一株胃癌干细胞功能性单克隆抗体的鉴定及功能研究  

Identification and Functional Study of a Functional Monoclonal Antibody Against Gastric Cancer Stem Cells

作　　者：矫婕[1] 舒雄[2] 刘辉琦[2] 孙力超[2] 冉宇靓[2] 遇珑[2] 孙立新[2] 杨治华[1] 

机构地区：[1]广西医科大学,广西南宁530021 [2]中国医学科学院北京协和医学院肿瘤研究所,分子肿瘤学国家重点实验室,北京100021

出　　处：《肿瘤学杂志》2016年第4期284-290,共7页Journal of Chinese Oncology

基　　金：国家863计划青年科学家专题(2014AA020537)

摘　　要：[目的]从抗人胃癌干细胞单克隆抗体杂交瘤库中筛选鉴定识别胃癌干细胞的单克隆抗体,并证明其具有抑制胃癌干细胞自我更新能力和侵袭功能的功能性单抗,为靶向人胃癌干细胞治疗胃癌提供有治疗潜能的单抗候选药物。[方法]采用无血清培养、PKH26染色及流式细胞术等方法确定人胃癌细胞系SNU-5中存在肿瘤干细胞,可作为研究抗人胃癌干细胞单抗的细胞模型。应用双色免疫荧光、流式细胞分选等技术分析鉴定单克隆抗体25G5是识别胃癌干细胞的单克隆抗体。用肿瘤干细胞的成球生长实验、Transwell侵袭实验及耐药性实验研究分析25G5单抗对胃癌干细胞功能的影响。[结果]SNU-5细胞在无血清培养基中存活并增殖成球形生长,SNU-5球体细胞中表达胃癌干细胞标志物CD44^+和CD90^+的细胞比例分别为72.4%和8.55%,较亲本细胞分别提高了21.3倍和2.4倍,表明SNU-5中存在有CD44^+、CD90^+的胃癌干细胞。细胞免疫荧光结果显示25G5单抗识别的抗原分子与CD44、CD90胃癌干细胞标志物共染表达于胃癌干细胞膜上。流式细胞术分选的25G5^+细胞的成球率为(21.4±0.3)%,显著高于25G5-细胞(12.3±0.7)%和亲本细胞(16.1±1.0)%。Transwell侵袭实验、细胞耐药性实验和动物体内致瘤实验结果显示25G5^+细胞的侵袭能力和耐药能力也显著高于25G5-细胞和亲本细胞,表明25G5单抗识别的细胞是具有自我更新、高侵袭、高耐药性特征的肿瘤干细胞。体外功能研究发现,单抗25G5能显著抑制SNU-5胃癌细胞在无血清培养液中的成球生长,抑制率可达46.4%,也能显著地抑制SNU-5成球细胞(Sphere细胞)的侵袭,抑制率达58.4%。单抗25G5是一株能显著抑制SNU-5中肿瘤干细胞自我更新和侵袭能力的功能性抗人胃癌干细胞单抗。[结论]单克隆抗体25G5是一株抗胃癌干细胞的功能性单抗,为胃癌干细胞靶向治疗的候选抗体药物。[Objective] To screen and identify monoclonal antibody（m Ab） which recognize gastric cancer stem cells from m Ab hybridoma library which against human gastric cancer stem cells,and to test inhibit self-renew and invasive ability of gastric cancer stem cells,so as to provide candidate antibody drugs for stem cell targeted therapy of gastric cancer. [Methods] Cancer stem cells in SNU-5 cell line proved with serum-free medium,PKH26 stain and flow cytometry. Dual-fluorescent,fluorescence-activated cell sorting（FACS） were used to identify 25G5 recognize gastric cancer cells. Effect of 25G5 on gastric cancer stem cells were detected by sphere forming,transwell assay and drug resistance analysis. [Results] SUN-5 cells could survive,proliferate and form sphere cells in serum-free medium. The percentage of CD44^＋and CD90^＋cells population in sphere cells were 72.4% and 8.55%,respectively. Those percentage increased by 21.3 and 2.4folds compared to SUN-5 parent cells. Cell immunofluorescence showed that antigen recognized by 25G5 co-expression with CD44 and CD90 on cell membrane. Then we applied the FACS to isolate 25G5^＋cells for sphere culture,invasion assay,chemosensitivity and tumorigenicity test.The sorted 25G5^＋cell presented stem cell-like features,including self-renewal [sphere formation rate：（21.4±0.3）% vs（12.3±0.7）%],invasion ability [the number of invasive cells：（244.5±13.5）vs（122.4±7.2）] and tumorigenic capacity and chemosensitivity. All these results demonstrated that 25G5^＋cells were cancer stem cells featured with strong self-renewal,invasive and drug resistance. In vitro functional assay showed that m Ab 25G5 significantly suppressed the sphere formation ability of SUN-5 cells,with the inhibitory rates being 46.4%. MAb 25G5 significantly suppressed the invasive ability of SNU-5 cells,with the inhibitory rates being 58.4%. [Conclusion]MAb 25G5 is a functional antibody which against gastric cancer stem cells and could be the candidate antibody drug targeting cancer st

关 键 词：人胃癌细胞 肿瘤干细胞 单克隆抗体 CD44 CD90 

分 类 号：R735.2[医药卫生—肿瘤]