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

作　　者：吴晓红 陈诗雨 陈敏 叶明君 陈淑君 吴阳培 李白坤[1] 李庆林[3] WU Xiaohong;CHEN Shiyu;CHEN Min;YE Mingjun;CHEN Shujun;WU Yangpei;LI Baikun;LI Qinglin(School of Traditional Chinese Medicine,Anhui University of Chinese Medicine,Hefei 230012,Anhui,China;School of Pharmacy,Anhui University of Chinese Medicine,Hefei 230012,Anhui,China;Research and Technology Center,Anhui University of Chinese Medicine,Hefei 230038,Anhui,China)

机构地区：[1]安徽中医药大学中医学院,安徽合肥230012 [2]安徽中医药大学药学院,安徽合肥230012 [3]安徽中医药大学科研技术中心,安徽合肥230038

出　　处：《中国临床药理学与治疗学》2024年第8期861-869,共9页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：安徽省自然科学基金面上项目(2208085MH279)。

摘　　要：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,

关 键 词：肝癌 血管生成 血小板 人脐静脉内皮细胞 华蟾素 

分 类 号：R735.7[医药卫生—肿瘤]