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目的:将抗精子蛋白17(anti-Sp17)单克隆抗体(3C12)与多柔比星(DOX)偶联,并考察偶联物的体内外抗卵巢癌作用。方法:以二琥珀酰亚胺基酒石酸盐(DST)为交联剂制备3C12-DOX偶联物;免疫荧光法检测3C12-DOX的内化作用;MTT法测定3C12-DOX对Sp17+卵巢癌细胞的杀伤作用,流式细胞仪检测细胞的凋亡率。建立高表达Sp17荷瘤裸鼠模型,分为5组给药,分别经尾静脉注入生理盐水、3C12、S2D11-DOX、3C12-DOX和DOX,定期测量肿瘤体积和裸鼠体质量;实验结束时检测血液AST、LDH和CK-MB,检测心肌组织MDA和SOD,比较3C12-DOX和DOX对心脏的毒性。各组肿瘤称取质量,并检测肿瘤组织中细胞的凋亡率。结果:Sp17单克隆抗体(3C12)与DOX成功偶联,形成的3C12-DOX偶联物能够与Sp17+的细胞结合,并内化至细胞内;MTT结果显示,3C12-DOX偶联物能明显杀伤SK-OV3和HO8910细胞,加速细胞凋亡。在体实验中,注射3C12-DOX偶联物组的肿瘤体积明显小于其他对照组(DOX组除外)。而与DOX组对比,3C12-DOX毒副作用明显降低。结论:3C12-DOX是潜在的靶向治疗卵巢癌的药物。
OBJECTIVE: To conjugate anti-Sp17 monoclonal antibody (3C12) with doxorubicin (DOX) and investigate the anti-ovarian cancer effect of the conjugate in vitro and in vivo. METHODS: 3C12-DOX conjugates were prepared by disuccinimidyl tartrate (DST) as crosslinking agent. The internalization of 3C12-DOX was detected by immunofluorescence assay. The effect of 3C12-DOX on the expression of Sp17 + Cytotoxicity was detected by flow cytometry. The nude mouse model of Sp17 tumor-bearing mice was established and divided into five groups. The mice were injected with normal saline, 3C12, S2D11-DOX, 3C12-DOX and DOX through the tail vein respectively to measure tumor volume and nude mice weight regularly. The levels of AST, LDH and CK-MB in blood were measured. The levels of MDA and SOD in myocardium were measured. The toxicity of 3C12-DOX and DOX to heart were compared. Tumors in each group weighed, and the rate of apoptosis in the tumor tissue was measured. RESULTS: The Sp17 monoclonal antibody (3C12) was successfully coupled with DOX and the formed 3C12-DOX conjugate was able to bind to Sp17 + cells and internalize into the cells. The MTT assay showed that the 3C12-DOX conjugate could obviously kill SK-OV3 and HO8910 cells, accelerate apoptosis. In vivo experiments, the tumor volume of the injected 3C12-DOX conjugate group was significantly less than that of the other control groups (except DOX group). Compared with DOX group, 3C12-DOX toxicity was significantly reduced. Conclusion: 3C12-DOX is a potential drug for ovarian cancer.