人源化YKL-40中和抗体(Rosazumab)体外阻断血管再生  

Inhibition of a Humanized YKL-40 Neutralizing Antibody(Rosazumab)on Angiogenesis in Vitro

作　　者：韩笑 朱博文 罗静[1] 陈慧[1] 虞海惠 邵荣 HAN Xiao;ZHU Bo-wen;LUO Jing;CHEN Hui;YUHai-hui;SHAO Rong(Department of pharmacology and chemical biology,College of Basic Medical Sciences,Shanghai Jiao Tong University,Shanghai,200025,China;Shanghai Key Laboratory of Biliary Tract Disease Research,Xinhua Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai,200092,China)

机构地区：[1]上海交通大学基础医学院药理学与化学生物学系,上海200025 [2]上海市胆道疾病重点实验室,上海交通大学医学院附属新华医院,上海200092

出　　处：《现代生物医学进展》2021年第16期3001-3006,共6页Progress in Modern Biomedicine

基　　金：国家自然科学基金面上项目(81772512);上海市科委科研基金项目(20S11901700);上海交通大学附属新华医院基金项目(J2PI201716)。

摘　　要：目的:血管再生是实体肿瘤的生长和恶性转移的一个必须病理过程,阻断这一过程能够有效阻止恶性肿瘤的发生发展。YKL-40是血管再生因子,能刺激肿瘤的血管再生。本文探讨了一个原创性人源化抗YKL-40单克隆抗体(Rosazumab,命名为洛沙单抗)阻断YKL-40血管生成的体外功能。方法:Western Blot检测洛沙单抗对分泌和重组YKL-40蛋白的特异性结合;Western Blot及考马斯亮蓝染色检测该抗体的抗体特异性和纯度;Live/Dead染色试验检测抗体的细胞毒性。以人微血管内皮细胞(Human microvascular endothelial cells,HMVECs)为研究对象,引入重组YKL-40蛋白或脑胶质瘤细胞(Glioblastoma serum-differentiated cells,GSDCs)的条件培养基进行培养。Transwell试验检测该抗体对HMVECs迁移力的影响,并用Matrigel试验检测对微管形成的作用。结果:洛沙单抗可特异性结合YKL-40,考马斯亮蓝染色进一步证明其纯度及特异性。体外血管再生试验包括细胞迁移力和微管形成证明该抗体可以有效中和重组YKL-40蛋白及肿瘤细胞条件培养基中的YKL-40,抑制HMVECs的迁移和血管形成。结论:洛沙单抗是首创的人源化中和YKL-40的抗体,其特异性高,细胞毒性低,能显著抑制YKL-40血管再生功能,为下一步体内试验奠定基础。本研究可为YKL-40诱导的肿瘤血管生成及恶性肿瘤转移提供一种新的治疗手段。Objective:Angiogenesis is a key pathologic process for the development and metastasis of solid tumors.Blocking this event can effectively prevent tumor development and malignant transformation.A secreted protein YKL-40 is an angiogenic factor.In this study,we created a new humanized anti-YKL-40 monoclonal antibody,named Rosazumab,and investigated its effects on angiogenesis induced by YKL-40 in vitro.Methods:Western blot was engaged to define the specific binding of Rosazumab to YKL-40.The specificity and purity of Rosazumab were also analyze by Western blot and Coomassie blue staining.Live/Dead staining was used to determine cytotoxicity of this antibody.Rosazumab was introduced to human microvascular endothelial cells(HMVECs)in Transwell to detect the effect on the cell migration,and in Matrigel to detect the effect on microtubule formation in the presence of recombinant protein YKL-40 or conditioned medium from brain tumor cell line GSDC.Results:Western blot showed that Rosazumab specifically recognized YKL-40,Coomassie brilliant blue staining further confirmed its purity and specificity.Live/dead study did not exhibit the cytotoxicity of different doses of Rosazumab to the cells.Transwell assay showed that the antibody effectively blocked HMVEC migration and tube formation by neutralizing recombinant protein YKL-40 and the YKL-40 in the conditioned medium of GSDC.Conclusions:Rosazumab,the first humanized YKL-40 monoclonal antibody has high specificity to bind to YKL-40 and low cytotoxicity.The antibody can effectively inhibit angiogenesis induced by YKL-40 in vitro,which lays the foundation for the following studies in vitro.Therefore,our study may provide a new therapeutic tool targeting YKL-40-induced tumor angiogenesis and subsequent metastasis of malignant tumors in the future.

关 键 词：YKL-40 人源抗体 血管生成 HMVECs 肿瘤转移 

分 类 号：R-33[医药卫生] R730.231