三氧化二砷联合华蟾素抗裸鼠人肝癌移植瘤血管新生的作用

Anti-angiogenesis Effect of Arsenic Trioxide plus Cinobufacin on Human Hepatocarcinoma Transplantation Model Nude Mice

目的探讨三氧化二砷(arsenic trioxide,As2O3)联合华蟾素对裸鼠人肝癌移植瘤血管新生的作用及其相关机制。方法建立裸鼠肝癌原位移植瘤模型,随机分为4组,即生理盐水组(对照组)、As2O3组、华蟾素组和As2O3联合华蟾素组(联合组),每组8只,成瘤后腹腔内分别连续21天注射生理盐水、2.5mg/kg As2O3注射液、5mL/kg华蟾素注射液和2.5mg/kg As2O3注射液+5mL/kg华蟾素注射液。比较各组裸鼠一般状态、移植瘤大小、肿瘤微血管密度(microvessel density,MVD),采用免疫组化、光镜、透射电镜进行观察移植瘤血管内皮生长因子(vascular endothelial growth factor,VEGF)、表皮生长因子受体(epidermalgrowth factor receptor,EGFR)及移植瘤病理,并对裸鼠肝、肾组织病理学和血常规进行检测。结果 As2O3组、华蟾素组及联合组的平均瘤重(g,0.65±0.25、0.70±0.27、0.42±0.16)及平均瘤体积(cm3,0.44±0.14、0.46±0.19、0.26±0.11)均低于对照组平均瘤重(1.06±0.25)及平均瘤体积(0.67±0.17,P<0.05);两药相互作用系数(coefficient of drug interaction,CDI)值计算结果为:瘤重CDI(0.97)<1,体积CDI(0.86)<1,表明两药有协同抑制移植瘤生长的作用。As2O3和华蟾素均能抑制移植瘤VEGF、EG-FR的表达,并可降低肿瘤MVD,两药联合后上述作用均显著增强(P<0.05)。裸鼠原位移植瘤的光镜和电镜下观察结果显示As2O3、华蟾素及两药联合均可抑制原位移植瘤组织细胞生长。联合用药并未增加裸鼠肝、肾和造血系统的毒性。结论 As2O3联合华蟾素协同抑制裸鼠人肝癌移植瘤的血管新生,能有效抑制移植瘤生长;同时联合用药对于荷瘤裸鼠的肝、肾和造血系统未见明显毒性。

Objective To study the anti-angiogenesis effect and toxicity of arsenic trioxide （As2O3） plus cinobufacin on transplanted human hepatocarcinoma in nude mice,and the acting mechanism of the treatment was explored as well. Methods Human hepatocarcinoma was transplanted in nude mouse,and the modeled mice were divided at random into 4 groups,8 in each group. They were treated respectively with normal saline（GA）,2.5 mg/kg As2O3 （GB）,5 mL/kg cinobufacin （GC） and 2.5 mg/kg As2O3 ＋ 5 mL/kg cinobufacin （GD）,by intraperitoneal injection for 21 days. The anti-tumor effects was evaluated by estimating general condition of nude mice,tumor size,microvessel density（MVD） level. Expressions of vascular endothelial growth factor （VEGF） and epidermal growth factor receptor（EGFR） in tumor,in tumor tissue of mice as well as pathology of tumor were detected by immunohistochemistry assay,optical microscope,transmission electron microscope （TEM）,respectively. Moreover,blood routine and pathological examinations of liver and kidney were performed. Results The tumor weight and volume were 0.65±0.25 g and 0.44±0.14 cm3 in GB,0.70±0.27 g and 0.46±0.19 cm3 in GC,0.42±0.16 g and 0.26±0.11 cm3 in GD,all significantly lower than those in GA （1.06±0.25 g and 0.67±0.17 cm3,P0.05）. The coefficient of drug interaction （CDI） on tumor weight was 0.97 and that on tumor size was 0.86,all less than 1,showing the synergistic action between the two drugs. Expressions of VEGF and EGFR in tumor as well as the MVD were decreased in GB and GC,and the decreasing of these indices were even more significant in GD. Pathologic examination showed the growth of tumor in GB,GC and GD were all inhibited significantly. No obvious toxicity of the treatments to the hepatic,renal and hematopoietic systems in the nude mice was observed. Conclusions As2O3 and cinobufacini showed synergistic action in inhibiting human hepatocarcinoma in nude mice and the angiogenesis in tumor. Combined use of the two had no obvious toxicity to the hepatic,renal and hematopoietic systems.