目的：筛选识别肝癌干细胞的单克隆抗体并研究其体外的抗肿瘤作用，为肝癌干细胞靶向治疗提供候选抗体药物。方法：无血清悬浮培养及PKH26染色分析人肝癌细胞株MHCC97H中是否存在肝癌干细胞。流式细胞术检测MHCC97H细胞及其成球细胞中7种肿瘤干细胞标志物的表达，以及MHCC97H细胞中肝癌干细胞标志物CD90和不同杂交瘤单抗3G7、4F11、11C9、15B7、15D2识别抗原的共表达情况。无血清悬浮培养法和CCK8法检测单抗4F11对MHCC97H细胞及其成球细胞自我更新和增殖的影响，Transwell实验检测单抗4F11对MHCC97H细胞体外侵袭和迁移的影响。结果：PKH26染色实验显示， MHCC97H细胞球体由单个肝癌干细胞增殖分化形成。MHCC97H细胞球中CD90+MHCC97H细胞比例较亲本MHCC97H细胞显著增加\[（18.0?7.5）% vs（2.3?1.0）%，P<0.05\]。杂交瘤单抗4F11、3G7、11C9、15B7、15D2均能识别MHCC97H细胞中CD90+MHCC97H细胞，其中单抗4F11对CD90+MHCC97H细胞的识别比例为（47.2?4.4）%，其对MHCC97H成球细胞增殖的抑制率远大于对亲本MHCC97H细胞增殖的抑制率\[（29.4?3.8）% vs（12.0?2.2）%，P<0.05\]。单抗4F11抑制MHCC97H细胞成球，抑制率达（58.0?20.8）%。单抗4F11能显著抑制MHCC97H细胞体外侵袭和迁移，抑制率分别为（48.6?5.1）%和（47.6?3.6）%。结论：杂交瘤单抗4F11能特异性识别CD90+肝癌干细胞，抑制肝癌细胞的侵袭和迁移，可作为肝癌干细胞靶向治疗的候选抗体药物。

Objective:To screen the monoclonal antibody (McAb) recognizing liver cancer stem cells and study its anticancer effects in vitro so as to provide antibody drug candidates for liver cancer stem cell-targeted therapy. Methods: Serum-free suspension culture and PKH26 staining was used to determine whether there were liver cancer stem cells in human hepatoma cell line MHCC97H. Expressions of 7 cancer stem cell biomarkers in MHCC97H cells and MHCC97H sphere cells, and co-expressions of antigens recognized by CD90 and different McAbs (3G7, 4F11, 11C9, 15B7, 15D2) in MHCC97H cells were examined by flow cytometry assay. The effects of McAb 4F11 on self-renewal and proliferation of MHCC97H cells and MHCC97H sphere cells were identified by serum-free suspension culture and CCK-8 assay. The effects of McAb 4F11 on in vitro invasion and migration of MHCC97 cells was examined by Transwell assay. Results: PKH26 staining results demonstrated that each MHCC97H sphere cell was proliferated and differentiated from one single liver cancer stem cell. The positive rate of CD90 in MHCC97H sphere cells were significantly higher than that in parent MHCC97H cells（\[18.0?7.5\]% vs \[2.3?1.0\]%，P<0.05). McAbs 3G7, 4F11, 11C9, 15B7 and 15D2 all CD90+MHCC97H cells in MHCC97H cells with the recognition rate of McAb 4F11 in CD90+MHCC97H cells was (47.2?44)%. The inhibitory rate of McAb 4F11 on MHCC97H sphere cells was significantly higher than that on MHCC97H cells（\[29.4?3.8\]% vs \[12.0?2.2\]%，P<0.05), and McAb 4F11 extremely suppressed the sphere formation ability of MHCC97H cells and inhibitory rate was (58.0?20.8)%. McAb 4F11 also significantly inhibited invasion and migration of MHCC97H cells in vitro (inhibitory rate: \[48.6?5.1\]% and \[47.6?3.6\]%). Conclusion: Hybridoma McAb 4F11 can specifically recognize CD90+ liver cancer stem cells, and inhibit invasion and migration of liver cancer cells. McAb 4F11 could be a antibody drug candidate for liver cancer stem cell targeted therapy.

国家科技重大专项课题资助（No. 2011ZX09102-010-02, No. 2011ZX09401-030-4）；国家重点基础研究发展计划（973计划）课题资助（No. 2009CB521804）