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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MH-CC97H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 monoclonal antibodies against hepatocellular carcinoma (HCC) stem cells and to investigate their anti-tumor effect in vitro so as to provide candidate antibody drugs for targeted therapy of HCC stem cells. Methods: Serum-free suspension culture and PKH26 staining were used to analyze the existence of hepatocellular carcinoma stem cells in human hepatoma cell line MHCC97H. Flow cytometry was used to detect the expression of seven kinds of tumor stem cell markers in MHCC97H cells and their spheroids, as well as the identification of hepatoma stem cell marker CD90 in MHCC97H cells and the monoclonal antibodies 3G7, 4F11, 11C9, 15B7 and 15D2 in different hybridomas The co-expression of the situation. Serum-free suspension culture and CCK8 assay were used to detect the effect of mAb 4F11 on the self-renewal and proliferation of MHCC97H cells and their spheroids. Transwell assay was used to detect the effect of mAb 4F11 on the invasion and migration of MHCC97H cells in vitro. Results: PKH26 staining showed that the spheres of MHCC97H cells proliferated and differentiated from single hepatocellular carcinoma stem cells. The proportion of CD90 + MHCC97H cells in MHCC97H cells was significantly higher than that of the parental MHCC97H cells [(18.0 ± 7.5)% vs (2.3 ± 1.0)%, P <0.05]. The hybridoma McAbs 4F11, 3G7, 11C9, 15B7 and 15D2 could both recognize CD90 + MHCC97H cells in MHCC97H cells. The recognition rate of McAbs 4F11 to CD90 + MHCC97H cells was (47.2 ± 4.4)%, The inhibition rate of proliferation was far greater than that of the parental MHCC97H cells [(29.4 ± 3.8)% vs (12.0 ± 2.2)%, P <0.05]. Monoclonal antibody 4F11 inhibited the spheroidization of MHCC97H cells with the inhibition rate of (58.0 ± 20.8)%. Mab 4F11 could significantly inhibit the invasion and migration of MHCC97H cells in vitro with the inhibitory rates of (48.6 ± 5.1)% and (47.6 ± 3.6)%, respectively. CONCLUSION: The monoclonal antibody McAb 4F11 can specifically recognize CD90 + hepatocarcinoma stem cells and inhibit the invasion and migration of hepatocellular carcinoma cells. It can be used as a candidate antibody drug for liver cancer stem cell targeted therapy.