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目的:研究增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP)转染人胚胎视网膜前体细胞的转染效率及瞬时表达情况,建立人胚胎视网膜前体细胞的示踪方法,为视网膜前体细胞移植提供示踪依据。方法:在大肠杆菌中扩增pEGFP-N1质粒,通过磷酸钙介导法转染人胚胎视网膜前体细胞．应用流式细胞仪检测其转染效率,荧光显微镜和激光共聚焦显微镜观察其瞬时表达情况。结果:pEGFP-N1在基因转染24 h细胞转染率为28．98％,48 h为32．45％,72 h为30．40％,对照未转染细胞24 h、48 h分别为2．50％和2．04％:体外观察培养细胞转染24 h已有绿荧光表达,48-72 h细胞荧光强度最强,96 h荧光强度减弱。结论:pEGFP-N1质粒能有效转染人胚胎视网膜前体细胞,其转染效率可达到30％,是人胚胎视网膜前体细胞较为理想的瞬时表达载体和细胞示踪报告分子。
OBJECTIVE: To study the transfection efficiency and transient expression of enhanced green fluorescent protein (EGFP) transfected human embryonic retinal precursor cells and to establish a method for tracing human embryonic retinal precursor cells for retinal precursor Cell transplantation to provide evidence of tracing. Methods: The pEGFP-N1 plasmid was amplified in Escherichia coli and transfected into human embryonic retinal precursor cells by calcium phosphate mediated method. The transfection efficiency was detected by flow cytometry. The transient expression was observed by fluorescence microscopy and laser confocal microscopy. Results: The transfection rate of pEGFP-N1 was 28.98% at 24 h, 32.45% at 48 h, 30.40% at 72 h, and 24 h at 48 h .50% and 2.04%, respectively. In vitro, the green fluorescent protein was expressed 24 hours after transfection in cultured cells, the fluorescence intensity was the strongest at 48-72 hours, and weakened at 96 hours. CONCLUSION: Plasmid pEGFP-N1 can effectively transfect human embryonic retinal precursor cells and its transfection efficiency can reach 30%. It is an ideal transient expression vector and cell reporter molecule for human embryonic retinal precursor cells.