【作者】 陈明清； 王熙才； 伍治平； 陈艳； 王志强； 许岩； 杜伟；

【Author】 Chen Ming-Qing Wang Xi-Cai Wu Zhi-Ping Chen Yan Wang Zhi-qiang Xu Yan Du Wei Department of Surgical Oncology, The First Affiliated Hospital of Kunming Medical College,Kunming,650032 Tumor Institute of Yunnan Province,Kunming,650118

【机构】 昆明医学院第一附属医院肿瘤中心； 云南省肿瘤研究所；

【摘要】 目的:血管内皮生长因子(vascular endothelial growth factor,VEGF)及其主要受体血管内皮生长因子受体-2(vascular endothelial growth factor receptor-2, V E G F R-2)在肿瘤血管生成过程中起着重要作用。 VEGFR-2在鼠中为flk-1,本研究以减毒沙门氏菌SL3261 为载体制备了抗肿瘤血管生成口服DNA疫苗pcDNA3．1+／ flk-1(n1-7),研究该疫苗抗BALB／c小鼠结肠癌生长的作用,并探讨其可能的作用机制。方法:提取BALB／c胎鼠总 RNA,采用RT-PCR技术扩增出VEGFR2胞外cDNA全长序列flk-1(n1-7),并将其插入载体质粒pcDNA3．1+中,构建重组质粒pcDNA3．1+／flk-1(n1-7),经脂质体介导转染 COS-7细胞,Western blot检测蛋白表达。并进一步将重组质粒pcDNA3．1+／flk-1(n1-7)转化减毒沙门氏菌SL3261, 制备成以SL3261为载体的口服DNA疫苗。小鼠随机分组, 口服接种疫苗两周后,用结肠腺癌细胞CT-26系建立结肠癌皮下动物模型,接种肿瘤细胞28天后处死小鼠,测肿瘤体积, 称瘤重,免疫组化法观察肿瘤微血管密度。流式细胞仪分别于接种疫苗前、接种疫苗后、接种CT-26细胞后测血液CD4+ 细胞和CD8+细胞的变化。结果:成功地克隆出VEGFR-2 胞外cDNA序列flk-l(n1-7),构建成重组质粒pcDNA3．1+／ flk-1(n1-7),Western blot法检测到flk-1(n1-7)在COS- 7细胞中的蛋白表达,成功制备成以SL3261为载体的口服 DNA疫苗pcDNA3．1+／flk-l(n1-7)。疫苗组与对照组的肿瘤重量、体积和微血管密度,均有显著性差异(P<0．05),对照组之间的肿瘤重量、体积和微血管密度之间无显著性差异 (P>0．05)。CD4+细胞在接种疫苗前、后及接种肿瘤后三组之间没有明显差异,(P>0．05),CD8+细胞在疫苗接种前三组没有明显的区别,(P>0．05)。接种疫苗后,CD8+细胞在疫苗组明显的增高,与其他两组相比(P<0．05)。在建立肿瘤模型三周后,CD8+细胞在疫苗组明显比另外两组高,(P<0．05), 结论:构建了重组质粒pcDNA3．1+／flk-1(n1-7),制备成以 SL3261为载体的口服DNA疫苗pcDNA3．1+／flk-1(n1-7)。该疫苗能显著提高小鼠细胞免疫水平,抑制小鼠结肠腺癌的血管生成,进而抑制肿瘤细胞的生长。更多还原

【Abstract】 Objective: Vascular endothelial growth factor (VEGF) and its receptor(VEGFR-2) is a key regulator of vasculogenesis in tumor growth and metastasis. This study was to construct an oral DNA vaccine pcDNA3. l+/flk-l(nl-7) against tumor growth by inhibit angio-genesis and investigate the effects and mechanism of the vaccine on tumor development in vivo. Methods: 1. Extracellular domain of flk-1 was amplified by RT-PCR and was inserted into eucaryotic expression vector pcDNA3.1+. 2.The recombinant plasmid pcDNA3.1+/flk-l(nl-7) was transformed into attenuated Salmonella typhimurium SL3261 to develop an oral DNA vaccine against angiogenesis 3. Mice were randomly separated into deferent groups and orally immunized with the oral DNA vaccine respectively. Then mice was challenged subcutaneously with colonic adenocarcinoma cells in the right armpit. Tumor volume, weight, Microvessel density (MVD), CD4+ cells and CD8+ T cells were analyzed after 28 days. Results: 1. The extracellular domain of flk-1 cDNA was amplified and pcDNA3.1+/flk-l(nl-7) was successfully constructed. 2. An oral DNA vaccine carried with attenuated Salmonella typhimurium SL3261 was developed. 3. Tumor volume, weight and microvessel density were significantly higher in the empty vector group and saline group than that of the DNA vaccine group (P<0.05). CD8+ cells level was obviously higher in the DNA vaccine group than that of the vector group and the saline group (P<0.05). Conclusion: Oral DNA vaccine, extracellular domain of flk-1 cDNA, can induce a anti-cancer effect in colonic adenocarcinoma in mouse. The mechanism is related with against and stimulating cellular immunity.更多还原