表达VEGFR-2的减毒鼠伤寒沙门氏菌疫苗诱导特异性抗胶质瘤血管免疫应答

Specific Anti-glioma Angiogenesis Immune Response Induced by Attenuated Salmonella Typhimurium Vaccine Expressing Vascular Endothelial Growth Factor Receptor-2

背景与目的:血管内皮生长因子(vascular endothelialgrowth factor,VEGF)及其主要受体血管内皮生长因子受体-2(vascular endothelialgrowth factorreceptor-2,VEGFR-2)在肿瘤新生血管和肿瘤基质形成过程中起着重要作用。本研究的目的是观察表达鼠VEGFR-2的重组减毒沙门氏疫苗菌诱导的抗血管特异性免疫应答及抗胶质瘤作用。方法:构建真核表达载体pcDNA3.1-VEGFR2,通过电转化法将pcDNA3.1-VEGFR2导入减毒鼠伤寒沙门氏菌SL7207中,经由胃管饲予C57BL/6J小鼠,对小鼠进行基因免疫。采用ELISA法检测免疫小鼠血清中特异性抗VEGFR2-IgG抗体,分离免疫小鼠的脾细胞,分析重组疫苗菌免疫后小鼠体内的特异性细胞毒性T细胞(cytotoxic T lym phocyte,CTL)应答。用携带pcDNA3.1-VEGFR2的重组沙门氏菌免疫治疗胶质瘤荷瘤小鼠,通过测量荷瘤小鼠肿瘤大小,检测肿瘤微血管密度及肿瘤细胞凋亡,评价重组疫苗菌的抗血管及肿瘤生长抑制作用。结果:重组疫苗菌免疫后小鼠产生了高水平的抗VEGFR2-IgG抗体,诱导小鼠脾淋巴细胞产生针对VEGFR2的特异性CTL活性。重组疫苗菌的免疫能够明显抑制胶质瘤的生长。NaH CO3对照组、载体对照组、重组疫苗菌组的平均微血管密度分别为26.5±5.8、27.2±4.5、8.8±1.9,平均凋亡细胞数分别为4.41.2、3.

BACKGROUND & OBJECTIVE: Vascular endothelial growth factor (VEGF) and its receptor (VEGFR-2) play important roles in tumor angiogenesis. This study was to investigate anti-vasculature and anti-glioma effects of attenuated Salmonella typhimurium vaccine expressing VEGFR2 gene. METHODS: Plasmid pcDNA3.1-VEGFR2 was constructed, and electrotransfected into attenuated Salmonella typhimurium strain SL7207. C57BL/6J mice were immunized with gastrogavage of cDNA vaccine encoding VEGFR2 (vaccine group). C57BL/6J mice received gastrogavage of pcDNA3.1 or NaHCO3 were used as controls. Serum level of specific anti-VEGFR2-IgG antibody was detected by ELISA. Cytotoxic T lymphocytes (CTLs) activity was measured by MTT assay. Mouse models of intracranial Gl261 glioblastoma were treated with gastrogavage of attenuated Salmonella typhimurium expressing VEGFR2 gene. Tumor diameter was measured; microvessel density (MVD) was detected by immunohistochemistry; tumor cell apoptosis was detected by TUNEL. RESULTS: All mice immunized with the vaccine developed high levels of anti-VEGFR2-IgG antibody, and showed strong CTLs activities against VEGFR2. The vaccine substantially inhibited glioblastoma growth. MVD was significantly lower in vaccine group than in pcDNA3.1 group, and NaHCO3 group (8.8±1.9 vs. 27.2±4.5, and 26.5±5.8, P < 0.01); while apoptotic cell count per visual field was significantly higher in vaccine group than in the rest 2 groups (23.4±4.7 vs. 3.1±1.0, and 4.4±1.2, P < 0.01). CONCLUSION: Attenuated Salmonella typhimurium vaccine expressing VEGFR2 gene can break immunologic tolerance against self-VEGFR2 antigen, and specifically kill glioblastoma vascular endothelial cells by inducing specific anti-VEGFR2 immunoreaction.