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目的:观察人骨保护素(human osteoprotegerin,hOPG)在犬牙周韧带细胞(periodontal ligament cells,PDLCs)中的表达情况,为后期的牙周组织再生实验制备高表达目的基因hOPG的种子细胞。方法:体外构建携带hOPG基因的腺病毒载体Ad5-hOPG-EGFP,并将其转染Beagle犬PDLCs,通过流式细胞术、实时定量RT-PCR、ELISA以及Western Blot检测目的基因的转录和表达。结果:重组腺病毒Ad5-hOPG-EGFP成功转染犬PDLCs,转染72 h后转染效率达到80%以上,转染的细胞发出较强的绿色荧光;基因和蛋白水平检测表明,hOPG在犬PDLCs中得到了有效表达。结论:重组腺病毒介导的hOPG基因可以在PDLCs中有效表达。
OBJECTIVE: To observe the expression of human osteoprotegerin (hOPG) in periodontal ligament cells (PDLCs) and to prepare the seed cells with high expressed hOPG gene for the periodontal regeneration experiment. Methods: Ad5-hOPG-EGFP was constructed in vitro and transfected into PDLCs of Beagle dogs. The transcription and expression of target genes were detected by flow cytometry, real-time quantitative RT-PCR, ELISA and Western Blot. Results: The recombinant adenovirus Ad5-hOPG-EGFP was successfully transfected into canine PDLCs. After 72 h of transfection, the transfection efficiency reached more than 80%, and the transfected cells emitted strong green fluorescence. The results of gene and protein levels showed that hOPG PDLCs have been effectively expressed. Conclusion: Recombinant adenovirus-mediated hOPG gene can be efficiently expressed in PDLCs.