反转录病毒载体介导的EGFP基因在SK-N-SH神经母细胞瘤细胞中的表达  

Retroviral vector mediated expression of EGFP gene in SK-N-SH neuroblastoma cells

作　　者：刘朝晖[1] 杨宇[2] 庄鹏辉[3] 许杰华[1] 马延兵[1] 胡海涛[1] 

机构地区：[1]西安交通大学医学院人体解剖与组织胚胎学系,陕西西安710061 [2]吉林大学第一医院神经内科,吉林长春130021 [3]西安交通大学医学院第一附属医院胸外科,陕西西安710061

出　　处：《西安交通大学学报（医学版）》2007年第1期10-13,共4页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(No.30300395No.30400515);教育部博士点基金项目(No.20030698011)

摘　　要：目的构建携带报告基因增强型绿色荧光蛋白(EGFP)的反转录病毒载体,并且探讨病毒载体对SK-N-SH神经母细胞瘤细胞株的感染效率。方法使用基因重组技术构建携带EGFP基因的反转录病毒载体(RV/EGFP)。将稀释的RV病毒液感染NIH3T3细胞,计数NIH3T3荧光表达细胞的数量来确定病毒的浓度;使用RV载体感染SK-N-SH细胞,通过流式细胞学荧光检测明确RV在SK-N-SH细胞的感染效率。结果成功构建了基因转移载体RV/EGFP,确定重组病毒的浓度为8.3×106病毒颗粒/mL。在SK-N-SH细胞RV/EGFP稳定转染并有效表达。在SK-N-SH细胞EGFP的荧光表达显示RV的转移并且表达的效率达到30%以上。结论SK-N-SH细胞能被RV病毒载体有效感染,在转基因细胞株外源基因的表达长期稳定并且显示RV病毒载体具有良好的基因转移效率。Objective To construct the retroviral （RV） vector with report gene enhanced green fluorescent protein （EGFP） and to explore the gene transfection efficiency of RV on SK-N-SH neuroblastoma cells. Methods By the technique of genetic recombination, the recombinant RV/EGFP viruses had been constructed. The concentration of diluted RV virus solution was determined by counting the NIH3T3 cells with fluorescent expression. Then the RV/EGFP virus was used to transfer SK-N-SH neuroblastoma cells. The transfection efficiency in SK-N- SH cell was measured by flow cytometer. Results The gene transfer vector RV/EGFP had been successfully constructed. We obtained the recombinant RV/EGFP with a concentration of 8.3 × 10^6 viruses/mL. The RV/EGFP had been stably transferred and effectively expressed in SK-N-SH cell. The transfection and expression efficiency of RV was above 30% in SK-N-SH ceils determined by observing EGFP expression. Conclusion The SK-N-SH cells can be transferred effectively by recombinant RV vector. In transgenetic cells, the expression of transgene was stable, and moreover, RV vector had better genetically transfected efficiency than other methods.

关 键 词：SK—N—SH神经母细胞瘤细胞 增强型绿色荧光蛋白 反转录病毒载体 

分 类 号：R592[医药卫生—老年医学]