靶向人SET8单抗的制备及对肝癌细胞增殖、凋亡及细胞周期的影响  

Preparation of human SET8 monoclonal antibody and its effect on hepatocellular carcinoma cell proliferation,apoptosis,and cell cycle

作　　者：王英南[1] 吴建华[2] 吴忱思[1] 张风宾[1] 张瑞星[1] 郭占军[3] WANG Yingnan;WU Jianhua;WU Chensi;ZHANG Fengbin;ZHANG Ruixing;GUO Zhanjun(Department of Gastroenterology,the Fourth Hospital of Hebei Medical University,Shijiazhuang 050011,China;Animal Center,the Fourth Hospital of Hebei Medical University,Shijiazhuang 050011;Department of Rheumatology and Immunology,the Fourth Hospital of Hebei Medical University,Shijiazhuang 050011)

机构地区：[1]河北医科大学第四医院消化内科,石家庄050011 [2]河北医科大学第四医院动物中心,石家庄050011 [3]河北医科大学第四医院风湿免疫科,石家庄050011

出　　处：《中国比较医学杂志》2024年第12期70-76,I0001-I0018,共25页Chinese Journal of Comparative Medicine

基　　金：河北省级科技计划资助(21377759D)。

摘　　要：目的制备针对人SET8的单克隆抗体,并探索其对肝癌细胞增殖、凋亡及细胞周期的影响,同时评估其在肝癌小鼠模型中的抗肿瘤效果。方法通过人SET8多肽片段免疫小鼠,筛选并融合B细胞与骨髓瘤细胞,建立稳定分泌SET8单抗的杂交瘤细胞株。通过小鼠腹腔注射、腹水采集的方式生产并纯化。采用CCK-8法、流式细胞术及Western blot技术,分别分析SET8单抗对肝癌细胞增殖、凋亡、细胞周期及凋亡相关蛋白表达的影响。最后,构建人肝癌细胞移植瘤小鼠模型,体内评估SET8单抗对肿瘤生长的抑制作用。结果人SET8单抗在50μg/mL和100μg/mL浓度下显著抑制Huh-7和Mahlavu肝癌细胞活力(P<0.05),且浓度越高抑制效果越强。流式细胞术分析显示,相较于空白对照组,SET8单抗、紫杉醇及二者联合处理均显著提高Mahlavu细胞凋亡率,且联合组效果最佳(P<0.05)。同时,SET8单抗诱导Mahlavu细胞周期阻滞于S期和G2期,减少G1期细胞。Western blot分析显示,该单抗增加凋亡相关蛋白Bax和Caspase-3的表达(P<0.05)。体内实验显示,SET8单抗单独或联合紫杉醇均有效抑制肝癌细胞在裸鼠体内的增殖,联合组治疗效果最为显著(P<0.05)。结论制备的人SET8单抗有效抑制肝癌细胞增殖,促进肝癌细胞凋亡,并在动物体内展现出良好的抗肿瘤效果。ObjectiveTo prepare human SET8 monoclonal antibody and explore its effects on the proliferation,apoptosis,and cell cycle of hepatoma cells,and to evaluate its anti-tumor effect in mouse models of hepatocellular carcinoma.MethodsWe immunized mice with human SET8 polypeptide fragment and screened and fused B cells and myeloma cells to establish a hybridoma cell line that stably secreted SET8 monoclonal antibody.Production was expanded by intraperitoneal injection into mice and the collection and purification of ascites.We investigated the effects of SET8 monoclonal antibody on the proliferation,apoptosis,cell cycle,and apoptosis-related protein expression of hepatocellular carcinoma cells by CCK-8,flow cytometry,and Western blot,respectively.Finally,we constructed a mouse model of human hepatocellular carcinoma by cell transplantation to evaluate the inhibitory effect of SET8 monoclonal antibody on tumor growth in vivo.ResultsHuman SET8 monoclonal antibody significantly inhibited the viability of Huh-7 and Mahlavu hepatoma cells at concentrations of 50 and 100μg/mL,in a concentration-dependent manner(P<0.05).Flow cytometry analysis showed that SET8 monoclonal antibody,paclitaxel,and their combination significantly increased the apoptosis rate of Mahlavu cells compared with the blank control group,with the combination group having the greatest effect(P<0.05).SET8 monoclonal antibody also induced Mahlavu cell cycle arrest in S and G2 phases and reduced G1 phase cells.Western blot analysis showed that the monoclonal antibody increased the expression of the apoptosis-related proteins Bax and Caspase-3(P<0.05).SET8 monoclonal antibody,alone or in combination with paclitaxel,also effectively inhibited the proliferation of hepatocellular carcinoma cells in nude mice,with the combination therapy having the most significant effect(P<0.05).ConclusionsThe prepared human SET8 monoclonal antibody effectively inhibited the proliferation and promoted the apoptosis of hepatocellular carcinoma cells,and showed good anti-tumor effe

关 键 词：人SET8单抗 肝癌细胞 细胞增殖 细胞凋亡 细胞周期 动物模型 

分 类 号：R-33[医药卫生]