重组腺病毒Ad-APN-EGFP的构建与促进种植体周围成骨

Recombinant adenovirus Ad-APN-EGFP constructing and promoting osteogenesis around implants

目的构建重组腺病毒Ad-APN-EGFP,研究其转染效率和对种植体周围成骨的影响。方法构建含脂联素(APN)基因的的重组腺病毒Ad-APN-EGFP,并测序鉴定和酶切鉴定。体外转染大鼠骨髓间充质干细胞(BMSCs),检测转染效率和APN基因mRNA表达;建立大鼠股骨干骺端羟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Ad-APN-EGFP悬浮液;对照组注入10μL的PBS。4周后取材,光镜下观察种植体周围成骨。结果测序鉴定和酶切鉴定表明,成功构建重组腺病毒Ad-APN-EGFP;转染大鼠BMSCs效率可达90%以上,并能高表达APN基因mRNA;体内实验显示实验组种植体周围成骨明显高于对照组。结论重组腺病毒Ad-APN-EGFP可以提高大鼠BMSCs的APN基因表达,具有促进种植体周围成骨的作用。

Objective We aimed to construct a recombinant adenovirus Ad-APN-EGFP and to evaluate the transfection efficiency and effect of the adenovirus promoting osteogenesis around implants. Methods Recombinant adenovirus Ad-APNEGFP containing adiponectin（APN） was constructed by DNA sequencing and restriction enzyme digestion. Rat bone marrow stem cells（BMSCs） were transfected in vitro. Transfection efficiency and APN mRNA expression were tested. Animal models of rat femoral epiphysis and hydroxyapatite（HA）-coated implants were established. The Ad-APN-EGFP adenovirus at 10 μL was injected into the defect around HA-coated implants in the experimental group, whereas the same volume of phosphate-buffered saline was injected into the defect in the control group. Osteogenesis around implants was observed by hematoxylin–eosin staining four weeks after implantation. Results Ad-APN-EGFP was successfully constructed by DNA sequencing and restriction enzyme digestion. The transfection efficiency of Ad-APN-EGFP to BMSCs was up to ≥90%. APN mRNA expression of BMSCs transfected with Ad-APN-EGFP was higher than that of the control group. Osteogenesis in the experimental group was more evident than that in the control group in vivo. Conclusion Ad-APN-EGFP could be transfected into BMSCs and express APN mRNA at a high level. Ad-APN-EGFP could improve osteogenesis around implants in vivo.