【作者】 赵静；

【导师】 李敏； 王金穗；

【作者基本信息】 兰州大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 目的（1）将人肝癌细胞株HepG₂与正常肝细胞进行三维培养，对比观察其能否形成血管生成拟态（Vasculogenic Mimicry,VM）；并进一步探讨Cyclooxygenase-2（COX-2）和Matrix metalloproteinase-2（MMP-2）对三维培养中拟态血管形成的影响。（2）观察人肝细胞肝癌（Hepatocellular carcinoma，HCC）组织中是否存在血管生成拟态；检测COX-2，MMP-2在HCC中的表达，探讨COX-2，MMP-2与血管生成拟态以及肿瘤分化程度之间的关系。方法（1）应用三维细胞培养技术，建立肝癌细胞株HepG₂的三维培养模型；同时三维培养正常肝细胞对其生成拟态血管的能力进行检测；在HepG₂的三维培养模型中加入COX-2，MMP-2抗体进行抗体抑制，PAS染色以及免疫组化染色，观察拟态血管生成能力的变化。（2）收集人肝细胞肝癌标本31例存档石蜡包埋组织进行连续切片，HE染色观察；并应用免疫组织化学和PAS双重染色的方法检测肿瘤组织CD31，Ferritin的表达以及拟态血管的形成；运用免疫组织化学染色方法检测COX-2，MMP-2在肿瘤组织中的表达，进行统计学分析。结果（1）肝癌细胞三维培养两天时伸出细长突起，培养七天时癌细胞部分区域逐渐消失，剩余癌细胞彼此之间相互连接形成网络样、环状结构。而正常肝细胞在培养第一天可以见到个别细胞伸出细小突起，培养三天时细胞开始变圆变小，胞浆减少，胞核固缩，不能形成网环状结构。（2）抗体抑制实验中加入COX-2抗体的培养皿在培养三天后固定进行PAS染色，可见细胞伸出突起，有形成网络样结构的趋势，但所形成的网状结构没有未加抗体的HepG₂三维培养明显；而加入MMP-2抗体的只有少数区域有形成网状结构的趋势，大部分区域细胞呈分散状态；加入COX-2，MMP-2两种抗体的基本上不能形成网环状结构，细胞分散生长。（3）免疫组化COX-2染色阳性强于MMP-2；加入COX-2抗体进行抗体抑制的细胞MMP-2染色阳性强度明显弱于未加抗体的细胞MMP-2染色。（4）31例人肝细胞肝癌标本中检测到VM的有22例。（5）COX-2蛋白在31例肝细胞肝癌中均有表达，有VM形成组COX-2阳性表达强度高于无VM形成组（P＜0.05）。（6）MMP-2蛋白在30例肝细胞肝癌中有表达，有VM形成组MMP-2阳性表达强度高于无VM形成组（P＜0.05）。（7）不同分化程度的肝细胞肝癌COX-2，MMP-2的表达差别有统计学意义（P＜0.05）。（8）COX-2与MMP-2在肝细胞肝癌组织中的表达呈正相关。结论（1）体外培养人肝癌细胞HepG₂具有形成拟态血管的能力。人正常肝细胞不具备形成拟态血管的能力。（2）COX-2在肿瘤细胞VM形成过程中发挥着重要的调节作用，抑制COX-2可以抑制肿瘤细胞拟态血管的形成。（3）COX-2能够通过上调MMP-2的表达抑制肿瘤细胞拟态血管的形成。（4）人肝细胞肝癌组织中有拟态血管生成。（5）MMP-2、COX-2在人肝细胞肝癌组织中过表达，COX-2的表达和肝细胞肝癌的拟态血管生成密切相关，MMP-2的表达强度与COX-2的表达强度正相关。（6）COX-2，MMP-2的表达强度与肝细胞肝癌的病理分级呈正相关。更多还原

