重组慢病毒载体的构建及其感染人脐带间充质干细胞的研究

Construction of recombinant lentiviral vector and its infection of human umbilical cord mesenchymal stem cells

目的 利用包装制备的重组慢病毒载体感染人脐带间充质干细胞,为后续的细胞重编程奠定基础.方法 利用组织块贴壁法分离、培养、扩增出人脐带间充质干细胞（HUMSC）.携带目的基因（oct4、sox2、klf4、c-myc）及绿色荧光蛋白的质粒经验证、扩增后,转染HEK-293T细胞,制备携带上述基因的慢病毒上清液.荧光显微镜下梯度稀释法检测病毒滴度.慢病毒液感染3～5代生长良好的HUMSC,荧光显微镜及流式细胞术检测病毒感染力,RT-PCR及定量PCR检测目的基因mRNA水平.结果 成功验证、扩增携带目的基因的质粒.制备的新鲜病毒液滴度达5×108U/mL,冻存后降为5×106 U/ml.重组慢病毒感染HUMSC的感染力高于80％,4种目的基因的表达均明显高于对照.结论 重组慢病毒载体可以在体外高效的转染HUMSC,并稳定表达目的基因,是HUMSC重编程的有效基因转移载体.

Objective To construct recombinant lentiviral vectors with the aim of infecting human umbilical cord mesenchymal stem cells （HUMSC） for subsequent cell reprogramming.Methods A tissue adherent method was performed to isolate,culture and expand HUMSC.Four different plasmids containing one factor：Oct4,Sox2,Klf4 and c-Myc （OSKM） with Green Fluorescent Protein （GFP）,were analyzed by gene sequencing and then amplified by competent E.Coli.Recombinant lentiviral particles were generated by co-transfecting the amplified expression plasmids along with a packaging plasmid into 293T lentiviral packaging cells.The viral titer was determined by concentration gradient method and the viral infection efficiency was also analyzed.HUMSC at the 3rd to 5th passage were infected with lentiviral particles.GFP expression was detected by fluorescent microscope and Flow cytometry.Moreover,gene expression of OSKM was confirmed by RT-PCR and qPCR.Results After packaging process,the virus titer reached 5 × 108 U/mL.However it decreased to 5 × 106 U/mL after cryopreservation.Three days after infection,fluorescence microscopy indicated that the infective efficiency of recombinant lentivirus in HUMSC was more than 80%.This result was confirmed and in good agreement with those of flow cytometry.HUMSC infected with the constructed lentiviral particles,highly expressed OSKM genes.Conclusion The recombinant lentiviral vectors can efficiently infect HUMSC which can stably express the protein of our interest in vitro,and it can be used as an effective gene delivery vector which is the base of cell reprogramming.