摘要
目的本研究构建含绿色荧光蛋白基因的重组慢病毒,并观察其在人椎间盘髓核细胞中的表达情况。方法应用分子克隆技术将绿色荧光蛋白(green fluorescent prote in,GFP)基因导入慢病毒载体质粒pLenti6/V5 TOPO,应用磷酸钙沉淀法将慢病毒载体三质粒系统(包括包装质粒、包膜蛋白质粒等)共转染入293细胞进行包装,48 h后在荧光显微镜下观察绿色荧光蛋白表达情况,72 h收集病毒上清并感染髓核细胞,在荧光显微镜下感染情况。结果重组慢病毒滴度测定约为107U/mL。感染人椎间盘髓核细胞后,荧光显微镜下观察到GFP的表达。结论成功构建了含GFP的慢病毒载体,且能成功将目的基因转入椎间盘髓核细胞并表达。
Objective To construct the Lenti-GFP carrying the green fluorescent protein （GFP） gene and to investigate the expression of GFP in human nucleus pulposus cells. Methods The GFP gene was inserted to lentiviral vector plasmid pLenti6/ V5 TOPO by molecular cloning technique. Human kidney 293 cells were co-transfected with the 3 plasmid system containing packaging plasmid and envelope plasmid by using calcium phosphate precipitation, and the expression of GFP was observed under fluorescence microscope after 48 h. The viral particles were collected 72 h after transfection, and were used to infect human nucleus pulposus cells. The expression of GFP in nucleus pulposus cells was observed by fluorescence microscopy. Results The results showed that the transfection efficacy was evident and the viral titer was 107 U/mL. Conclusion The lentiviral vector containing GFP gene is successfully constructed, and the transduction and expression of GFP gene is obvious in nucleus pulposus cells.
出处
《脊柱外科杂志》
2009年第4期213-215,共3页
Journal of Spinal Surgery
关键词
椎间盘
绿色荧光蛋白质类
遗传载体
转染
基因表达
Intervertebral disk
Green fluorescent proteins
Genetic vectors
Transfection
Gene expression
