慢病毒载体感染人口腔黏膜成纤维细胞稳定表达绿色荧光蛋白的研究

Investigation of the expression of green fluorescent protein in human oral fibroblasts mediated by lentiviral vector

目的:确立慢病毒载体高效感染人口腔黏膜成纤维细胞的感染条件,为进一步应用基因工程技术研究人口腔黏膜成纤维细胞奠定基础。方法:采用组织块法培养原代人口腔黏膜成纤维细胞,免疫组化SP法鉴定细胞类型,将携带绿色荧光蛋白基因的慢病毒载体以不同感染复数(multiplicity of infection,MOI)感染纯化细胞,并设置不同感染条件,感染4 d后于荧光显微镜下观察绿色荧光蛋白表达情况。结果:成功分离纯化得到人口腔黏膜成纤维细胞,当慢病毒感染复数为30,polybrene浓度为8μg/mL,并加入感染增强液时,可达到较高的感染效率,满足后续实验需要。结论:慢病毒载体可高效感染人口腔黏膜成纤维细胞,携带的绿色荧光蛋白基因可在成纤维细胞内稳定表达。

Objective：To investigate the efficiency of green fluorescent protein（GFP） gene expression mediated by lentiviral vector in the primary cultured human oral fibroblasts,which could establish the basis for the application of gene-labeling oral fibroblasts.Method：The human oral fibroblasts were initially obtained from tissue culture and were purified by trypsinization and identified by immunohistochemistry with SP method.Lentiviral vector encoding GFP infected these cells at diverse multiplicity of infection（MOI） values under different conditions.Four days after the infection,the expression of GFP in oral fibroblasts were observed under fluorescence microscope.Result：The purified human oral fibroblasts were obtained.The best MOI value for lentivirus infecting human oral fibroblasts was 30,and the enhanced infection solution and polybrene with the concentration of 8μg/ml were also needed in order to improve the infection efficiency.Conclusion：The expression of GFP gene in human oral fibroblasts mediated by lentiviral vector was high efficient and stable.