重组腺病毒载体Ad5-hOPG-EGFP介导hOPG基因在犬牙周韧带细胞中的表达检测

[摘要]目的：观察人骨保护素（humanosteoprotegerin，hOPG）在犬牙周韧带细胞（periodontalligamentcells，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WesternBlot检测目的基因的转录和表达。结果：重组腺病毒Ad5一hOPG—EGFP成功转染犬PDLCs，转染72h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。

Expression of hOPG in PDLCs of dog infected by recombinant adenoviral vector Ad5-hOPG-EGFP

Objective：To evaluate the expression of human osteoprotegerin （hOPG） in periodontal ligament cells （PDLCs） of Bea- gle dog in order to prepare seed cells with high expression of hOPG gene for periodontal tissue regeneration experiment in later stage. Methods：PDLCs of Beagle dogs were isolated and cultured. Then, adenovirus vector carrying hOPG gene was structured in vitro and transfected into canine PDLCs. Expression of hOPG was observed by flow cytometry, real-time RT-PCR, ELISA and Western Blot. Re- sults ： Recombinant adenovirus Ad5-hOPG-EGFP was successfully transfected PDLCs of Beagle dogs. More than 80 per cent of transfect- ed cells positively expressing the green fluorescene protein could be observed under fluorescence microscope and detected by flow cy- tometry. Effective expression of hOPG was also observed by real-time RT-PCR,ELISA and Western Blot,which proved the successful transfect hOPG into canine PDLCs. Conclusions ：hOPG was successfully expressed in PDLCs of Beagle dog which were infected by re- combinant adenoviral vector Ad5-hOPG-EGFP.