【摘要】 目的探讨血清2型腺相关病毒(rAAV2)介导增强型绿色荧光蛋白(EGFP)基因转染神经干细胞后绿色荧光蛋白(GFP)的表达规律及转染对神经干细胞生物学特性的影响。方法应用荧光显微镜观察转染神经干细胞的绿色荧光蛋白表达情况。传代后第10天,比较转染组和对照组子代细胞形成的神经球数;分化后第14天行免疫组织化学检查,比较转染组和对照组神经元和胶质细胞的比例。结果转染后第3天,可观察到神经球发出绿色荧光,强度逐渐增强,并在第9天达到稳定状态(90%的神经球发出强弱不等的荧光)。传代后第10天,转染组子代细胞每井形成的神经球数为(17.83±1.35)个,与对照组比较无显著性差别(P>0.05)。分化后第14天,转染组神经元、胶质细胞的胞体与纤细突起均可见绿色荧光,转染组神经元与胶质细胞比例分别为35.3%±3.1%和55.4%±7.3%,与对照组比较无显著性差别(P>0.05)。结论rAAV2-EGFP可以稳定、高效转染神经干细胞,且不影响神经干细胞的生物学特性,是神经干细胞理想的基因标记方法。更多还原

【Abstract】 Objective To observe the expression of green fluorescence protein(GFP) in neural stem cells(NSCs) with serum type 2 recombinant adeno-associated viral vector-mediated enhanced GFP gene(rAAV2-EGFP) and the changes in the biological characteristics of NSCs in relation to the transfection.Methods rAAV2-EGFP was transfected into NSCs,and the expression of GFP was observed with fluorescence microscope every day.The number of neurospheres between the transfected group and control group was compared on day 10 of cell passage,and the percentages of neurons and glial cells between the two group were compared on day 14 following cell differentiation by immunohistochemical methods.Results The neurospheres emitted green fluorescence 3 days after the transfection with rAAV2-EGFP,and the intensity increased and stabilized on day 9 with 90% of the neurospheres emitting fluorescence.On day 10 of cell passage,the progeny cells formed 17.83 ± 1.35 neurospheres per well in the transfected group,as was comparable to that in the control group.On day 14 following cell differentiation,the cell body and fine dendrites of the neurons and glial cells in transfected group emitted green fluorescence.The percentage of neurons and glial cells in the transfected group was 35.3% ± 3.1% and 55.4% ± 7.3% respectively,showing no significant difference from that of the control group.Conclusion rAAV2-EGFP can efficiently and stably transduce NSCs without causing changes in their biological characteristics,and therefore is an ideal method for genetic labeling of NSCs.更多还原