摘要
目的研究重组腺病毒载体感染小鼠神经瘤细胞(N2a)的感染效率及重组腺病毒介导的人鞘氨醇激酶-1(sphingosine kinase-1,SPK1)基因在N2a细胞表达特性。方法用流式细胞仪检测感染效率,用激光共聚焦显微镜观察绿色荧光蛋白的表达;用Western blot方法检测SPK1蛋白的表达。结果 N2a细胞的感染效率和绿色荧光蛋白表达量在一定范围内随着病毒感染复数(Multiplicity of infection,MOI)的增加而增高,MOI为100时感染效率达峰值为(99.57±0.133)%。以MOI为25的重组腺病毒感染N2a细胞,感染效率随时间变化(0-72h)而增高,72h峰值为(98.03±0.721)%,到96h有所降低(P<0.05)。SPK1蛋白的表达量较感染空病毒组和未感染病毒组明显升高。结论重组腺病毒在适宜的MOI下,在N2a细胞中96小时内均有较高的感染效率,且重组腺病毒介导的人SPK1基因在体外能够得到有效的表达。
Objective To investigate infection efficiency of recombinant adenovirus to N2a cells and the expression of SPK1 mediated by recombinant adenovirus.Methods Infection percentage was detected by flow cytometry.Green fluorescent protein expression was observed by Laser scanning confocal microscope.The expression of SPK1 was detected by Western blot.Results The results showed that infection efficiency of Ad-CMV-EGFP to N2a cell was increased due to the increase of MOI（0-100）.It reached its peak when MOI is 100 pfu/cell,（99.57±0.133）% cell are green.N2a cell was infected with Ad-CMV-EGFP at a MOI of 25 and infection efficiency was increased due to the increase of time（0-72 h.）,which reached its peak at 72 hours,（98.03±0.721）% cell are green and however decreased in 96 hours（P0.05）.The expression of SPK1 is higher than empty virus group and uninfected group.Conclusion There is higher infection efficiency of Ad-CMV-EGFP in N2a cell within 96 hours with optimal MOI,SPK1 can be expressed in vitro mediated by recombinant adenovirus.
出处
《中国实验诊断学》
2012年第11期1969-1972,共4页
Chinese Journal of Laboratory Diagnosis
基金
国家自然科学基金资助项目(81070879)
