental group, while the same volume PBS was used in control group. Osteogenesis was observed by HE staining 4 weeks after implantation. Results： Western blot results showed that hOPG adenovirus transfection improved expression of hOPG in protein level. In alizarin red S staining, two groups showed similar density of mineralized nodules and extraction spectrophotometric detection after dyeing. No difference could be found between the two groups. Osteogenesis in experimental group was more obvious than that in control group in vivo. Conclusion： AdS --hOPG--EGFP could be transfected into BMSCs and secret hOPG at a high level. Transfection dose of Ad5- hOPG--EGFP has no obvious effect on the osteogenic activity of BMSCs. OPG could improve osteogenesis around implant in vivo.