【Abstract】 Objective （1） This experiment is to study if Vasculogenic Mimicry exists in Hepatocellular carcinoma （HCC） by constructing tumor cell HepG₂ tri-dimension culture system and comparing it with tri-dimension cell culture of normal hepatocyte. We detect the expression of COX-2 and MMP-2 protein in tumor cells to reveal its effect to Vasculogenic Mimicry in tri-dimension culture system.（2） To observe Vasculogenic Mimicry in Hepatocelllar carcinoma, and detect the expression of COX-2 and MMP-2 in human Hepatocellular carcinoma for investigating its effect to Vasculogenic Mimicry and tumor differentiated degree.Methods （1） This experiment constructs tumor cell HepG₂ tri-dimension culture system. We cultured HepG₂ cells and normal hepatocytes for comparing the capacity of them to form Vasculogenic Mimicry. COX-2 and MMP-2 antibodies were added in HepG₂ tri-dimension culture system for study of antibody inhibition experiment, stained with PAS and immunohistochemistry for observing the capacity of HepG₂ cells to form VasculogenicMimicry.（2） 31 samples of Hepatocellular carcinoma were collected. All cases were observed byserial section and HE staining, detected the expression of Ferritin and CD31 proteins in the cases by immnohistochemistrial and PAS staining, and detected the expression of COX-2 and MMP-2 proteins by immnohistochemistrial staining.Results （1） Hepatocellular carcinoma cells （HepG₂） formed distinctive loops or networks after 2 days of three-dimensional cultures in collogen matrix, and the cells linked each other to form wreath and net work structures at the seventh day. But normal hepatocytes stretched out leptos tubers. At the third day, the tumor cells began to atrophy. The cells turned round and small, and the nuclei pycnosys. The normal hepatocytes could not form wreath and net work structures.（2） In antibody inhibition experiment, culture dishes which had been added COX-2 antibody were fixed and stained with PAS after cultured for three days. The tumor cells could be observed to stretch out tubers, and had the tendency to form net work structures. But the network structures were less than those tumor cells which not added antibody. Culture dishes which had been added MMP-2 antibody had a kind of tendency to form network structure only in a few part of cells, more cells were dispersal. The culture dishes which have been added COX-2 and MMP-2 antibodies could not form network structure. The tumor cells grew scattered.（3） The expression of COX-2 proteins was stronger than MMP-2 proteins in immunohistochemical staining. The expression of MMP-2 proteins was weaker in the cells added MMP-2 antibody than not added MMP-2 antibody. Added COX-2 proteins could inhibit the expression of MMP-2.（4） Vasculogenic Mimicry could be observed in 22 simples of Hepatocellular carcinoma.（5） COX-2 proteins were detected in all 31 cases of Hepatocellular carcinoma. The expression of COX-2 proteins in Hepatocellular carcinoma with VM was significantly higher than that group without VM （P<0.05）.（6） MMP-2 proteins were detected in 30 cases of hepatocellular carcinoma. The expression of MMP-2 proteins in hepatocellular carcinoma with VM was significantly higher than that group without VM （P<0.05）.（7） The expression of COX-2 and MMP-2 proteins showed differentiated in different depth groups of differentiation （P<0.05）.（8） The expression of COX-2 and MMP-2 proteins was positive correlation in hepatocellular carcinoma.Conclusions （1） Hepatocellular carcinoma cells can form vasculogenic mimicry in vitro, but normal hepatocytes can not.（2） HepG₂ can produce COX-2 proteins. It is very important for the formation of Vasculogenic Mimicry. VM can be abrogated by using inhibitors of COX-2 proteins in tri-dimension cell culture system.（3） COX-2 proteins can inhibit the formation of Vasculogenic Mimicry by mediating the up-regulation of MMP-2.（4） Vasculogenic Mimicry exists in human Hepatocellular carcinoma.（5） The expression of COX-2 and MMP-2 proteins all can be detected in Hepatocellular carcinoma. Expression of COX-2 proteins is related with VM in Hepatocellular carcinoma and the expression of COX-2 and MMP-2 proteins is positive correlation.（6） As the differentiated degree degrades, the expression of COX-2 and MMP-2 proteins is positive correlation.更多还原