目的:探讨血小板(PLT)对肝癌血管生成的影响以及华蟾素的干预作用。方法:先筛选PLT与肝癌细胞的适宜共孵育比例，制备条件培养基，确定华蟾素(CBG)的半数抑制浓度;然后，设对照组[人脐静脉内皮细胞(EC)+常规培养基]、串扰组[EC+CM_HP(HUH7与PLT串扰制备的条培)]、干预组(EC+CM_HP+CBG)，采用划痕实验、小管形成实验、出芽实验和ELISA实验分别评估EC的迁移力、成管力、出芽能力以及共培养上清中血管内皮生长因子(VEGF)水平;运用West-ern blot检测VEGFR2、p-VEGFR2的表达，并采用抑制剂进行了反向验证。建立肝癌裸鼠皮下移植瘤模型，设 Model 组、Model+CBG 组和 Model+Apa组，通过Masson染色观察移植瘤的胶原表达情况，免疫荧光检测血管内皮标志物CD31和CD34的表达水平。结果:PLT:HUH7=200时HUH7活力最强，HUH7与PLT串扰明显促进EC的增殖力(P＜0。01)。与对照组相比，串扰组的迁移、成管、生芽能力增强，干预组的低于串扰组(P＜0。01)。串扰组上清中VEGF的表达水平高于对照组，而干预组的低于串扰组(P＜0。01)。串扰组p-VEGFR2蛋白的表达量较对照组显著增多，而干预组的表达量较串扰组少(P＜0。01)。Model组可见大片胶原纤维沉积，CBG干预显著降低移植瘤组织胶原纤维沉积。Model组的肝癌移植瘤组织存在CD31和CD34表达，CBG干预显著降低肝癌移植瘤组织CD31和CD34的表达(P＜0。01)。结论:PLT能增强肝癌的血管生成，CBG可能经由VEGF/VEGFR2通路抑制其成管能力。
Study on platelet enhancement of angiogenesis in hepatocellular car-cinoma and intervention effect of Cinobufagin
AIM:To investigate the effect of plate-let(PLT)on angiogenesis in hepatocellular carcino-ma and the intervention effect of Cinobufagin(CBG).METHODS:Firstly,we screened the suitable co-incubation ratio of PLT and hepatocellular carci-noma cells,prepared conditioned medium,and de-termined the half inhibitory concentration of Cinob-ufagin;then,we set up a control group(human um-bilical vein endothelial cells(EC)+conventional me-dium),a crosstalk group(EC+CM_HP(strip culture prepared by crosstalk of HUH7 and PLT)),and an in-tervention group(EC+CM_HP+CBG).The migration,tube-formation and sprouting capacity of EC and the level of vascular endothelial growth factor(VEGF)in co-cultured supernatant were evaluated by scratch assay,tube-formation assay,budding as-say and ELISA assay.Western blot was used to de-tect the expression of VEGFR2 and p-VEGFR2,and reverse verification was performed with inhibitors.A subcutaneous transplantation tumour model of hepatocellular carcinoma in nude mice was estab-lished,with Model group,Model+CBG group and Model+Apa group.The collagen expression of the transplantation tumour was observed by Masson staining,and the expression levels of vascular endo-thelial markers CD31 and CD34 were detected by immunofluorescence.RESULTS:When PLT:HUH7=200,the activity of HUH7 was the strongest,and the crosstalk between HUH7 and PLT significantly promoted the proliferation of EC(P＜0.01).Com-pared with Control group,the migration,tube-for-mation and budding ability of Crosstalk group were enhanced,and those of Intervention group were lower than those of Crosstalk group(P＜0.01).The expression level of VEGF in the supernatant of Crosstalk group was higher than that of Control group,while that of Intervention group was lower than that of Crosstalk group(P＜0.01).The expres-sion level of p-VEGFR2 protein in Crosstalk group was significantly higher than that of Control group,but the expression level of Intervention group was lower than that of Crosstalk group(P＜0.01).Large collagen fibre deposition was seen in the Model group,and CBG intervention significantly reduced collagen fibre deposition in the transplanted tu-mour tissues.CD31 and CD34 expression was pres-ent in the hepatocellular carcinoma transplanted tumour tissues in the Model group,and CBG inter-vention significantly reduced the expression of CD31 and CD34 in the liver cancer transplanted tu-mour tissues(P＜0.01).CONCLUSION:PLT enhances angiogenesis in hepatocellular carcinoma,and CBG may inhibit its tube-forming ability via the VEGF/VEGFR2 pathway.
万方数据
维普
