转移性胃癌干细胞单抗6H6靶向治疗胃癌的实验研究

Experimental study on targeted therapy of gastric cancer with metastatic gastric cancer stem cell monoclonal antibody 6H6

目的:研究一株识别转移性胃癌干细胞的单克隆抗体6H6的体外功能实验,为靶向治疗胃癌提供候选抗体药物。方法:流式细胞术分选胃癌细胞系SNU-16中CD44(+)细胞,双色细胞免疫荧光染色检测CD44与6H6单抗识别的抗原蛋白在SNU-16细胞中的表达。无血清悬浮培养检测流式分选SNU-16中CD44(+)细胞的自我更新能力及6H6单抗对CD44(+)细胞自我更新能力的影响。Transwell小室法检测SNU-16中CD44(+)细胞的侵袭能力及6H6单抗对CD44(+)细胞侵袭能力的影响,CCK8法检测单抗6H6对SNU-16中CD44(+)细胞顺铂耐药性的影响。裸鼠体内治疗实验分析6H6单抗联合顺铂对SNU-16移植瘤生长的抑制作用。结果:免疫荧光显示单抗6H6识别的抗原能与CD44在SNU-16细胞上共定位。与CD44(-)细胞相比较,成球实验和Transwell实验证明SNU-16中CD44(+)细胞具有更强的自我更新和侵袭能力。抗体的体外功能研究发现,单抗6H6能显著抑制SNU-16中CD44(+)细胞的成球率和侵袭率,抑制率分别达35.8%(P<0.01)和31.6%(P<0.01)。单抗6H6能够有效降低SNU-16中CD44(+)细胞顺铂耐药性,实验组与对照组的IC 50分别为0.76μg/mL和1.98μg/mL。此外,体内治疗实验发现单抗6H6可明显抑制SNU-16移植瘤的生长,当高浓度(40 mg/kg)时,其抑制作用高达81.9%,抗体抑制作用存在剂量依赖关系。结论:单抗6H6体外能够显著抑制转移性胃癌干细胞的干性特征,为胃癌靶向治疗提供有价值的候选靶向药物。

Objective:To identify a monoclonal antibody(McAb)6H6 against metastatic gastric cancer stem cell,to provide candidate antibody drug for targeted therapy of gastric cancer.Methods:Flow cytometry was used to separate CD44(+)cells from SNU-16 to detect the expression of CD44 and antigen recognized by 6H6,which was detected by immunofluorescence.Serum-free suspension culture and Transwell assay were used to detect the self-renewal and invasion ability of CD44(+)cells from SNU-16 sorted by flow cytometry and CD44(+)cells treated by 6H6.The effect of 6H6 on cisplatin resistance was examined by CCK8.The inhibition of SNU-16 implanted tumor growth 6H6 combined with cisplatin was studied by tumor treatment experiments in nude mice.Results:Immunofluorescence showed that the antigen recognized by 6H6 could co-localize with CD44 on 6H6 cells.CD44(+)cells formed a significantly higher number of spheroid colonies compared with CD44(-)cells from SNU-16,which exhibited significantly increased invasion ability.6H6 suppressed the sphere formation of CD44(+)cells from SNU-16(the inhibitory rate 35.8%)(P<0.01).Meanwhile,6H6 can inhibit the invasive ability of CD44(+)cells from SNU-16(the inhibitory rate 31.6%)(P<0.01).6H6 also inhibited the drug-resistance of CD44(+)cells from SNU-16.The IC 50 was 0.76μg/mL in 6H6 group,and 1.98μg/mL in control group.The experiment in vivo showed that 6H6 significantly inhibit the growth of transplanted tumor.When the concentration was high,its inhibitory effect reached 81.9%,and the antibody inhibitory effect was dose-dependent.Conclusion:The monoclonal antibody 6H6 can significantly inhibit the stem characteristics of metastatic gastric cancer stem cells in vitro,providing a valuable candidate targeted drug for gastric cancer.