组织工程与重建外科杂志2011，Vol.7Issue(5)：248-253,6.DOI:10.3969/j.issn.1673-0364.2011.05.003
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
摘要
Abstract
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 efficiencies 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. Transfection 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 transfection 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Key words
Enhanced Green Fluorescent Protein/ Retrovirus/ Cell labeling/ Tissue engineering分类
生物科学引用本文复制引用
吕晓杰,周广东,马俐君,刘霞,曹谊林..增强型绿色荧光蛋白转染活细胞效率的研究[J].组织工程与重建外科杂志,2011,7(5):248-253,6.基金项目
国家自然科学基金(30300353). （30300353）
