采用肝癌球体细胞筛选鉴定识别肝癌干细胞的单抗  

Identification of monoclonal antibodies against hepatoma stem cells by screening for hepatoma spheroid cells

作　　者：孙力超 杨婧 孙立新 张媛 杨治华 冉宇靓 SUN Lichao;YANG Jing;SUN Lixin;ZHANG Yuan;YANG Zhihua;RAN Yuliang(State Key Laboratory of Molecular Oncology,National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100021,China)

机构地区：[1]国家癌症中心/国家肿瘤临床医学研究中心/中国医学科学院北京协和医学院肿瘤医院/分子肿瘤学国家重点实验室

出　　处：《实用肿瘤学杂志》2019年第3期193-199,共7页Practical Oncology Journal

基　　金：国家自然科学基金资助项目(编号:81773170);中国医学科学院医学与健康科技创新工程(编号:2016-I2M-3-013)

摘　　要：目的采用肝癌球体细胞筛选鉴定识别肝癌干细胞的单抗,为靶向肿瘤干细胞治疗肝癌提供候选治疗单抗。方法采用无血清悬浮培养的方法富集培养肝癌干细胞。通过细胞免疫荧光、顺铂耐药实验、Real-timePCR、裸鼠皮下成瘤实验等方法筛选、鉴定抗肝癌干细胞单抗。采用免疫组织化学的方法鉴定单抗识别的抗原在肝癌组织中的表达情况。质谱鉴定抗原。结果MHCC97L细胞能在无血清悬浮培养条件下形成细胞球,并能被PKH26染料标记。流式细胞检测发现MHCC97L球体细胞中CD90表达比例较亲本细胞提高了3.4倍。无血清成球抑制实验获得6株显著抑制MHCC97L细胞在无血清成球的单抗,抑制率分别为54.67%,50.33%,45.73%,42.26%,39.11%,37.63%。细胞免疫荧光结果显示单抗28C10和CD90在MHCC97L细胞中能共定位。Real-TimePCR检测结果显示MHCC97L28C10^+细胞Sox-2和Oct-4表达显著高于MHCC97L28C10-细胞。流式细胞检测,在MHCC97L及其sphere细胞中28C10^+细胞比例分别为7.98%和10.7%,28C10^+细胞比例提高了1.34倍。流式细胞术分选获得的28C10^+细胞的体外成球能力、侵袭能力显著高于28C10-细胞。CCK-8法检测结果显示28C10^+细胞较28C10-细胞显示出对化疗药物顺铂的耐药,IC50分别为1.96μg/mL和1.16μg/mL。裸鼠致瘤实验结果显示,28C10^+细胞裸鼠皮下接种2×10^4cells/只,2个月可形成肿瘤,成瘤率为40%。另外1只未形成肿瘤的裸鼠形成了肺部转移灶(1/5)。免疫组化检测显示单抗28C10的靶抗原阳性率为72.0%(77/107),而在癌旁组织中低表达,差异有统计学意义。质谱结果显示28C10识别的抗原为HSP90α。结论采用MHCC97L球体细胞模型成功鉴定一株特异识别肝癌干细胞的单抗,为靶向肝癌干细胞的抗体治疗奠定基础。ObjectiveThe objectives of this study were to screen and identify monoclonal antibodies against hepatoma stem cells by screening for hepatoma spheroid cells,and to provide candidate therapeutic monoclonal antibodies for targeting cancer stem cells to treat hepatic cancer.MethodsHepatic cancer stem cells were enriched by serum-free suspension culture.Immunofluorescence,cisplatin resistance assay,Real-time qPCR,subcutaneous tumor formation in nude mice,and other methods were used to screen and identify anti-hepatocarcinoma stem cell monoclonal antibodies.Immunohistochemistry was used to identify the expression of antigen recognized by monoclonal antibody in liver cancer tissues.The antigen was identified by mass spectrometry.ResultsMHCC97L cells were able to form cell spheres in serum-free suspension culture and were labeled with PKH26 dye.Flow cytometry showed that the expression of CD90^+ in MHCC97L spheroid cells was 3.4 times higher than that in the parental cells.In the inhibition experiment of serum-free spheroid,6 monoclonal antibodies significantly inhibited MHCC97L cells in serum-free medium,and inhibitory rates were 54.67%,50.33%,45.73%,42.26%,39.11%,and 37.63%,respectively.The results of immunofluorescence showed that monoclonal antibodies 28C10 and CD90 were colocalized in MHCC97L cells.The results of real-time qPCR showed that the expression of Sox-2 and Oct-4 in MHCC97L 28C10 ^+ cells was significantly higher than those of MHCC97L 28C10 - cells.Flow cytometry showed that the ratio of 28C10 ^+ in MHCC97L cells and its sphere cells were 7.98% and 10.7%,respectively.The ratio of 28C10 + cells was increased by 1.34 times.The in vitro globing ability and invasive ability of 28C10^+ cells obtained by flow cytometry were significantly higher than those of 28C10 - cells.The results of CCK-8 assay showed that 28C10 ^+ cells were resistance to cisplatin in 28C10 - cells,which are 1.96 g/ml and 1.16 g/ml,respectively.Tumorigenic assay showed that 28C10 ^+ cells were inoculated subcutaneously with 2×10^ 4 cells

关 键 词：肝癌 肿瘤干细胞 单克隆抗体 CD90 HSP90Α 

分 类 号：R735.7[医药卫生—肿瘤] R392.11[医药卫生—临床医学]