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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: The growth, metastasis and neovascularization of tumors are closely related. Vascular endothelial growth factor (VEGF) is the most potent and specific angiogenic factor known. In this study, phage display technology was used to construct a recombinant human vascular endothelial growth factor T7 bacteriophage vaccine, and the inhibitory effect of the vaccine on Lewis lung cancer in mice was examined. METHODS: Human VEGF165 gene was cloned from human lung cancer tissue by reverse transcriptase polymerase chain reaction (RT-PCR). Human VEGF165 gene and T7Select10-3b phage gene were recombined to construct T7Select10-3b_VEGF bacteriophage vaccine. The phage titer reached 1 x 1013 pfu/ml. The C57BL/6J mice were randomly divided into 3 groups: 10 in the T7-VEGF vaccine group, 10 in the blank phage group (T7), and 10 in the saline group (NS). Each group of samples was mixed with an equal volume of Freund’s adjuvant. Each group of mice was immunized with 1×10 12 pfu/200×l recombinant phage vaccine, blank phage, and saline each week for 4 weeks. Lewis lung cancer cells were inoculated to observe the growth of the tumor and the state of the mice. After inoculation of the tumor for 14 days, the mice were sacrificed and the tumors were weighed to measure the specific anti-VEGF antibody titer in the mouse serum and the microvessel density (MVD). . Results: The tumor weights of T7-VEGF vaccine group, T7 group, and saline group were (0.543±0.259) g, (0.982±0.359) g, and (1.169±0.460) g, respectively. The tumor weight of the vaccine group was significantly smaller than that of the saline group (P<0.01). The serum of the vaccine group produced an anti-VEGF antibody titer of 1:1000. The MVD of the vaccine group, blank phage group, and saline group were: 8.5±0.8, 16.4±1.3, 18.5±1.6. The MVD in the vaccine group was significantly smaller than that in the saline group. CONCLUSION: The xenogeneic vascular endothelial growth factor gene recombinant T7 bacteriophage vaccine can interfere with the body’s immune tolerance to autologous VEGF, produce higher levels of specific anti-VEGF antibodies, and have the effect of inhibiting the growth of mouse Lewis tumors.