摘要
目的利用同源重组技术获得插入p53基因的重组1型单纯疱疹病毒(HSV-1),并在体外培养细胞和荷瘤小鼠中研究其溶瘤特性。方法将含p53基因的真核表达框克隆人含HSV-1同源重组臂的pK05/BN,获得重组穿梭质粒pK05/p53,其电转含pHSV△IR—BAC的大肠杆菌后,筛选获得含有p53的重组DNApHSVAIR—BAC/053。pHSV△IR-BAC/053转染Vero细胞获得重组病毒,经Southem印迹和Western印迹鉴定正确后命名为MH1004。分别检测MH1004感染多种肿瘤细胞24h后的滴度,分析其在肿瘤细胞中的复制特性。建立小鼠黑色素瘤模型,于0、3、6d瘤内分别注射2×106 PFU的MH1004、未插入p53基因的重组HSV-1(MH1001)、HSV-1wt和PBS,观察肿瘤大小和小鼠生存时间并作统计学分析。结果Western印迹检测表明MHl01M所携带的p53基因可在感染细胞中表达;在同种肿瘤细胞中,MHIOIM和HSV-1wt的复制水平差异无统计学意义(P〉O.05);MH1004在SK—N.SH和U251细胞中的复制水平显著高于其在其他肿瘤细胞中的复制水平;治疗15d后,HSV-1 wt、MH1001和MHl004组小鼠的肿瘤体积分别为(6180±751)、(5760±267)和(4850±532)mm3,显著小于PBS对照组(9860±91)mm3(均P〈0.01),MHl004组肿瘤体积小于MH1001组和HSV.1wt组,但差异均无统计学意义(均P〉0.05)。MH1004组小鼠生存率(5/6)显著高于PBS组(3/6)、HSV-1wt组(3/6)和MHl001组(3/6)。结论MH1004在神经肿瘤细胞中的复制水平较高,能够显著抑制荷瘤小鼠肿瘤生长,并延长荷瘤小鼠的生存期。
Objective To obtain the recom'binant herpes simplex virus 1 (HSV-1) inserted p53 gene with homologous recombination technology and investigate the virus' replication ability and oncolytic property in vitro and in vivo. Methods A eukaryotic expression case with p53 gene was cloned into pKO5/BN. The pKO5/p53 was constructed and transfected to the E. coli with pHSVAIR-BAC by electroporation. Then the recombinant pHSV△IR-BAC/p53 was obtained and transfected into Vero cells. Recombinant virus (MH1004) was identified by Southern blot and Western blot. Then the virus' replication abilities in several human tumors cells were tested by plaque assay. A murine melanoma model was established by subcutaneous inoculation of B16 cells. A dosage of 2 ~ 106 PFU (plaque forming unit) of MH1004, MH1001, HSV-I wt or PBS was injected 3 times intratumorally in every 3 days. The tumor volume and survival rate were measured twice a week. Results The results of Western blot showed that the p53 protein can be detected from the Vero cells infected by MHIO04. The replication abilities of MHIO04 and HSV-1 wt in the same tumor cell was insignificant (P 〉 0. 05 ). And MH1004's replication abilities in SK-N-SH and U251 was significantly higher than other cancer cells. The tumors volume of group HSV-I wt, MHIO01 (HSValR)and MH1004 were (6 180 ±751), (5 760 ±267) and (4 850 ±532) mm3 compared with PBS group (9 860 ± 91 )mm3 , the difference of reduction of tumors volume was significant ( P 〈 0. 01 ). And the tumors volume of MH1004 group was smaller than HSV-1 wt and MH1001 group, but without significant difference ( P 〉 0. 05 ). And the survival rate of MH1004 treated mice (5/6) was greatly higher than PBS (3/6), HSV-1 wt ( 3/6 ) and MH1001 ( 3/6 ). Conclusion The replication abilities of MH1004 in neural tumor are very high and MHI004 can inhibit the growth of tumor so that prolong the survival of mice bearing murine melanoma.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2016年第5期370-374,共5页
National Medical Journal of China
基金
国家自然科学基金(31140018)
新疆维吾尔自治区高技术研究发展项目(2010016)
关键词
疱疹病毒I型
人
染色体
人工
细菌
基因
p53
溶瘤病毒
Herpesvirus 1, human
Chromosomes, artificial, bacterial
Genes, p53
Oncolytic viruses