中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2012年
2期
89-95
,共7页
施桂兰%庄秀芬%韩香萍%李洁%张郁%张叔人%刘滨磊
施桂蘭%莊秀芬%韓香萍%李潔%張鬱%張叔人%劉濱磊
시계란%장수분%한향평%리길%장욱%장숙인%류빈뢰
溶瘤病毒%疱疹病毒2型,人%人粒细胞-巨噬细胞集落刺激因子%肿瘤生物治疗%合胞体
溶瘤病毒%皰疹病毒2型,人%人粒細胞-巨噬細胞集落刺激因子%腫瘤生物治療%閤胞體
용류병독%포진병독2형,인%인립세포-거서세포집락자격인자%종류생물치료%합포체
Oncolytic virus%Herpesvirus 2,human%Human granulocyte-macrophage colony-stimulating factor%Biological therapy%Synscytia
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(oHSV2hGM-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oHSV2hGM-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(oHSV1 hGM-CSF)或oHSV2hGM-CSF(感染复数=1)感染24、48 h后,显微镜下观察细胞病变.以表达HSV感染受体的C57 BL/6小鼠黑色素瘤细胞B16R为动物模型,当肿瘤生长至7～8 mm3时,瘤内分别注射2.3×106 PFU的oHSV1hGM-GSF和oHSV2hGM-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.oHSV1hGM-CSF和oHSV2hGM-CSF感染肿瘤细胞24h后,即能观察到细胞病变,目溶瘤谱较广,oHSV2hGM-CSF感染的肿瘤细胞合胞体现象多于oHSV1hGM-CSF感染者；感染48 h后,oHSV2hGM-CSF对HeLa、HepG2、SK-Mel-28、B16R、U87-MG细胞的溶瘤效果优于oHSV1hGM-CSF.动物实验显示,肿瘤接种后第15天,PBS组、oHSV1 hGM-CSF治疗组和oHSV2hGM-CSF治疗组小鼠的肿瘤体积分别为( 374.7±128.24) mm3、( 128.23±45.32) mm3和( 10.06±5.10) mm3,oHSV2hGM-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22 d后全部死亡；oHSV1 hGM-CSF治疗组和oHSV2hGM-CSF治疗组小鼠在接种B16R细胞110 d时,存活率分别为60％和80％(P＞0.05).与PBS组相比,oHSV1 hGM-CSF治疗组和oHSV2hGM-CSF治疗组小鼠的生存期明显延长(P＜0.01).结论 oHSV2hGM-C-SF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效应,具有较好的开发前景.
目的 構建錶達人粒細胞-巨噬細胞集落刺激因子(hGM-CSF)的2型溶瘤單純皰疹病毒(oHSV2hGM-CSF),初步驗證其體外溶瘤效果及其對小鼠B16R黑色素瘤模型的體內療效.方法 採用PCR、分子剋隆及同源重組技術,從HG52野生病毒株的基因組中剔除ICP34.5和ICP47基因,併插入hGM-CSF錶達序列,構建oHSV2hGM-CSF.將HeLa、Eca-109和PG等16種腫瘤細胞分彆接種到24孔闆,用1型溶瘤單純皰疹病毒(oHSV1 hGM-CSF)或oHSV2hGM-CSF(感染複數=1)感染24、48 h後,顯微鏡下觀察細胞病變.以錶達HSV感染受體的C57 BL/6小鼠黑色素瘤細胞B16R為動物模型,噹腫瘤生長至7～8 mm3時,瘤內分彆註射2.3×106 PFU的oHSV1hGM-GSF和oHSV2hGM-CSF,共3次,每次間隔3d,同時測量腫瘤大小,併觀察生存期.結果 PCR檢測和DNA序列分析結果證實,各目的基因已被剔除,hGM-CSF錶達盒插入到ICP34.5的基因位點.oHSV1hGM-CSF和oHSV2hGM-CSF感染腫瘤細胞24h後,即能觀察到細胞病變,目溶瘤譜較廣,oHSV2hGM-CSF感染的腫瘤細胞閤胞體現象多于oHSV1hGM-CSF感染者；感染48 h後,oHSV2hGM-CSF對HeLa、HepG2、SK-Mel-28、B16R、U87-MG細胞的溶瘤效果優于oHSV1hGM-CSF.動物實驗顯示,腫瘤接種後第15天,PBS組、oHSV1 hGM-CSF治療組和oHSV2hGM-CSF治療組小鼠的腫瘤體積分彆為( 374.7±128.24) mm3、( 128.23±45.32) mm3和( 10.06±5.10) mm3,oHSV2hGM-CSF明顯延緩瞭荷瘤小鼠腫瘤的生長.PBS組小鼠在接種B16R細胞22 d後全部死亡；oHSV1 hGM-CSF治療組和oHSV2hGM-CSF治療組小鼠在接種B16R細胞110 d時,存活率分彆為60％和80％(P＞0.05).與PBS組相比,oHSV1 hGM-CSF治療組和oHSV2hGM-CSF治療組小鼠的生存期明顯延長(P＜0.01).結論 oHSV2hGM-C-SF對人腫瘤細胞溶瘤譜較廣,對荷B16R黑色素瘤瘤內註射有較好的抗腫瘤效應,具有較好的開髮前景.
