摘要
目的通过将将携带有绿色荧光蛋白基因的重组腺病毒(Ad-GFP)转染兔离体基底动脉环,观察GFP表达部位及表达量的变化。方法获取兔基底动脉环,置入含DMEM的96孔板中;加入1.8×106pfu/μl的Ad-GFP转染1 h;转染后即刻3、5、7 d行冰冻切片,荧光显微镜观察亮绿色物质。另外,将不同病毒浓度(分别为1.2×105、1.2×106、1.8×106和2.4×106pfu/μl)转染离体血管环1 h后,培养3 d后行冰冻切片,用荧光显微镜观察结果。结果荧光显微镜下观察发现,基底动脉环管壁各层均表达GFP,但主要位于血管内皮层,1 000倍下在内皮细胞内中明确观察到荧光颗粒。1.8×106pfu/μl的Ad-GFP转染后发现,GFP在转染3d后表达最明显,5 d次之,而7 d后表达最弱;不同浓度Ad-GFP转染3 d后发现,浓度为1.2×105pfu/μl时,管壁有较弱绿色荧光,浓度不断升高亮度有所增强,浓度为1.8×106pfu/μl时达到最高峰,浓度继续增加时荧光亮度未见进一步增强。结论 Ad-GFP可以成功转染入离体血管环中,以内皮层为主;3 d为病毒转染后最佳培养时间。
ObjectiveTo observe the expression sites and the quantity of the green fluorescent protein（GFP） in order to providethe base for recombinant adenovirus endothelial nitrogen oxide synthase transfection in vitro and in vivo.MethodsThe basilar arteryrings derived from the rabbits were cultured in the media containing the same concentration of recombinant adenovirus encoding greenfluorescent protein（Ad-GFP） for 0, 3, 5 and 7 days respectively and in the media containing the different concentrations of Ad-GFP for 3 days respectively. The expressions of GFP in the basilar artery rings were observed in all groups.ResultsThe GFP could be detectedin all the vascular walls, and the most significant expression was in the endothelia. When the concentration of Ad-GFP in the mediaincreased, GFP expression quality also increased. The unchanged expression was not observed until the concentration of Ad-GFP in themedia arrived at 1.8×106pfu/μl. The expression of GFP were the strongest and the weakest respectively in 3 and 7 days groups among allthe groups in which the basilar artery rings were cultured in the media containing the same concentration of Ad-GFP for different days.ConclusionsAd-GFP may be successfully transfected into the vascular walls, especially in the endothelia. It is suggested that theexpression of GFP is best in the basilar artery rings which are cultured for 3 days in the medium with Ad-GFP concentration of 1.8×106pfu/μl.
出处
《中国临床神经外科杂志》
2015年第10期621-623,共3页
Chinese Journal of Clinical Neurosurgery
