血管内皮生长因子2型受体胞外1～3区核酸疫苗的构建及对H22肿瘤生长的抑制作用  

Construction of a DNA Vaccine against Extracellular Domain 1-3 of Flk1 and Its Inhibitory Effect on Growth of Liver Cancer Cell Line H22

作　　者：吕凡[1] 秦兆寅[1] 黎一鸣[1] 齐颖新[1] 刘彦仿[2] 杨文彬[1] 

机构地区：[1]西安交通大学第二医院普通外科,陕西西安710004 [2]第四军医大学病理学教研室,陕西西安710061

出　　处：《癌症》2004年第12期1616-1621,共6页Chinese Journal of Cancer

基　　金：陕西省自然科学基金(No.2003C254)~~

摘　　要：背景与目的:大多数实体肿瘤的生长转移都需要新生血管的支持,血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)在肿瘤的血管生成中居中心地位,而flk1(fetalliverkinase1)是VEGF发挥作用的关键。阻断VEGF同flk1的结合可望达到抑制肿瘤生长的目的。我们构建了小鼠flk1胞外1～3区核酸疫苗,并检验疫苗对肝癌细胞H22小鼠移植瘤生长的抑制作用。方法:RT-PCR法克隆flk1胞外1～3区基因片段,将片段插入质粒pcDNA3.1(+),构建核酸疫苗pcDNA3.1(+)-flk1-Domain1-3;脂质体转染COS7细胞,Westernblot法检测flk1-Domain1-3的真核表达;采用标准4h51Cr释放实验检验疫苗免疫小鼠特异性CTL。30只小鼠分为疫苗接种组,pcDNA3.1(+)接种组和生理盐水接种组。股四头肌肌肉丰厚处接种10天后再皮下接种H22细胞,记录肿瘤的生长情况、体积、湿重、微血管密度、小鼠死亡时间,观察疫苗对小鼠H22皮下移植瘤生长的抑制作用。结果:PCR扩增出1252bp大小的基因片段,测序证明所获基因序列阅读框架与GenBank所公布的flk1胞外1～3区一致。Westernblot显示转染疫苗的细胞中有分子量约为44kDa的蛋白表达,与flk1胞外1～3区蛋白大小一致;51Cr释放实验提示,疫苗可引起针对内皮细胞的特异性CTL反应。疫苗对肿瘤的预防作用实验发现,肿瘤出现时?BACKGROUND &OBJECTIVE: Angiogenesis plays an important role in the growth,invasion,and metastasis of most solid tumors. Vascular endothelial growth factor (VEGF) and its receptor flk 1 play a key role in tumor angiogenesis. Blocking VEGF flk1 pathway may inhibit tumor growth. This study was to construct a DNA vaccine against extracellular domain 1 3 of flk1,and test its inhibitory effect on growth of liver cancer cell line H22. METHODS: Extracellular domain 1 3 of flk1 was cloned by reverse transcriptase polymerase chain reaction (RT PCR),and inserted into plasmid pcDNA3.1(+) to construct vaccine pcDNA3.1(+) flk1 domain 1 3. The vaccine was transfected into COS7 cells,and protein expression of flk1 domain 1 3 was detected by Western blot. Standard 4 h 51Cr releasing test was used to detect specific cytotoxic T lymphocyte (CTL) activity in vaccine inoculated mice. To test vaccines preventive effect,mice were divided into V,P,and S groups,treated with vaccine,pcDNA3.1(+),and saline,respectively. H22 cells were inoculated into mice 10 days later. The tumor size,tumor weight,mice survival time,tumor latent period,and microvessel density were recorded and analyzed. RESLUTS: Extracellular domain 1 3 of flk1 was cloned,and vaccine against it was constructed,both have been proved by DNA sequencing and comparing with data in GenBank. A protein of 44 kDa,which is consisted with flk1 domain 1 3 protein,expressed in COS7 cells inoculated with the DNA vaccine,specific CTL activity in these cells raised. After inoculated with H22 cells,tumor latent time of V group was (5.2±0.9) d,of P group was (4.0±0.7) d,of S group was (3.8±0.6) d; survival time of V group was (24.5±3.2) d,of P group was (14.7±2.6) d,of S group was (14.3±2.0) d; microvessel density of V group was 10.1±1.7,of P group was 27.3±3.3,of S group was 25.3±4.6; tumor weight of V group was (1.4±0.1) g,of P group was (1.8±0.2) g,of S group was (1.8±0.2)g. In comparison among groups,all data in V group were significantly different from P

关 键 词：VEGF FLK1 核酸疫苗 肝肿瘤 H22 

分 类 号：R735.7[医药卫生—肿瘤]