hVEGF-GnRH3疫苗降低C57BL/6小鼠B16F10黑色素瘤血管生成及增强炎症免疫调节作用  

hVEGF-GnRH3 Vaccine Reduces B16F10 Melanoma Angiogenesis and Enhances Inflammatory Immunomodulation in C57BL/6 Mice

作　　者：吴越 方越 韩淳致 李虹霖 王晨璐 王睿[1] 曹荣月[1] WU Yue;FANG Yue;HAN Chun-zhi;LI Hong-lin;WANG Chen-lu;WANG Rui;CAO Rong-yue(School of Life Science and Technology,China Pharmaceutical University,Nanjing 211198 China)

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京211198

出　　处：《药物生物技术》2022年第2期132-140,共9页Pharmaceutical Biotechnology

基　　金：The National Natural Science Foundation of China(No.81673340);The National Training Programs of Innovation and Entrepreneurship for Undergraduates(No.J1310032;202010316014G、202110316102、202210316256);2020 China College students’“Internet+”Innovation and Entrepreneurship Incubation Programme(No.3312000657)。

摘　　要：本研究旨在研究融合蛋白促性腺激素释放激素(Gonadotropin-releasing hormone,GnRH)和h VEGF-GnRH3(VG)对C57BL/6J小鼠B16F10黑色素瘤的抑制作用,并对其抗肿瘤作用机制进行初步研究。GnRH和VG的靶向抗原过量表达可以促进黑色素瘤的转移。且有研究证明,VG疫苗能引起针对前列腺癌的免疫炎症反应。因此,作者假设VG也有防止黑色素瘤血管生成、进展和复发的潜力。通过大肠杆菌表达系统获取重组蛋白h VEGF-GnRH3,并通过DEAE阴离子交换色谱法纯化,制备蛋白疫苗。雄性C57BL/6J小鼠皮下注射B16F10黑色素瘤细胞(每只小鼠1×10^(6)个细胞),构建黑色素瘤模型。荷瘤小鼠用VG蛋白疫苗免疫三个周期。接着,从肿瘤生长、血管生成和肿瘤组织学、外周和脾细胞体液、细胞和细胞内免疫调节等方面,对疫苗的抑瘤效果进行了评估。结果显示,VG组黑色素瘤的体积和血管生成受到明显抑制(P<0.01)。与PBS组相比,VG提高了小鼠血清中抗GnRH、抗VEGF Ig G和循环干扰素-γ(IFN-γ)的水平,而IL-6水平明显降低。在脾脏相关实验中,通过MTT法检测脾细胞增殖情况。结果显示,VG疫苗能促进脾细胞增殖,并增强脾细胞的细胞毒作用。同时,对细胞内细胞因子水平进一步分析,结果显示CD3^(+)CD4^(+)IFN-γ^(+)细胞明显增加。综上所述,VEGFGnRH3是一种很有前景的治疗黑色素瘤的疫苗。This study aimed at investigating the inhibitory effects and the anti-tumor mechanisms of co-administration of fusion proteins gonadotropin-releasing hormone(GnRH)and h VEGF-GnRH3(VG)on B16F10 melanoma in C57BL/6J mice model.The metastasis of melanoma can be promoted by overexpression of the targeting antigens GnRH and VG.VG vaccine has been shown to elicit an immune inflammatory response against prostate cancer.Hence the authors hypothesized VG also has potential to prevent melanoma angiogenesis,progression and recurrence.Recombinant protein h VEGF-GnRH3 was extracted by E.coli expression system and purified by DEAE anion exchange chromatography to prepare protein vaccine.B16F10 melanoma cells were transplanted into male C57BL/6J mice(1×10^(6)cells per mouse)to construct a melanoma model.The tumor-bearing mice were immunized with VG protein vaccine for three cycles.Thereafter,the tumor suppressing effect of the vaccine was evaluated from the aspects of tumor growth,angiogenesis and tumor histology,periphery and splenocytes humoral,cellular,and intracellular immunomodulation also included.The results showed that the volumes and angiogenesis of melanoma were significantly inhibited in VG group(P<0.01).Compared to the PBS group,VG increased the levels of anti-GnRH,anti-VEGF Ig G and circulating interferon-gamma(IFN-γ)in mice serum,while the level of IL-6 was significantly decreased.In the spleen-related experiments,the MTT assay showed that VG vaccine promoted splenocytes prolife-ration and cytotoxicity.At the same time,further analysis of intracellular cytokines revealed a significant increase in CD3^(+)CD4^(+)INF-γ^(+)cells.The results above indicate that VEGF-GnRH3 is a promising vaccine for the treatment of melanoma.

关 键 词：B16F10黑色素瘤 重组疫苗 促性腺激素释放激素(GnRH) 人类血管内皮生长因子(h VEGF) 干扰素-γ(INF-γ) 免疫治疗 

分 类 号：Q78[生物学—分子生物学]