抗肿瘤转移肝素酶特异性抗体的筛选及鉴定-第三军医大学学报.DOCVIP

抗肿瘤转移肝素酶特异性抗体的筛选及鉴定第三军医大学西南医院全军消化病研究所1放射科2摘要目的 通过鉴定制备的人肝素酶单克隆抗体，筛选出具有抗肿瘤转移特异性的单克隆抗体株，并大量制备。 方法 采用Western blot技术，以HepG2、SGC-7901、MKN45、SW480、U2OS、MCF-7细胞总蛋白为样本，对已制备的3株人肝素酶单克隆抗体进行初步鉴定，筛选出能够与肝素酶蛋白特异性结合的抗体，并采用免疫细胞化学技术进一步验证筛选出的抗体特异性，然后通过Transwell体外侵袭实验观察筛选出的肝素酶单克隆抗体对肿瘤转移的抑制作用。 结果 已制备的3株人肝素酶单克隆抗体均能与不同肿瘤细胞肝素酶蛋白结合，其中12号抗体与商品化肝素酶单克隆抗体在MCF-7细胞中均检测出肝素酶弱阳性表达，其特异性最佳；免疫细胞化学结果也显示在SGC-7901、HepG2、MKN45、SW480和U2OS细胞中12号抗体能检测出肝素酶阳性表达，而在MCF-7细胞中检测出肝素酶弱阳性表达Transwell体外侵袭实验12号抗体SGC-7901、HepG2、MKN45、SW480和U2OS细胞P＜0.05。结论 纯化制备的人肝素酶单克隆抗体能够与肿瘤细胞表达的肝素酶蛋白特异性结合，并通过抑制肝素酶蛋白达到抑制肿瘤细胞的扩散与转移。 肝素酶；单克隆抗体；抗肿瘤转移Screening and identification of anti-heparanase monoclonal antibody against tumour metastasis in vitro Li Ning1, Chen Wei2, Zhang Yafei1, Li Chuan1, Wu Yuyun1, Wang Shuming1, Fang Dianchun1, Yang Shiming1(1 Institute of Gastroenterology of PLA, Southwest Hospital, Third Military Medical University； 2 Department of Radiology, Southwest Hospital, Third Military Medical University) 【Abstract】 Objective To screening the monoclonal antibody against tumour metastasis specifically from anti-heparanase monoclonal antibodies which have been prepared previously. Methods Western blot was used to identify the antibody that could bind to heparanase specifically in three prepared anti-heparanase antibodies, then immunocytochemistry was carried out to demonstrated the specificity of the identified antibody in further, and in the end the effect of the identified antibody on tumour metastasis was observed by transwell test. Results All the three anti-heparanase antibodies could bind to heparanase proteins in different tumour cells. But Number 12 antibody and the purchased anti-heparanase monoclonal antibody detected little expression of heparanase in MCF-7 cells, which meant Number 12 antibody had the best specificity. Immunohistochemisty results also showed Number 12 antibody detected more heparanase expressions in SGC-7901, HepG2, MKN45, SW480 and U2OS cells than in MCF-7 cells. Furthermore, Number 12 ant