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目的 :建立可以稳定表达红色荧光蛋白(red fluorescent protein,RFP)的人胚胎干细胞(human embryonic stem cells,h ESCs)系。方法:优化核转染方法后,将RFP表达载体p EF1α-Ds Red-Express2导入h ESCs中。利用碱性磷酸酶染色、RT-PCR、免疫荧光染色和类胚体形等方法,比较核转染前后h ESCs的干细胞特性。结果:核转染后h ESCs的碱性磷酸酶染色结果为阳性。核转染前后h ESCs的Oct4、Sox2、Klf4、Nanog基因的表达未出现明显差异。RFP-h ESCs具有三胚层分化潜能,并且可稳定传30代以上。结论:成功建立稳定表达红色荧光蛋白的h ESCs,干细胞特性不受RFP的影响,可用于后续的实验研究。
Objective: To establish human embryonic stem cells (h ESCs) lines that stably express red fluorescent protein (RFP). METHODS: After optimization of the transfection method, RFP expression vector pEF1α-Ds Red-Express2 was introduced into hESCs. The stem cell characteristics of h ESCs before and after nuclear transfection were compared by alkaline phosphatase staining, RT-PCR, immunofluorescence staining and embryoid body morphology. Results: The results of alkaline phosphatase staining of hESCs after transfection were positive. The expression of Oct4, Sox2, Klf4 and Nanog genes in h ESCs before and after nuclear transfection did not show any significant difference. RFP-h ESCs have the potential to differentiate into three germ layers and are stable for over 30 generations. Conclusion: The hESCs stably expressing red fluorescent protein were successfully established. The characteristics of stem cells were not affected by RFP and could be used in subsequent experimental studies.