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[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 against human gastric cancer stem cell monoclonal antibody hybridoma and to prove that it has the function of inhibiting self-renewal and invasion of gastric cancer stem cell monoclonal antibody, targeting human gastric cancer stem cells Treating stomach cancer provides therapeutic potential monoclonal antibody candidates. [Method] Using serum-free culture, PKH26 staining and flow cytometry to determine the existence of cancer stem cells in human gastric cancer cell line SNU-5 can be used as a cell model for the study of anti-human gastric cancer stem cell monoclonal antibody. Application of two-color immunofluorescence, flow cytometry and other technical analysis identified monoclonal antibody 25G5 is a monoclonal antibody to identify gastric cancer stem cells. The effect of 25G5 McAb on the function of gastric cancer stem cells was analyzed by the growth of tumor stem cells, transwell invasion assay and drug resistance experiment. [Results] SNU-5 cells survived in serum-free medium and proliferated into a spherical shape. The percentage of cells expressing SND markers CD44 + and CD90 + in SNU-5 spheroids was 72.4% and 8.55%, respectively The parental cells were increased 21.3 times and 2.4 times respectively, indicating that there are CD44 +, CD90 + gastric cancer stem cells in SNU-5. Immunofluorescence results showed that the antigen molecules recognized by 25G5 mAb were co-transfected with CD44 and CD90 gastric cancer stem cell markers on the membrane of gastric cancer stem cells. The percentage of scaffolds of 25G5 ~ + cells sorted by flow cytometry was (21.4 ± 0.3)%, significantly higher than that of 25G5-cells (12.3 ± 0.7)% and parent cells (16.1 ± 1.0)%. Transwell invasion assay, cell resistance assay and in vivo tumorigenicity assay showed that the invasiveness and drug resistance of 25G5 ~ + cells were also significantly higher than that of 25G5-cells and parental cells, indicating that the cells identified by 25G5 mAbs are self-renewal , High invasion, high resistance characteristics of cancer stem cells. In vitro functional studies showed that McAb 25G5 significantly inhibited the growth of SNU-5 gastric cancer cells in serum-free medium with a suppression rate of 46.4% and a significant inhibition of SNU-5 spheroid cells Invasion, the inhibition rate of 58.4%. McAb 25G5 is a functional anti-human gastric cancer stem cell monoclonal antibody that significantly inhibits the ability of tumor stem cells to self-renew and invade in SNU-5. [Conclusion] Monoclonal antibody 25G5 is a functional monoclonal antibody against gastric cancer stem cells and is a potential antibody drug for the targeted therapy of gastric cancer stem cells.