血小板增强肝癌血管生成及华蟾素的干预作用研究

Study on platelet enhancement of angiogenesis in hepatocellular carcinoma and intervention effect of Cinobufagin

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Western 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。

AIM:To investigate the effect of platelet(PLT)on angiogenesis in hepatocellular carcinoma and the intervention effect of Cinobufagin(CBG).METHODS:Firstly,we screened the suitable co-incubation ratio of PLT and hepatocellular carcinoma cells,prepared conditioned medium,and determined the half inhibitory concentration of Cinobufagin;then,we set up a control group(human umbilical vein endothelial cells(EC)+conventional medium),a crosstalk group(EC+CM_HP(strip culture prepared by crosstalk of HUH7 and PLT)),and an intervention 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 assay and ELISA assay.Western blot was used to detect 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 established,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 endothelial 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).Compared with Control group,the migration,tube-formation 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 expression 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 tumour tissues.CD31 and CD34 expression was present in the hepatocellular carcinoma transplanted tumour tissues in the Model group,and CBG intervention significantly reduced the expression of CD31 and CD34 in the liver cancer transplanted tumour tissues(P<0.01).CONCLUSION:PLT enhances angiogenesis in hepatocellular carcinoma,and CBG may inhibit its tube-forming ability via the VEGF/VEGFR2 pathway.