慢病毒介导的增强型绿色荧光蛋白报告基因对间充质干细胞的标记  

Enhanced green fluorescence protein reporter gene labeling of mesenchymal stem cells mediated by lentivirus

作　　者：李丹[1] 朱康顺[1] 周斌[1] 王劲[1] 颜荣华[1] 李征然[1] 孟晓春[1] 黄明声[1] 姜在波[1] 单鸿[1] 

机构地区：[1]中山大学附属第三医院放射科分子影像学实验室,广州510630

出　　处：《中华医学杂志》2010年第19期1357-1361,共5页National Medical Journal of China

基　　金：国家自然科学基金（30670594,30770628）;广东省科技厅科学事业费计划项目（2008B06060034,2009B030801026）;广东省自然科学基金（8151008901000066）

摘　　要：目的 探讨慢病毒介导的增强型绿色荧光蛋白（EGFP）标记间充质干细胞（MSC）是否影响其细胞生物学特性,以及EGFP基因能否持久稳定表达.方法 以不同病毒感染复数（MOI）实行EGFP慢病毒对MSC的感染,通过流式细胞分析法（FACS）检测EGFP的阳性率,并在荧光显微镜下观察EGFP在MSC的表达情况;通过锥虫蓝染色、MTT比色法、Hoechst染色和FACS检测细胞活力、增殖、凋亡和周期;EGFP慢病毒感染MSC体外持续培养2、4、8、16周时通过FACS检测EGFP的阳性率和荧光强度,以评价EGFP基因在MSC表达的稳定性.结果 EGFP慢病毒以MOI=20感染MSC 96 h,EGFP阳性率达97.39%±0.68%;与对照MsC相比,EGFP慢病毒感染MSC时,对细胞活力、增殖、凋亡和周期均没有影响（P＞0.05）;EGFP-MSC在体外持续培养2,4、8、16周,EGFP阳性率分别为97.50%±0.54%、97.32%±0.51%、97.39%±0.11%、97.48%±0.13%,荧光强度（A值）分别为440 ±13、445±12、458±13、456±16,均能够保持稳定水平.结论 EGFP慢病毒能够高效标记MSC,并且不影响其生物学特性,EGFP基因在MSC能够持久稳定表达,可以用于下一步的细胞示踪研究.Objective To explore the effect of enhanced green fluorescence protein（EGFP） labeling mediated by lentivirus on the biophysical properties of mesenchymal stem cells（MSC）,and whether the EGFP gene expression is permanent and stable.Methods MSC were infected with EGFP lentivirus at different virus multiplicity of infection（MOI）.EGFP positive rate was measured with fluorescent-activated cell scanning（FACS）analysis,and EGFP expression in MSC was investigated under a fluorescence microscope.Cell viability,proliferation,apoptosis and cell cycle were detected with trypan blue stain,MTT ciorimetric assay,Hoechst stain and FACS analysis respectively.To evaluate the stability of EGFP expresion,EGFP lentivirus infected MSC were harvested after cultured continuously in vitro for 2.4,8 or 16 weeks,and EGFP positive rate and fluorescence strength were detected with FACS analysis.Results After infected with EGFP lentivirus（MOI=20）for 96 h,EGFP positive rate of MSC was 97.39%±0.68%.Cell viability,proliferation,apoptosis and cell cycle of MSC infected with EGFP lentivirus were unaffected,as compared with control MSC（P〉0.05）.When cultured in vitro continuously for 2,4,8 or 16 weeks,EGFP positive rates of EGFP-MSC were 97.50%±0.54%,97.32%±0.51%,97.39%±0.11%,and 97.48%±0.13% respectively,while EGFP fluorescence strength were 440 ±13,445 ±12.458 ±13 and 456 ±16 respectively.Both EGFP positive rate and fluorescence strength kept in a stable level.Conclusion EGFP lentivirus can efficiently label MSC and has no significant effect on the biophy8ical Droperties of MSC.EGFP gene expression in MSC is permanent and stable.EGFP-MSC can be used for further cell tracing researth.

关 键 词：干细胞 慢病毒属 增强型绿色荧光蛋白 

分 类 号：R3[医药卫生—基础医学]