摘要
目的构建表达人粒细胞-巨噬细胞集落刺激因子（hGM-CSF）的2型溶瘤单纯疱疹病毒（oHSV2^hGM-CSF），初步验证其体外溶瘤效果及其对小鼠B16R黑色素瘤模型的体内疗效。方法采用PCR、分子克隆及同源重组技术，从HG52野生病毒株的基因组中剔除ICP34．5和ICP47基因，并插入hGM．CSF表达序列，构建oHSV2^hGM-CSF。将HeLa、Eca．109和PG等16种肿瘤细胞分别接种到24孔板，用1型溶瘤单纯疱疹病毒（oHSV2^hGM-CSF）或oHSV2^hGM-CSF”（感染复数=1）感染24、48h后，显微镜下观察细胞病变。以表达HSV感染受体的C57BL／6小鼠黑色素瘤细胞B16R为动物模型，当肿瘤生长至7-8mm’时，瘤内分别注射2．3×10^6PFU的oHSV2^hGM-CSF和oHSV2^hGM-CSF，共3次，每次间隔3d，同时测量肿瘤大小，并观察生存期。结果PCR检测和DNA序列分析结果证实，各目的基因已被剔除，hGM-CSF表达盒插入到ICP34．5的基因位点。oHSV2^hGM-CSF和oHSV2^hGM-CSF感染肿瘤细胞24h后，即能观察到细胞病变，且溶瘤谱较广，oHSV2^hGM-CSF感染的肿瘤细胞合胞体现象多于oHSV2^hGM-CSF感染者；感染48h后，oHSV2^hGM-CSF对HeLa、HepG2、SK-Mel-28、B16R、U87-MG细胞的溶瘤效果优于oHSV2^hGM-CSF。动物实验显示，肿瘤接种后第15天，PBS组、oHSV2^hGM-CSF’治疗组和oHSV2^hGM-CSF。”治疗组小鼠的肿瘤体积分别为（374．74-128．24）mm’、（128．23±45．32）mm。和（10．06±5．10）mm3，oHSV2^hGM-CSF明显延缓了荷瘤小鼠肿瘤的生长。PBS组小鼠在接种B16R细胞22d后全部死亡；oHSV2^hGM-CSF治疗组和oHSV2^hGM-CSF治疗组小鼠在接种B16R细胞110d时，存活率分别为60％和80％（P〉0．05）。与PBS组相比，oHSV2^hGM-CSF治疗组和oHSV2^hGM-CSF治疗组小鼠的生存期明显延长（P〈0．01）。结论oHSV2^hGM-CSF对人肿瘤细胞溶瘤谱较广，对荷B16R黑色素瘤瘤内注射有较好的抗肿瘤神府目．右掂砰的开学前景．
Objective The aim of this study was to construct a new oncolytic virus oHSV2^hGM-CSF and evaluate its oncolytic activity in vitro and in vivo in parallel with oHSV2^hGM-CSF Methods oHSV2^hGM-CSF was a replication-competent, attenuated HSV2 based on the HG52 virus （an HSV2 strain ）. It was engineered to be specific for cancer by deletion of the viral genes ICP34.5 and ICP47 and insertion of the gene encoding hGM-CSF. To measure the in vitro killing effect of the virus, 15 human tumor cell lines （HeLa, Eca-109, PG, HepG2, SK/FU, CNE-2Z, PC-3, SK-OV3, A-549, 786-0, MCF-7, Hep-2, HT- 29, SK-Mel-28, U87-MG） and mouse melanoma （B16R） cell line were seeded into 24-well plates and infected with viruses at MOI = 1 （ multiplicity of infection, MOI）, or left uninfected. The ceils were harvested 24 and 48 hours post infection, and observed under the microscope. For animal studies, the oncolytic viruses were administered intratumorally （at 3-day interval） at a dose of 2.3 x 106 PFU （plaque forming unit, PFU） for three times when the tumor volume reached 7-8 mm3. The tumor volume was measured at 3-day intervals and animal survival was recorded. Results Both oHSV2^hGM-CSF and oHSV1hGM-csvinduced widespread cytopathic effects at 24 h after infection. oHSV2^hGM-CSF, by contrast, produced more plaques with a syncytial phenotype than oHSV2^hGM-CSF. In the in vitro killing experiments for the cell lines HeLa, HepG2, SK-Mel-28, B16R and U87-MG, oHSV2^hGM-CSF eradicated significantly more cells than oHSV2^hGM-CSF under the same conditions. For the mouse experiments, it was observed that oHSV2^hGM-CSF significantly inhibited the tumor growth. At 15 days after B16R tumor cells inoculation, the tumor volumes of the PBS, oHSV2^hGM-CSF and oHSV2^hGM-CSF groups were （374.7±128.24） mm3, （ 128.23 ±45.32） mm3 （ P 〈 0.05, vs. PBS group） or （ 10.06±5.1 ） mm3 （ P 〈 0.01, vs. PBS group）, respectively （ mean±error）. The long term therapeutic effect of oHSV2^hGM-CSF on the BItR animal model was
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2012年第2期89-95,共7页
Chinese Journal of Oncology
