肝癌诱导特异性血管新生实验方案的建立  

An investigative and evaluative experimental scheme of antiangiogenesis of hepatocellular carcinoma

作　　者：叶庆[1,2] 殷晓进[3] 刘艳红[2] 丰俊东[1] 吴穷[1] 曲文书[1] 秦叔逵[1] 

机构地区：[1]解放军八一医院全军肿瘤中心,210002 [2]南京大学医学院附属鼓楼医院病理科,210008 [3]江苏先声药物研发有限公司,210042

出　　处：《临床肿瘤学杂志》2014年第9期783-789,共7页Chinese Clinical Oncology

摘　　要：目的建立一套用于研究和评价抗肝癌血管新生的实验方案。方法以人脐静脉内皮细胞(HUVEC)为细胞模型,以正常胎肝细胞系L02为对照,应用荧光定量的Boyden小室、划痕实验检测肝癌条件培养基(HCM)、肝癌细胞系HepG2等诱导作用下对HUVEC迁移的影响;应用荧光定量的粘附实验检测上述诱导作用对HUVEC的粘附作用;应用CFSE/PKH-26染色流式细胞术检测上述诱导作用对HUVEC增殖的作用;应用小管形成实验、Matrigel栓实验检测上述诱导作用对HUVEC小管形成行为及对SCID小鼠体内血管新生的影响。结果体外实验显示,HCM能够比正常条件培养基(NCM)更能诱导HUVEC的趋化和迁移运动,差异有统计学意义(P<0.05);HepG2较L02更容易粘附HUVEC细胞,差异有统计学意义(P<0.05);HCM较NCM更能促进HUVEC的增殖,与L02相比,HepG2和HUVEC共培养更能促进人脐静脉内皮的增殖,差异有统计学意义(P<0.05);HCM和/或HepG2与HUVEC共培养均有利于HUVEC形成管型,而NCM和/或L02与HUVEC共培养对HUVEC形成管型无明显影响。Matrigel栓实验显示,HepG2较L02更能促进SCID小鼠体内的血管新生。结论以上一系列改良的体内、外经典实验能够用于抗肝癌血管新生药物的研究和评价。Objective To develop a set of methods for studying and evaluating the angiogenesis in hepatocellular carcinoma （HCC）. Methods Human umbilical vein endothelial cells （HUVEC） was employed as the model and normal hepatoeyte line L02 was control. A series of classical in vivo and in vitro experiment were modified and then applied during the research to actually and precisely describe the phenomenon of HCC induced angiogenesis, such as Boyden chamber invasion assay, fluorescence quantitative adherence assay, scratch test,flow cytometry, quantitative analysis using Image-Pro Plus and animal experiment of matrigel-plug. Results HCC conditioned media （HCM） was more potent than normal hepatocyte conditioned media （ NCM ） in inducing migration of HUVEC with significant difference（P〈0.05）. HepG2adhered to HUVEC easier than L02 did（ P〈0. 05）. Co-culture of HepG2 and HUVEC promoted proliferation of HUVEC significantly in contrast to L02（ P〈0.05 ）. Co-culture system of HCM, HepG2 and HUVEC facilitated HUVEC to form net-like structure, while that of NCM, L02 and HUVEC did not. In vivo experiment also revealed that HCC was more potent than normal hepatocyte on angiogenesis. Conclusion The results indicated that this set of modified classical in vivo and in vitro experiments can be applied for the study and evaluation of antiangiogenic agents in HCC-induced angiogenesis.

关 键 词：肝细胞癌 血管新生 生物学行为 血管内皮细胞 

分 类 号：R735.7[医药卫生—肿瘤]