靶向胃癌干细胞单克隆抗体2C12的鉴定及功能研究

目的:研究胃癌干细胞的功能性单克隆抗体2C12体外功能实验，为胃癌干细胞的靶向治疗提供候选单抗药物。方法:以胃癌细胞系SNU-5为细胞模型，流式细胞术检测其亲本细胞和通过无血清悬浮培养的球体细胞中2C12+细胞的表达水平，双色细胞免疫荧光检测单抗2C12和CD90识别的抗原在SNU-5细胞中的表达情况。流式细胞术分选SNU-5细胞中2C12+和2C12-细胞，采用无血清悬浮培养成球实验和Transwell小室法分别检测其自我更新能力和侵袭能力。用单抗2C12处理SNU-5的球体细胞后，检测其对SNU-5的球体细胞自我更新能力和侵袭能力的影响。结果:2C12+细胞在SNU-5的球体细胞中的表达水平明显高于亲本细胞。免疫荧光结果显示单抗2C12识别的抗原分子能与CD90在SNU-5细胞上共定位。流式细胞术分选结果表明SNU-5细胞中2C12+细胞成球数明显高于2C12-细胞（103.3±9.07 vs 50.67±5.86）（P<0.01），侵袭能力也明显较高（209.3±12.9 vs 99.0±3.61）（P<0.01）。对2C12单抗的体外功能研究发现，不同浓度的单抗2C12（0 μg/mL、20 μg/mL、40 μg/mL）能显著抑制SNU-5的球体细胞的成球能力（122.0±5.66、83.0±5.66、59.0±4.24），抑制率达到31.97%和51.64%，同时侵袭能力也显著降低（178.0±8.49、106.5±5.0、78.0±2.83），抑制率达到40.17%和56.18%。结论:单抗2C12是一株抗胃癌干细胞的功能性单抗，能够显著抑制胃癌干细胞的干性相关特征，为胃癌干细胞靶向治疗的候选抗体药物。

Identification and functional study of 2C12 monoclonal antibody against gastric cancer stem cells

Objective:To identify functional monoclonal antibody (2C12) that can target gastric cancer stem cells (CSCs) and provide antibody candidate drugs for targeted therapy of gastric CSCs.Methods:Taking gastric cancer cell line SNU-5 as a cell model,the expression of 2C12+ cells from parent cells and sphere cells was detected by flow cytometry.The expression of antigen protein,which was identified by both monoclonal antibody 2C12 and CD90 in SNU-5 cells,was detected by two-color immunofluorescence.Flow cytometry was used to separate 2C12+ cells and 2C12- cells.The spheroidization experiment in serum-free suspension culture and Transwell method were used to detect their self-renewal ability and invasion ability.After SNU-5 sphere cells were treated with 2C12,SNU-5 sphere cells' ability of self-renewal and invasion was detected.Results:The expression of 2C12+ cells from sphere cells was significantly higher than that of parent cells from SNU-5 cells.The cell immunofluorescence analysis showed that the antigen identified by 2C12 and CD90 could be co-localized in SNU-5 cells.Sphere formation abilities in 2C12+ and 2C12- SNU-5 cells were identified (103.3±9.07 vs 50.67±5.86)（P<0.01）.The invasion ability of 2C12+ cells was significantly higher than that of 2C12- cells (209.3±12.9 vs 99.0±3.61)（P<0.01）.The study on functional properties of antibody in vitro illustrated that different concentrations of 2C12 (0 μg/mL,20 μg/mL,40 μg/mL) could significantly inhibit sphere formation of sphere cells from SNU-5 cells (122.0±5.66，83.0±5.66，59.0±4.24),and the inhibition rate reached 31.97% and 51.6%,meanwhile,2C12 could inhibit the invasion ability of sphere cells from SNU-5 cells (178.0±8.49，106.5±5.0，78.0±2.83),and the inhibition rate was 40.17% and 56.18%.Conclusion:2C12 monoclonal antibody is one of functional monoclonal antibodies targeting gastric CSCs,which can significantly inhibit the stem-related characteristics of gastric CSCs,and could be a candidate drug for the targeted therapy of gastric CSCs.