增强型绿色荧光蛋白转染活细胞效率的研究  

The Effectiveness of Labeling Technique of Enhanced Green Fluorescent Protein by Transfection into Living Cell

作　　者：吕晓杰[1] 周广东[2] 马俐君[3] 刘霞 曹谊林[2,4] 

机构地区：[1]中国人民解放军第二炮兵总医院,北京市100088 [2]上海交通大学医学院附属第九人民医院整复外科,上海市200011 [3]上海交通大学医学院附属仁济医院,上海市200001 [4]中国医学科学院整形外科医院,北京市100044

出　　处：《组织工程与重建外科杂志》2011年第5期248-253,共6页Journal of Tissue Engineering and Reconstructive Surgery

基　　金：国家自然科学基金(30300353)

摘　　要：目的研究影响增强型绿色荧光蛋白(EGFP)逆转录病毒表达系统转染活细胞效率的主要相关参数,建立一种稳定高效的活细胞EGFP标记技术。方法培养、扩增携带EGFP逆转录病毒载体的包装细胞PG13,分别于12 h、24 h、36 h和48 h收集病毒液,转染骨髓基质细胞(BMSCs),通过流式细胞仪和荧光显微镜检测不同时间点病毒转染效率。选择转染率最高时间点的病毒液,连续转染BMSCs两次,小剂量嘌呤霉素筛选6 h,观察病毒转染率及细胞活力。再以上述方法转染脂肪基质细胞(ADSCs)和成纤维细胞(FBs),观察这种方法标记其他干细胞或成熟分化细胞的可行性。结果根据流式细胞仪及荧光显微镜观察结果,24 h收集的病毒液转染BMSCs效率最高(64.56+2.53)%,36 h次之(56.98+3.22)%,12 h(47.39±1.82)%与48 h(37.84±1.77)%相对较低。连续转染两次并经短暂筛选后,BMS(s转染率可达80%以上,且细胞活力不受影响。采用相同方法转染脂肪基质细胞(ADSCs)和成纤维细胞(FBs),转染率均能达到80%,且细胞形态、增殖能力均未见明显改变。结论本实验建立了一种稳定高效的活细胞EGFP转染技术,为研究组织工程种子细胞的体内、外转归及细胞间相互作用提供了稳定且直观的检测手段。Objective To explore the vital parameters which affect transfection efficiency of Enhanced Green Fluorescent Protein （EGFP） retrovirus expression system in living cells and to establish a stable and effective labeling technique of living cells. Methods PG13 cells with EGFP retrovirus vector were expanded in vitro and the supernatant was respectively collected at 12 h, 24 h, 36 h, 48 h for the retrovirus transfection of BMSCs. The transfection efficieneies of retrovirus in BMSCs were detected by flow cytometer and fluorescent microscope to confirm the optimal collection time of the retrovirus supernatant. BMSCs were transfected twice by the optimal supernatant and then were selected by low-dose puromycin for 6 h. Transfeetion efficiency and cell vitality were detected to evaluate the effect of transfection. ADSCs and FBs were transfected with optimal condition to explore the feasibility of labeling technique in other stem cells or differentiated cells. Results According to the results of flow cytometer and fluorescent microscope, the retrovirus supernatant collected at 24 h reached the highest transfection efficiency of （64.56±2.53）%. The high transfeetion efficiency was also reached （56.98±3.22）% at 36 h while relatively low at 12 h and 48 h, （47.39±1.82）% and （37.84±1.77）%. After transfected twice and selected, over 80% BMSCs expressed EGFP with fine cell vitality and morphology. Using this transfection methods, ADSCs and FBs were transfected likewise and both of them reached high transfection efficiencies of over 80%. Conclusion This study established a stable and effective cell abeling technique of EGFP, which can be widely used for the related research of cell tracing and cell interaction in tissue engineering.

关 键 词：增强型绿色荧光蛋白 逆转录病毒 细胞标记 组织工程 

分 类 号：Q786[生物学—分子生物学]