【摘要】 目的研究吡唑啉酮镉（Ⅱ）配合物1-苯基-3-甲基-4-丙酰基-5-吡唑啉酮缩水杨酰肼-镉（Ⅱ）（Cd-PMPP-SAL）体内外对小鼠黑素瘤B16细胞的抗肿瘤作用及其作用机制。方法以Cd-PMPP-SAL1.0,1.5,3.0,5.0和10.0 mg·L^-1分别作用小鼠黑素瘤B16细胞24,48和72 h,采用MTT法检测B16细胞存活率;Cd-PMPP-SAL 6.25,12.50和25.00 mg·L^-1作用B16细胞24 h,用Hoechst33258染色观察B16细胞形态,AnnenxinⅤ/PI双染色法检测B16细胞凋亡率;胱天蛋白酶活性检测试剂盒检测B16细胞内胱天蛋白酶活性。C57BL/6J小鼠皮下接种B16细胞制备荷瘤模型,5 d后分别瘤内注射Cd-PMPP-SAL 6.25,12.50和25.00 mg·kg^-1,每天1次,连续12 d。每天检测体质量,给药结束后处死小鼠,测量瘤体积并测瘤质量,计算抑瘤率。HE染色法观察瘤体、肝和肺组织病理变化;免疫组织化学法检测肿瘤组织中血管内皮生长因子（VEGF）和成纤维细胞生长因子2（FGF2）蛋白表达;TUNEL法检测移植瘤组织内的细胞凋亡。结果Cd-PMPP-SAL抑制B16细胞存活,IC₅₀为4.946 mg·L^-1,95%置信限为4.24⁵.65 mg·L^-1;Cd-PMPP-SAL12.50和25.00 mg·L^-1作用24 h,B16细胞凋亡率为（12.8±1.4）%和（18.4±0.4）%,显著高于细胞对照组（1.7±0.1）（P<0.01）;Cd-PMPP-SAL 25.00 mg·L^-1组胱天蛋白酶3和9活性与细胞对照组比较显著增高（P<0.01）,胱天蛋白酶3/7活性变化不明显。瘤内注射Cd-PMPP-SAL 12.50和25.00 mg·kg^-1治疗组,从治疗第8天起瘤体积与模型组相比明显减小（P<0.01）,对小鼠体质量无明显影响;Cd-PMPP-SAL 12.50和25.00 mg·kg^-1治疗组小鼠移植瘤组织有不同程度的坏死,肝、肺组织无明显病理变化;与模型组比较,Cd-PMPP-SAL 12.50和25.00 mg·kg^-1治疗组移植瘤组织VEGF和FGF2蛋白表达显著下降（P<0.05）,凋亡细胞明显增加（P<0.05）。结论 Cd-PMPP-SAL体内外可有效地抑制B16细胞生长,该作用可能与诱导细胞凋亡及抑制肿瘤内血管生成有关。更多还原

【Abstract】 OBJECTIVE To investigate the antitumor effect of cadmium（Ⅱ） complex of pyrazolone derivatives 1-phenyl-3-methyl-4-propionyl-5-pyrazolone salicyloyl hydrazide-cadmium（Ⅱ）（Cd-PMPPSAL） on the murine melanoma B16 cells in vitro and in vivo and its mechanisms. METHODS B16 cells were incubated with Cd-PMPP-SAL at 1.0,1.5,3.0,5.0 and 10.0 mg·L^-1for 24,48 or 72 h. The proliferation rate of B16 cels was evaluated by MTT assay. B16 cels were incubated with Cd-PMPP-SAL at 6.25,12.50 and 25.00 mg·L^-1for 24 h,while cell morphology was observed by Hoechst33258 staining. Apoptosis of B16 cells was detected by Annexin Ⅴ-FITC/PI staining. The activity of caspases in B16 cells was detected by caspase activity assay. C57BL/6J mice were inoculated subcutaneously with B16 cells to establish a tumor-bearing model. Five days later,Cd-PMPP-SAL at 6.25,12.50 and 25.00 mg·kg^-1was injected into tumors of C57BL/6J mice once a day for 12 d. The body mass was recorded daily.One day after the last administration,all the mice were killed and the tumor was harvested. Tumor volume and mass were measured,and the tumor inhibitory rates were calculated. Pathological changes of the tumor,liver and lung were observed under a microscope. The expressions of vascular endothelial growth factor（VEGF） and fibroblast growth factor 2（FGF2） in tumor tissues were detected by immunohistochemistry. The apoptotic cells in transplanted tumor tissues were detected by TUNEL. RESULTS Cd-PMPP-SAL inhibited the proliferation of B16 cells. The IC₅₀ was 4.946 mg·L^-1,and 95% confidence interval was 4.24-5.65 mg·L^-1. The apoptosis rates（12.8±1.4）% and（18.4±0.4）% of Cd-PMPP-SAL12.50 and 25.00 mg·L^-1groups were significantly higher than those of control group（1.7±0.1）%（P<0.01）. The activity of caspase 3 and 9 of Cd-PMPP-SAL 25.00 mg·L^-1group was significantly higher than that of control group（P<0.01）,but there was no significant difference in caspases 3/7. The relative tumor volumes of Cd-PMPP-SAL 6.25,12.50 and 25.00 mg·kg^-1treated groups from the 8thday of treatment were significantly decreased compared with the model group（P<0.01）. The result of paraffin sections showed that the transplanted tumor tissues in Cd-PMPP-SAL 12.50 and 25.00 mg · kg^-1groups exhibited different degrees of necrosis,but there was no significant pathological damage to the liver or lung tissues of mice. Compared with model group,expressions of VEGF and FGF2 in Cd-PMPP-SAL12.50 and 25.00 mg·kg^-1treated groups were significantly inhibited（P<0.05）,and apoptotic cell rates were significantly higher（P<0.05）. CONCLUSION Cd-PMPP-SAL can inhibit growth of B16 cells in vivo and in vitro,which may be associated with induction of tumor cell apoptosis and inhibition of tumor angiogenesis.更多还原