【作者】 王顺祥； 吴晓慧； 高峰； 宋西进； 李建坤； 彭利； 唐瑞峰； 张萌； 肖燕； 张风瑞；

【Author】 Wang Shun-Xiang Wu XiaoHui Gao Feng Department of Hepatobiliary Surgery, The Fourth Hospital of Hebei medical university & Hebei provincial tumor hospital , Shijiazhuang, 050011, China

【机构】 河北医科大学第四医院暨河北省肿瘤医院肝胆外科；

【摘要】 目的:乙酰肝素酶是一种葡萄糖醛酸内切酶,也是目前发现的唯一可以降解硫酸乙酰肝素活性的酶,它通过切断硫酸乙酰肝素蛋白多糖侧链降解细胞外基质和血管基底膜, 并且能释放和活化硫酸乙酰肝素蛋白多糖结合型的生长因子诱导血管生成,从而促进肿瘤细胞的侵袭和转移。目前乙酰肝素酶表达的调控机制不清。研究表明HPA在大部分易侵袭转移的缺氧恶性肿瘤中高表达、高活性状态,且仅在酸性环境中发挥作用,而缺氧状态又造就了酸性环境,这提示我们缺氧可能参与了肿瘤细胞中HPA基因表达及其活性的调控。本研究探讨肝细胞癌中HPA和HIF—1 α表达的相关性以及缺氧对人肝癌细胞株SMMC-7721细胞HPA表达的影响。方法:免疫组织化学方法检测肝细胞癌标本中HPA和HIF-1 α的表达,常氧和缺氧培养肝细胞癌细胞株SMMC-7721,采用逆转录-聚合酶链式反应(RT-PCR)技术,检测常氧培养和缺氧培养后SMMC-7721细胞Hpa mRNA表达的变化, 以β-actin作为内参照标准,对扩增产物通过凝胶扫描仪进行DNA电冰条带的密度值分析,将Hpa与β-actin比值作为Hpa表达水平的参数,对Hpa产物相对定量,以Western blot方法检测SMMC-7721细胞Hpa蛋白表达的变化,并进行相对定量分析。结果:HPA蛋白在肝癌组织中的阳性表达率(92％)显著高于癌旁(71．4％)及正常肝组织(8．3％)(P<0．05), HIF-1 α蛋白在肝癌组织中阳性表达率(88％)显著高于癌旁 (60．71％)及正常肝组织(8．33％)(P<0．05),肝癌组织中HPA和 HIF-1 α的表达呈显著正相关(r=0．295,P=0．038),经缺氧培养20小时后,SMMC-7721细胞HPA mRNA表达量为6． 234±0．457,常氧培养组HPA mRNA表达量为2．910±0． 137,缺氧培养组细胞HPA mRNA表达量显著高于常氧培养组(P<0．05),缺氧培养后SMMC-7721细胞HPA蛋白表达量 (65KD为1．437±0．067,50KD为1．706±0．066)显著高于常氧培养组HPA蛋白表达量(65KD为1．192±0．060．50KD为 1．580±0．265)(P<0．05)。结论:缺氧可以通过促进HIF—1 α的表达,从而上调HPA的表达。更多还原

【Abstract】 Objective: To investigate the relationship between HPA and HIF-1 α protein expression in hepa-tocellular Carcinoma, and to document the effect of hy-poxia on HPA expression of human liver cancer cell line SMMC-7721. Methods: Immunohistochemistry was used to detect the expression of HPA and HIF-1 α protein. Human hepatocellular carcinoma cell line SMMC-7721 cultured in normoxic and hypoxic conditions. The heparanase gene and protein expression level were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and Western blot respectively. Beta-actin was used as an internal standard. The relative expression level of Hpa was represented with the ratio between produces of Hpa and that of beta-actin. Results: In HCC thirty-one of fifty tumor tissues (62.0%) expressed Hpa and thirty (60%) expressed HIF-1 α. In contrast, only six of twenty-eight tumor-surrounding tissues (21.4%) were Hpa positive and four (14.3%) HIF-1 α positive. Two of fourteen normal liver tissues (14.3%) expressed Hpa and three (21.4%) expressed HIF-1 α. Statistical analysis revealed that both Hpa and HIF-1 α expression were higher in tumor tissues than that in tumor-surrounding tissues and in normal liver tissues (P<0.05). There was a positive relationship between the expression of HPA and HIF-1 α protein (r=0.295) (P<0.05). In SMMC-7721 cell cultured in hypoxic group, the expression level of HPA mRNA is 6.234 ± 0.457, 65kD protein is 1.437 ± 0.067 and 50kD is 1.706 ± 0.066, but in normoxic group, it is 2.910 ± 0.137,1.192 ± 0.060,1.580 ± 0.265 respectively. HPA mRNA and protein expression level increased significantly compared with those in normoxic group (P<0.05). Conclution: Hypoxia can up-regulates heparanase expression through accelerating HIF-1 α expression. It may be a mechanism of HP A regulation.更多还原