목적 구건표체인립세포-거서세포집락자격인자(hGM-CSF)적2형용류단순포진병독(oHSV2hGM-CSF),초보험증기체외용류효과급기대소서B16R흑색소류모형적체내료효.방법 채용PCR、분자극륭급동원중조기술,종HG52야생병독주적기인조중척제ICP34.5화ICP47기인,병삽입hGM-CSF표체서렬,구건oHSV2hGM-CSF.장HeLa、Eca-109화PG등16충종류세포분별접충도24공판,용1형용류단순포진병독(oHSV1 hGM-CSF)혹oHSV2hGM-CSF(감염복수=1)감염24、48 h후,현미경하관찰세포병변.이표체HSV감염수체적C57 BL/6소서흑색소류세포B16R위동물모형,당종류생장지7～8 mm3시,류내분별주사2.3×106 PFU적oHSV1hGM-GSF화oHSV2hGM-CSF,공3차,매차간격3d,동시측량종류대소,병관찰생존기.결과 PCR검측화DNA서렬분석결과증실,각목적기인이피척제,hGM-CSF표체합삽입도ICP34.5적기인위점.oHSV1hGM-CSF화oHSV2hGM-CSF감염종류세포24h후,즉능관찰도세포병변,목용류보교엄,oHSV2hGM-CSF감염적종류세포합포체현상다우oHSV1hGM-CSF감염자；감염48 h후,oHSV2hGM-CSF대HeLa、HepG2、SK-Mel-28、B16R、U87-MG세포적용류효과우우oHSV1hGM-CSF.동물실험현시,종류접충후제15천,PBS조、oHSV1 hGM-CSF치료조화oHSV2hGM-CSF치료조소서적종류체적분별위( 374.7±128.24) mm3、( 128.23±45.32) mm3화( 10.06±5.10) mm3,oHSV2hGM-CSF명현연완료하류소서종류적생장.PBS조소서재접충B16R세포22 d후전부사망；oHSV1 hGM-CSF치료조화oHSV2hGM-CSF치료조소서재접충B16R세포110 d시,존활솔분별위60％화80％(P＞0.05).여PBS조상비,oHSV1 hGM-CSF치료조화oHSV2hGM-CSF치료조소서적생존기명현연장(P＜0.01).결론 oHSV2hGM-C-SF대인종류세포용류보교엄,대하B16R흑색소류류내주사유교호적항종류효응,구유교호적개발전경.
Objective The aim of this study was to construct a new oncolytic virus oHSV2hGM-CSF and evaluate its oncolytic activity in vitro and in vivo in parallel with oHSV1hGM-CSF.Methods oHSV2hGM-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 cells 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 × 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 oHSV2hGM-CSFand oHSV1 hGM-CSFinduced widespread cytopathic effects at 24 h after infection.OHSV2hGM-CSF,by contrast,produced more plaques with a sTncytial phenotype than oHSV1hGM-CSF.In the in vitro killing experiments for the cell lines HeLa,HepG2,SK-Mel-28,B16R and U87-MG,oHSV2hGM-CSF eradicated significantly more cells than oHSV1hGM-CSF under the same conditions.For the mouse experiments,it was observed that oHSV2hGM-CSFsignificantly inhibited the tumor growth.At 15 days after B16R tumor cells inoculation,the tumor volumes of the PBS,oHSV1hGM-CSF and oHSV2hGM-CSFgroups 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 oHSV2hGM-CSF on the B16R animal model was evaluated by recording animal survival over 110 days after tumor cells inoculation whereas all the mice in the PBS group died by day 22 (P＜0.01).The anti-tumor mechanism of the newly constructed oHSV2hGM-CSF against B16R cell tumor appeared to include the directly oncolytic activity and the induction of anti-tumor immunity to some degree.Conclusion The findings of our study demonstrate that the newly constructed oHSV2hGM-CSF has potent anti-tumor activity in vitro to many tumor cell lines and in vivo to the transplanted B16R tumor models.
