目的： 探索抗HSP90单克隆抗体28C10通过靶向肿瘤干细胞促进顺铂（cisplatin，DDP）对人胃癌细胞PAMC82恶性生物学行为的抑制效果及其可能的作用机制。 方法: 28C10单独或与DDP联合处理人胃癌细胞PAMC82，采用不同实验方法检测该细胞的无血清成球能力、迁移和侵袭能力与克隆形成能力，CCK-8法检测28C10对PAMC82细胞恶性生物学行为和协同DDP抗癌能力的影响。采用细胞免疫荧光及流式细胞术检测PAMC82细胞中HSP90及eHSP90 （extracellular HSP90）的表达、定位、eHSP90 + 亚群比例，以及28C10处理后对ALDH + 、CD44 + 、eHSP90 + 细胞亚群的影响。采用WB实验检测28C10作用后PAMC82细胞中HSP90、干性相关蛋白以及PI3K/AKT/mTOR信号通路蛋白表达的变化。 结果： 胃癌细胞PAMC82膜表面表达eHSP90，具有2%~3%的eHSP90 + 细胞亚群，且eHSP90 + 细胞多为与ALDH + 或CD44 + 共阳性细胞。28C10处理能显著抑制PAMC82细胞的成球、克隆形成、增殖、耐药、迁移及侵袭能力，而且和DDP联用的效果更明显（P<0.05或P<0.01）。流式细胞术分析发现28C10处理显著抑制PAMC82细胞的eHSP90 + 、ALDH + 和CD44 + 亚群数量（均P<0.01）。免疫荧光实验发现28C10作用后eHSP90发生内吞，WB实验结果显示eHSP90、CD44、ALDH和干性相关蛋白OCT4、SOX2表达量均降低（P<0.05或P<0.01）。 结论 ：抗HSP90单克隆抗体28C10可靶向胃癌PAMC82细胞的ALDH + 、CD44 + 肿瘤干细胞相关亚群、内化eHSP90且降低细胞总HSP90的水平、抑制PI3K/AKT/mTOR信号通路，从而有效地抑制PAMC82细胞的干性、耐药和其他恶性生物学行为，协同DDP显著提高抗癌效果。

Objective: To explore the promotive effect of anti-HSP90 monoclonal antibody mAb 28C10 on Cisplatin (DDP) inhibiting malignant biological behavior of human gastric cancer PAMC82 cells by targeting tumor stem cells and the possible mechanisms of action. Methods: Human gastric cancer PAMC82 cells were treated with 28C10 alone or in combination with DDP. Then, the serum- free pellet-forming ability, clone-forming ability, migration and invasion ability of PAMC82 cells were detected by different experiments, and the effects of 28C10 on the malignant biological behaviors of PAMC82 cells and the synergistic anti-cancer effect of 28C10 and DDP were detected by CCK-8 method. The expression, localization, eHSP90 + subgroup ratio of HSP90 and eHSP90 (extracellular HSP90) in PAMC82 cells and the effects of 28C10 on ALDH + , CD44 + , eHSP90 + subgroups of PAMC82 cells were detected by cellular immunofluorescence and flow cytometry. The changes in HSP90, stemness-related proteins and PI3K/AKT/mTOR signaling pathway in PAMC82 cells treated with 28C10 were detected by WB. Results: eHSP90 was expressed on the membrane surface of gastric cancer PAMC82 cells, and there were about 2%-3% of eHSP90 + subgroup cells. eHSP90 + cells were mostly co-positive with ALDH + or CD44 + cells. 28C10 significantly inhibited the ability of pellet formation, clone formation, proliferation, drug resistance, migration and invasion of PAMC82 cells; moreover, the effect was more obvious when combined with DDP (all P<0.05 or P<0.01). Flow cytometry analysis showed that 28C10 treatment significantly inhibited the number of eHSP90 + , ALDH + and CD44 + subgroups of PAMC82 cells (all P<0.01). Immunofluorescence assay showed that endocytosis of eHSP90 occurred after 28C10 treatment. WB results showed that the expression levels of eHSP90, CD44, ALDH and the expression of stemness-related proteins OCT4 and SOX2 were decreased (P<0.05 or P<0.01). Conclusion: 28C10 can target ALDH + and CD44 + tumor stem cell-related subgroups of gastric cancer PAMC82 cells, internalize eHSP90, reduce the level of total HSP90, and inhibit PI3K/AKT/mTOR signaling pathway, thereby effectively inhibiting stemness, drug resistance and other malignant biological behaviors of PAMC82 cells and synergistically improving the anticancer effect of DDP.

R735.2；R730.554；R730.53

国家自然科学基金面上项目资助（No.81773170；No.82073278）；中国医学科学院医学与健康科技创新工程资助项目（No. 2021-I2M-1-067）；北京自然科学基金面上项目资助（No.7222012）