CAJ | 학술논문

目的制备携带GFP基因的逆转录病毒，以简便、快速标记活细胞。方法将编码GFP的cDNA片段插入逆转录病毒载体pLNCX构建重组逆转录病毒载体pLNCX-GFP，借助脂质体转染单嗜性及双嗜性逆转录病毒包装细胞，G418筛选抗性克隆，然后利用荧光显微镜选出GFP表达最高的克隆。取含病毒颗粒的上清液感染NIH3T3及多种肿瘤或血管内皮细胞。结果重组逆转录病毒载体pLNCX-GFP转染包装细胞后，包装细胞可表达GFP并产生GFP逆转录病毒。尽管GFP逆转录病毒对动物及人的肿瘤细胞或血管内皮细胞的感染能力各不相同，但由于逆转录病毒能整合进入宿主细胞基因组DNA内，经短期G418筛选后容易获得持续、稳定、高水平表达GFP的阳性细胞。表达GFP的肿瘤细胞仍可在同系动物体内生长并持续表达GFP。结论携带GFP基因的逆转录病毒能简便、快速介导稳定的基因转移，遗传标记多种细胞，比表达质粒等更优越。
목적제비휴대GFP기인적역전록병독，이간편、쾌속표기활세포。방법장편마GFP적cDNA편단삽입역전록병독재체pLNCX구건중조역전록병독재체pLNCX-GFP，차조지질체전염단기성급쌍기성역전록병독포장세포，G418사선항성극륭，연후이용형광현미경선출GFP표체최고적극륭。취함병독과립적상청액감염NIH3T3급다충종류혹혈관내피세포。결과중조역전록병독재체pLNCX-GFP전염포장세포후，포장세포가표체GFP병산생GFP역전록병독。진관GFP역전록병독대동물급인적종류세포혹혈관내피세포적감염능력각불상동，단유우역전록병독능정합진입숙주세포기인조DNA내，경단기G418사선후용역획득지속、은정、고수평표체GFP적양성세포。표체GFP적종류세포잉가재동계동물체내생장병지속표체GFP。결론휴대GFP기인적역전록병독능간편、쾌속개도은정적기인전이，유전표기다충세포，비표체질립등경우월。
Objective　To prepare retrovirus which carry GFP gene and are able to label living cells simply and rapidly. Methods　The recombinant retroviral vector pLNCX-GFP was constructed by inserting 780?bp GFP cDNA fragment into the MCS site of retroviral plasmid pLNCX. Both ecotropic packaging cell line ΦX-Eco and amphotropic packaging cell line ΦX-Ampho and PA317 were transfected by pLNCX-GFP with liposome. The supernate collected from transfected packaging cells was used to infect a variety of tumor cell lines and vascular endothelia cell lines. Results　When packaging cells were transfected by retroviral vector pLNCX-GFP, the GFP expression could be observed in 25％～40％ of cells and GFP retrovirus then could be detected, however G418 resistant clones showed more stable GFP expression and higher retrovirus titer. The GFP retrovirus from different packaging cell line showed variant ability to infect tumor cell lines and vascular endothelia cell lines and the tumor cells infected by GFP retrovirus showed stable GFP expression in vitro. GFP transduced tumor cells could grow in syngenic animal and continue expressing GFP. Conclusion　Using GFP retrovirus to label target cells represent an important advantage over conventional plasmid because they can efficiently transfer GFP gene into target cells and GFP can be stably expressed in target cells no matter in vitro or in vivo.