一株靶向CD90+MHCC97-L细胞单克隆抗体治疗肝癌的实验研究

目的:研究抗人肝癌干细胞单抗28C10体内外功能，为肝癌干细胞的靶向治疗提供有应用价值的候选治疗剂。方法:采用无血清成球实验、侵袭实验和CCK-8方法等检测分析28C10单抗对MHCC97-L sphere的细胞自我更新、侵袭和耐药的影响。裸鼠体内治疗实验研究单抗28C10联合顺铂对MHCC97-L移植瘤生长的作用。Western-Blot方法鉴定该单抗识别抗原的分子量。结果:流式细胞检测结果显示单抗28C10能够识别MHCC97-L中CD90阳性细胞的比例为2.75%。体外功能实验结果显示单抗28C10能显著抑制MHCC97-L sphere细胞的无血清成球能力和侵袭能力，抑制率分别达33.33%和53.8%。28C10能显著抑制MHCC97-L sphere的顺铂耐药能力，其IC50为0.74 μg/ml，而对照组的IC50为1.42 μg/ml。抗体体内治疗实验结果显示，低、高剂量抗体28C10均能抑制肝癌移植瘤的生长，抑制率分别达到了24.0%和67.7%，单独顺铂组肝癌移植瘤的抑制率为35.9%，而顺铂联合高剂量抗体28C10组对肝癌移植瘤的抑制率达到70.1%。Western-Blot结果显示单抗28C10识别的抗原蛋白分子量约100 kD。结论:筛选获得了1株抗肝癌干细胞的功能性单抗，为靶向肝癌干细胞治疗肝癌奠定了重要的基础。

The study of monoclonal antibody targeting CD90+MHCC97-L cell for liver cancer treatment

Objective:To study the biological characteristics and function of the liver cancer stem cells mAb 28C10 in vivo and in vitro，and to provide therapeutic antibody targeting liver cancer stem cell.Methods:Flow cytometry was used to detect the expression of CD90 and 28C10 in MHCC97-L cells.The effects of mAb 28C10 on self-renewal，invasion and chemosensitivity to cisplatin of MHCC97-L cells were identified by mammosphere formation in serum-free medium and CCK-8 assay.The roles of 28C10 combining with cisplatin on growth of MHCC97-L xenograft in vivo were studied by tumor treatment experiment.Molecular weight of antigen was identified by Western-Blot.Results:Flow cytometry assay showed that mAb 28C10 could recognize cells which also were partly co-stained with CD90，and the positive rate was 2.75%.Functional assay indicated that mAb 28C10 significantly suppressed the sphere formation of MHCC97-L cells in serum-free medium with the inhibitory rate being 33.33%.Moreover，mAb 28C10 could inhibit the invasion of MHCC97-L，and the inhibitory rate was 53.8%.mAb 28C10 could sensitize MHCC97-L to cisplatin，and IC50 was 0.74 μg/ml in mAb 28C10 treatment group，and 1.42 μg/ml in control group.Antibody treatment experiment demonstrated that 2.5 mg/kg and 40 mg/kg-dose of 28C10 can significantly inhibit the tumor growth，and the inhibitory rate was 24.0% and 67.7%，respectively.Furthermore，the inhibitory rate of cisplatin without mAb 28C10 was 35.9% and the inhibitory rate of cisplatin with high-dose of mAb 28C10 was 70.1%.Western-Blot revealed that the molecular weight of the antigen recognized by mAb 28C10 was about 100 kD．Conclusion:We successfully obtained a functional monoclonal antibody 28C10 targeting liver cancer stem cells，which might be the candidate antibody drug treating liver cancer.