异种血管内皮生长因子基因重组T7噬菌体疫苗对小鼠Lewis肺癌的抑制作用

Antitumor Effect of Recombinant T7 Phage Vaccine Expressing Xenogenic Vascular Endothelial Growth Factor on Lewis Lung Cancer in Mice

背景与目的:肿瘤的生长、转移和新生血管生成有密切关系,血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)是已知作用最强、最专一的促血管生成因子。本实验采用噬菌体展示技术,构建人血管内皮生长因子重组T7噬菌体疫苗,并检验疫苗对小鼠Lewis肺癌的抑制作用。方法:用反转录聚合酶链反应(RT-PCR)从人肺癌组织克隆出人VEGF165基因,将人VEGF165基因与T7Select10-3b噬菌体基因重组,构建T7Select10-3b_VEGF噬菌体疫苗,制备噬菌体效价达到1×1013pfu/ml。将C57BL/6J小鼠随机分为3组,T7-VEGF疫苗组10只、空白噬菌体组(T7)10只、生理盐水组(NS)10只。将各组样品与等体积弗氏佐剂混合,各组小鼠每只每周分别免疫1×1012pfu/200!l重组噬菌体疫苗、空白噬菌体、生理盐水,共免疫4周。接种Lewis肺癌细胞,观察肿瘤的生长情况和小鼠状态.接种肿瘤14天后,处死小鼠,剥离肿瘤称重,检测小鼠血清中特异性抗VEGF抗体滴度、肿瘤微血管密度(microvesseldensity,MVD)。结果:T7-VEGF疫苗组,T7组、生理盐水组肿瘤均重分别为:(0.543±0.259)g、(0.982±0.359)g、(1.169±0.460)g。疫苗组肿瘤重量明显小于生理盐水组(P<0.01)。疫苗组血清产生抗VEGF抗体滴度为1∶1000。疫苗组、空白噬菌体组、生理盐水组MVD分别为:8.5±0.8,16.4±1.3,18.5±1.6。疫苗组MVD明显小于生理盐水组。结论:异种血管内皮生长因子基因重组T7噬菌体疫苗可干扰机体对自身VEGF的免疫耐受,可产生较高水平的特异性抗VEGF抗体,并具有抑制小鼠Lewis肿瘤生长的效应。

BACKGROUND ＆ OBJECTIVE- Angiogenesis plays an important role in growth and metastasis of tumors. Vascular endothelial growth factor （VEGF） is considered as a fundamental regulator for angiogenesis. This study was to construct a recombinant T7 phage vaccine expressing xenogenic VEGF on the capsid, and test its inhibitory effect on Lewis lung cancer cells in mice. METHODS：VEGF gene was cloned by reverse transcription-polymerase chain reaction （RT-PCR） from human lung cancer tissues, and inserted into phage using T7 Select10-3b kit to construct T7 Select10-3b_VEGF vaccine. The titer of prepared phage reached 1×10^13 pfu/ml. C57BL/6J mice were randomly divided into 3 groups： T7 Select10- 3b_VEGF vaccine group （T7-VEGF）, T7 phage （T7） group, normal saline （NS） group （10 mice/group）. Each mouse was injected with Freund＇s adjuvant mixed with 1 ×10^12 pfu/200μl T7 Select10-3b_VEGF, or T7, or normal saline once a week for 4 weeks. Lewis lung carcinoma model （LL/2） was established in C57BL/6J mice after 4-week immunization. Tumor growth and mouse＇s physical status were observed during immunization. Tumor weight and serum level of specific anti-VEGF antibody were measured by enzyme-linked immunosorbent assay （ELISA）. Microvessel density （MVD） of tumors was detected by immunohistochemistry 14 days after the inoculation of tumor cells. RESULTS： Tumor weight of T7-VEGF vaccine group,T7 group, and NS group were （0.543 ±0.259）g, （0.982±0.359）g, （1.169±0.460）g, respectively. Tumor weight of T7-VEGF vaccine group was significantly lower than that of NS group （P〈0.01）. Serum anti-VEGF antibody level in T7-VEGF vaccine group was 1：1 000. MVD was significantly lower in T7-VEGF vaccine group than in NS group（8.5±0.8 vs 18.5±1.6, P〈0.05）. MVD in T7 group was 16.4±1.3. CONCLUSION, Recombinant T7 phage vaccine expressing xenogenic VEGF can break immunologic tolerance against self-VEGF and inhibit the growth of Lewis lung cancer cells.