摘要
BACKGROUND： At present, a universal method and vector for transfecting enhanced green fluorescent protein （EGFP） into neural stem cells does not exist. The traditional use of liposome to transfect GFP shows low labeling efficiency and short labeling time. However, there is an increasing number of reports in recent years utilizing adeno-associated virus （AAV） transfection of neural stem cells. OBJECTIVE： To compare differences of neural stem cell transfection via rAAV-2-EGFP or liposome, with regard to transfection efficiency, stability, and safety. DESIGN, TIME AND SETTING： A parallel, controlled experiment at a cellular molecular level was performed in the Central Laboratory, Clinical Neuromedicine Research Center, Tongji Medical College, Huazhong University of Science and Technology, between June 2007 and March 2008. MATERIALS： Liposome 2000 was purchased from Invitrogen, USA; rAAV-2-EGFP was offered from Beijing AGTC Gene Technology, China. METHODS： Cerebral cortical cells from embryonic day 12 C57BL/6 mouse embryo were isolated and cultivated, and the logarithmically growing neural stem cells were divided into three groups. Liposome transfection： neural stem cells were transfected with liposome/EGFP plasmid mixture comprising 2 pg pcDNA-3.0-EGFP plasmid and 12 μg Liposome 2000 in complete culture solution. AAV transfection： neural stem cells were transfected with virus transfection solution comprising rAAV-2-EGFP and complete culture solution at multiplicity of infection = 10^5. Negative control： physiological saline was used instead of virus transfection solution. MAIN OUTCOME MEASURES： At different time points after transfection （36 hours, 1 week, 2 weeks, 1 month, and 6 months）, the proportion of green fluorescent cells was quantified under fluorescent microscopy. Transfection efficiency and proliferative activity of the transfected neural stem cells were detected with flow cytometry and 3-（4,5）-dimethylthiahiazo （-z-yl）-3,5-di- phenytetrazoliumremide, respectively. RESULTS： The neural stem cells began to express green fluorescence 36 hours after transfection with rAAV-2-EGFP. Transfection efficiency reached a peak （61.2%） at 1 week, and was higher than the liposome transfection group （38.7%; P 〈 0.05）. Green fluorescence was detectable for 6 months, with no weakness of expression, and rAAV-2-EGFP transfection showed no obvious effects on the proliferation activity of neural stem cells. In the liposome transfection group, green fluorescence was observed after 24 hours and reached a peak at 3 days. Fluorescence expression and proliferation activity disappeared at 2 weeks. CONCLUSION： rAAV-2-EGFP transfection of neural stem cells was superior to liposome transfection.
BACKGROUND： At present, a universal method and vector for transfecting enhanced green fluorescent protein （EGFP） into neural stem cells does not exist. The traditional use of liposome to transfect GFP shows low labeling efficiency and short labeling time. However, there is an increasing number of reports in recent years utilizing adeno-associated virus （AAV） transfection of neural stem cells. OBJECTIVE： To compare differences of neural stem cell transfection via rAAV-2-EGFP or liposome, with regard to transfection efficiency, stability, and safety. DESIGN, TIME AND SETTING： A parallel, controlled experiment at a cellular molecular level was performed in the Central Laboratory, Clinical Neuromedicine Research Center, Tongji Medical College, Huazhong University of Science and Technology, between June 2007 and March 2008. MATERIALS： Liposome 2000 was purchased from Invitrogen, USA; rAAV-2-EGFP was offered from Beijing AGTC Gene Technology, China. METHODS： Cerebral cortical cells from embryonic day 12 C57BL/6 mouse embryo were isolated and cultivated, and the logarithmically growing neural stem cells were divided into three groups. Liposome transfection： neural stem cells were transfected with liposome/EGFP plasmid mixture comprising 2 pg pcDNA-3.0-EGFP plasmid and 12 μg Liposome 2000 in complete culture solution. AAV transfection： neural stem cells were transfected with virus transfection solution comprising rAAV-2-EGFP and complete culture solution at multiplicity of infection = 10^5. Negative control： physiological saline was used instead of virus transfection solution. MAIN OUTCOME MEASURES： At different time points after transfection （36 hours, 1 week, 2 weeks, 1 month, and 6 months）, the proportion of green fluorescent cells was quantified under fluorescent microscopy. Transfection efficiency and proliferative activity of the transfected neural stem cells were detected with flow cytometry and 3-（4,5）-dimethylthiahiazo （-z-yl）-3,5-di- phenytetrazoliumremide, respectively. RESULTS： The neural stem cells began to express green fluorescence 36 hours after transfection with rAAV-2-EGFP. Transfection efficiency reached a peak （61.2%） at 1 week, and was higher than the liposome transfection group （38.7%; P 〈 0.05）. Green fluorescence was detectable for 6 months, with no weakness of expression, and rAAV-2-EGFP transfection showed no obvious effects on the proliferation activity of neural stem cells. In the liposome transfection group, green fluorescence was observed after 24 hours and reached a peak at 3 days. Fluorescence expression and proliferation activity disappeared at 2 weeks. CONCLUSION： rAAV-2-EGFP transfection of neural stem cells was superior to liposome transfection.
基金
General Program of the National Natural Science Foundation of China,No.30500167
863 Project of the National Science and Technology Ministry of China,No. 2007AA03Z312
