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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 establish the infection conditions of human oral mucosal fibroblasts efficiently infected with lentiviral vector, and lay the foundation for further study on human oral mucosal fibroblasts using genetic engineering. Methods: Primary human oral mucosal fibroblasts were cultured by tissue block method. The cell types were identified by immunohistochemical SP method. The lentiviral vector carrying green fluorescent protein gene was infected with purified cells at different multiplicity of infection (MOI) And set different infection conditions, 4 days after infection under the fluorescence microscope to observe the expression of green fluorescent protein. RESULTS: Human oral mucosal fibroblasts were successfully isolated and purified. When the number of lentiviral infections was 30, the concentration of polybrene was 8μg / mL, and the infection enhancement fluid was added, the infection efficiency could be high, which could meet the needs of subsequent experiments. Conclusion: The lentiviral vector can efficiently infect human oral mucosal fibroblasts, and the green fluorescent protein gene can be stably expressed in fibroblasts.