HIF-1α纳米抗体的制备及其抑制黑素瘤生长的作用  

Effect on Preparation of HIF-1αNano-Antibody and Its Inhibition of Melanoma Growth

作　　者：李佳敏 贾琼 秦蓉芬 迟志端 王富明 范瑞文[1] LI Jiamin;JIA Qiong;QIN Rongfen;CHI Zhiduan;WANG Fuming;FAN Ruiwen(College of Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,China;Jinzhong City Zhuangzi Integrated Convenient Service Center,Jinzhong 030600,China)

机构地区：[1]山西农业大学动物医学学院,山西太谷030801 [2]晋中市庄子乡综合便民服务中心,山西晋中030600

出　　处：《山西农业科学》2023年第12期1435-1441,共7页Journal of Shanxi Agricultural Sciences

基　　金：“三晋学者”支持计划专项经费。

摘　　要：缺氧诱导因子1α(Hypoxia inducible factor 1α,HIF-1α)参与低氧微环境相关疾病的发生等过程,具有控制肿瘤生长和发展的功能。黑色素瘤是一种发生于人和动物恶性程度较高的肿瘤。为探明HIF-1α纳米抗体对黑色素瘤的影响,研究利用前期保存的羊驼源黑色素瘤细胞噬菌体文库筛选HIF-1α纳米抗体,经原核表达与纯化后,通过Western Blot和免疫组织化学验证HIF-1α纳米抗体与抗原的结合性;分别通过CCK-8法、划痕试验以及Western Blot法检测其对B16黑素瘤细胞的增殖和迁移能力及其相关分子表达的影响。结果表明,经表达和纯化获得的HIF-1α纳米抗体分子质量约为16 ku,没有跨膜结构,具有亲水性。通过Western Blot和免疫组织化学验证了其具有良好的抗原结合性。在增殖试验和划痕试验中,与对照组相比,HIF-1α纳米抗体抑制了B16细胞的增殖和迁移,下调了靶基因VEGF的表达,并使细胞增殖和迁移相关蛋白Ras、ERK、RAC和RAF的表达量下调。预测HIF-1α纳米抗体进入B16细胞内,与抗原结合,通过下游靶基因VEGF下调RAs、ERK、RAC、RAF的表达,从而对细胞增殖和迁移起抑制作用,可作为黑色素瘤治疗的新靶点。The hypoxia inducible factor 1α(HIF-1α)is involved in the occurrence of diseases related to hypoxia microenvironment and has the function of controlling tumor growth and development.As we known,melanoma is a highly malignant tumor occurring in animals and humans.To explore the effect of HIF-1αnano-antibody on melanoma,in this study,the phage library of alpaca-drived melanoma cells previously preserved in our laboratory was used to screen HIF-1αnanoantibodies.After prokaryotic expression and purification,the binding of HIF-1αnano-antibody and its antigen was verified by Western blot and immunohistochemistry.The effects of HIF-1αnano-antibody on the proliferation and migration of B16 melanoma cells and the expression of related molecules were detected by CCK-8,wound healing test,and Western blot methods.The results showed that HIF-1αnano-antibody obtained by expression and purification was hydrophilic protein without transmembrane structure and had a molecular weight of about 16 ku.Western blot and immunohistochemistry results showed that it had good antigenic binding.In the proliferation and wound healing experiments,HIF-1αnano-antibody inhibited the proliferation and migration of B16 cells,down-regulated the expression of target gene VEGF and the proliferation and migration related proteins Ras,ERK,RAC,and RAF,comparing with the control group.In Conclusion,it was predicted that HIF-1αnano-antibody entered B16 cells and combined with antigens and down-regulated the expression of RAs,ERK,RAC,RAF through the downstream target gene VEGF,which inhibited cell proliferation and migration,and could be used as a new target for melanoma treatment.

关 键 词：HIF-1Α 纳米抗体 B16细胞 Western Blot法 CCK-8法 细胞增殖 细胞迁移 

分 类 号：R739.5[医药卫生—肿瘤]