抗肿瘤转移肝素酶特异性抗体的筛选及鉴定

Screening and identification of specific anti-heparanase monoclonal antibody inhibiting tumor metastasis

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体外侵袭实验结果显示12号抗体(100μg,浓度约1 mg/ml)作用于SGC-7901、HepG2、MKN45、SW480和U2OS细胞48 h后,其穿膜细胞数与正常小鼠IgG对照组相比明显减少(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。

Objective To identify monoclonal antibodies against human heparanase and to screen a monoclonal antibody specifically inhibiting tumor metastasis.Methods Through Western blotting,three self-prepared monoclonal antibodies（No.7,No.11,and No.12） were subjected to heparanase specificity identification and screening with total proteins of HepG2,SGC-7901,MKN45,SW480,U2OS and MCF-7 cells as samples.Immunocytochemical assay was used to further verify the specificity of the screened antibody.Transwell cell invasion assay in vitro was used to observe the inhibitory effect of the screened antibody on tumor metastasis.Results All the three monoclonal antibodies could bind heparanase in different tumor cells.Monoclonal antibody No.12,as well as a commercial anti-heparanase monoclonal antibody as positive control,could detect weak positive expression of heparanase in MCF-7 cells,indicating the best specificity.Immunocytochemical assay results showed that monoclonal antibody No.12 could detect positive heparanase expression in SGC-7901,HepG2,MKN45,SW480 and U2OS cells,and weak positive heparanase expression in MCF-7 cells.Transwell cell invasion assay results demonstrated that the migrated cell numbers of SGC-7901,HepG2,MKN45,SW480 and U2OS cells treated by monoclonal antibody No.12（100 μg,1 mg/ml approximately） for 48 h were significantly lowered as compared with those treated by normal mouse IgG for 48 h（P0.05）.Conclusion A self-prepared monoclonal antibody can specifically bind heparanase in tumor cells and inhibit the invasion and metastasis of tumor cells by suppressing heparanase activity.