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目的:将质粒PAcGFP1-Golgi转染到中国仓鼠卵巢细胞(CHO细胞)并进行筛选纯化,得到单一克隆细胞株,从而构建能很好显示高尔基体形态、分布和功能的新模型。方法:将质粒PAcGFP1-Golgi以脂质体法转染CHO细胞,稳定后用G418筛选、平板稀释法克隆细胞;克隆完成后以激光共聚焦显微镜观察高尔基体形态及分布,动态监测荧光表达率,MTT法检测细胞增殖活性。结果:成功获得稳定表达质粒PAcGFP1-Golgi的CHO细胞株,激光共聚焦显微镜下高尔基体显示清晰,荧光表达率高达(99.31±0.26)%且表达稳定,细胞增殖活性与空白CHO细胞无异。结论:稳定表达PAcGFP1-Golgi质粒的CHO细胞株构建成功,为进一步研究高尔基体、COG复合体及其与遗传性糖基化障碍疾病之间的关系打下基础。
OBJECTIVE: To construct a new model of human Golgi apparatus that can display the morphology, distribution and function of Golgi well by transfection of plasmid PAcGFP1-Golgi into Chinese hamster ovary cells (CHO cells) and screening and purification. METHODS: CHO cells were transfected with PAcGFP1-Golgi by lipofectamine. After stabilization, the cells were screened by G418 and cloned by plate dilution method. After the cloning, the morphology and distribution of Golgi apparatus were observed by laser scanning confocal microscopy. MTT assay of cell proliferation activity. Results: The CHO cell line stably expressing plasmid PAcGFP1-Golgi was successfully obtained. The Golgi apparatus showed a clear expression under confocal laser scanning microscope (99.31 ± 0.26)% with stable expression. The cell proliferation activity was similar to that of blank CHO cells. CONCLUSION: The CHO cell line stably expressing the PAcGFP1-Golgi plasmid is successfully constructed, which lays the foundation for the further study on the relationship between Golgi apparatus, COG complex and inherited glycosylation disorder.