摘要
目的构建能同时表达人和鼠可溶性VEGFR2的双基因真核表达载体,并初步验证其功能。方法分别以pORF-hVEGFR2和pORF-mVEGFR2为模板,通过PCR将人和鼠的sVEGFR2基因定向克隆入真核细胞双顺反子载体pVITRO2的多克隆位点区,构建pVITRO2-hm-sVEGFR2双基因表达质粒。通过体外实验研究pVITRO2-hm-sVEGFR2的表达产物能否阻断VEGF对脐静脉血管内皮细胞的促增殖能力,体内实验建立小鼠B16肿瘤模型研究其抗肿瘤活性,CD31免疫组化染色检测其对肿瘤新生血管的抑制情况。结果 pVITRO2-hm-sVEGFR2双基因表达质粒成功构建,体外能阻断VEGF对血管内皮细胞的促增殖能力,能明显抑制肿瘤在小鼠体内的生长以及肿瘤组织内的新生血管生成,其抑制作用大于人或鼠可溶性VEGFR2单基因治疗方案。结论 pVITRO2-hm-sVEGFR2明显抑制新生血管生成活性,是一种新型的抗血管生成DNA疫苗,为抗肿瘤治疗研究提供了新的思路和方法。
Objective To develop a novel anti-angiogenesis strategy based on a DNA vaccine coding both human and mouse soluble VEGFR2.Methods The gene fragments coding human and mouse sVEGFR2 were amplified with PCR and cloned into pVITRO2 to generate pVITRO2-hm-sVEGFR2 recombinant.The in vitro VEGF blocking effect of the pVITRO2-hm-sVEGFR2 expression products on HUVEC cells were evaluated.The anti-tumor effect of pVITRO2-hm-sVEGFR2 was studied in mouse B16 model.The microvessels were stained by using CD31 antibody.Results The coexpressing vector pVITRO2-hm-sVEGFR2 was constructed successfully,confirmed by the restriction endonuclease digestion and sequencing.The expressing products of pVITRO2-hm-sVEGFR2 could obviously block the function of VEGF on promoting the proliferation of HUVEC in vitro.The tumor growth in mice was also significantly inhibited by pVITRO2-hm-sVEGFR2 expression.CD31 staining demonstrated that the microvessel density obviously decreased in tumor tissues treated with pVITRO2-hm-sVEGFR2.Both anti-tumor and anti-angiogenesis effects of pVITRO2-hm-sVEGFR2 were stronger than that of plasmids which coding only human or mouse sVEGFR2.Conclusion pVITRO2-hm-sVEGFR2 could be a novel DNA vaccine for the anti-tumor therapy by inhibiting angiogenesis.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2010年第4期563-566,共4页
Journal of Sichuan University(Medical Sciences)
基金
国家973计划(项目编号2004CB518800)资助
