【作者】 王健；

【导师】 易继林；

【作者基本信息】 华中科技大学 ， 普通外科， 2006， 博士

【摘要】 本文旨在研究缺氧诱导因子-2α（hypoxia inducible factor-2 alpha，HIF-2α）mRNA和蛋白在肝癌组织中的定性和定量表达，及其与肝癌组织临床病理指标的相关关系，探讨其在肝癌组织中表达的临床意义；通过检测并比较缺氧状态下肝癌SMMC-7721细胞中HIF-1α和HIF-2α在不同时相的mRNA和蛋白量以及两者的半衰期，探讨HIF-1α和HIF-2α的时相差异表达意义及其可能的调节机制；并通过阻断HIF-2α在肝癌SMMC-7721细胞中的表达，研究HIF-2α对肿瘤细胞生长、黏附和侵袭能力的影响。第一部分HIF-1α和HIF-2α在肝癌细胞中的时相差异表达目的：缺氧诱导因子2α与缺氧诱导因子1α结构功能相似却不可相互替代；两者在缺氧过程中的表达不一致。本研究在检测缺氧状态下肝癌细胞中HIF-2α和HIF-1α的时相表达差异并探讨其可能的调节机制和意义。方法：以1％低氧培养箱模拟细胞低氧环境，检测人肝癌SMMC- 7721细胞中HIF-1α和HIF-2αmRNA量和蛋白量，以及反义HIF-1α（natural antisense hypoxia inducible factor-1α，aHIF）的mRNA量。同时应用氯化钴（CoCl₂）和转录抑制剂5，6-二氯苯并咪唑1-beta-D-呋喃核糖苷（5，6-dichlorobenzimida-zole-1-beta-d-ribofuranoside，DRB）检测HIF-1α和HIF-2αmRNA的半衰期，评价HIF-1α和HIF-2αmRNA的稳定性。结果：急性缺氧迅速诱导HIF-1α蛋白和HIF-2α蛋白在细胞内堆积。随着缺氧时间延长HIF-1αmRNA的稳定性下降，其蛋白表达降低，而HIF-2αmRNA转录和蛋白表达以及aHIF mRNA持续增高。结论：在较长时相的缺氧过程中，针对于肿瘤细胞对缺氧的适应性调节，以及在肿瘤的放疗和化疗抗性以及与肿瘤侵袭和转移方面，HIF-2α可能比HIF-1α起着更为重要的作用。aHIF参与了HIF-1αmRNA的负反馈调节。第二部分HIF-2α在肝癌组织中的表达及其临床意义目的：研究缺氧诱导因子-2α（HIF-2α）mRNA及蛋白在肝癌组织中的表达。方法：半定量RT-PCR法和免疫组织化学SP法检测67例肝细胞肝癌（HCC）标本中HIF-2αmRNA转录和蛋白的表达。结果：在67例肝癌标本中，HIF-2α阳性表达率为40.3％（27／67），其阳性染色在肝癌细胞、肿瘤新生血管内皮中，主要在胞浆中表达，胞核中表达较少。在有肝癌坏死周边组织中表达阳性率较高。统计分析显示hif-2αmRNA转录和HIF-2α蛋白表达在大小肝癌之间、播散型与非播散型肝癌之间以及伴有坏死和无坏死肝癌之间均有显著性差异（P＜0.05）；但肝癌各病理组织分级之间以及是否有肝门淋巴结转移之间无显著性差异（P＞0.05）。相关分析显示hif-2αmRNA转录与HIF-2α蛋白表达有显著性相关（r=0.264，P＜0.05）。结论：HIF-2α在肝癌组织中的表达较为普遍，其表达增高与肝癌的恶性生长有关，同时亦可能是促进肝癌细胞浸润和转移的一个因素。第三部分HIF-2α对肝癌细胞生长和侵袭能力的影响目的：检测在缺氧条件下缺氧诱导因子2α对肝癌SMMC-7721细胞生长、黏附和侵袭能力的影响。方法：以1％低氧培养箱模拟细胞低氧环境下脂质体Lipofectamine介导HIF-2α脱氧寡核苷酸转染SMMC-7721肝癌细胞，测转染前后HIF-2αmRNA量和蛋白量检验转染效果；MTT法测定细胞生长和粘附能力变化；Transwell小室测定细胞的体外侵袭能力。结果：HIF-2αmRNA和蛋白表达在反义转染组显著低于正义组、随机组与对照组（P＜0.01）。HIF-2α表达阻断对细胞生长和活力无显著性影响（P＞0.05），但显著抑制体外粘附能力（抑制率16.99％～31.07％，P＜0.01）和侵袭能力（抑制率17.9％，P＜0.01）。结论：HIF-2α表达阻断可抑制SMMC-7721肝癌细胞的黏附和侵袭活性，对其生长无显著影响。更多还原

【Abstract】 The aims of this article were to study the expression of hypoxia inducible factor-2 alpha in the hepatoma tissue, and the influence of mionectic culture on the expression of hypoxia inducible factor-2 alpha, and another, the influence of hypoxia inducible factor-2 alpha on the proliferative and invasive abilities of hepatocellular carcinoma cells. To explore primarily the significance of hypoxia inducible factor-2 alpha in the hepatocellular carcinoma cells.PART ONE Expression of HIF-1α and HIF-2α in Hepatoma CellsObjectives: The constructions and functions of hypoxia inducible factor-2 alpha （HIF-2α） and hypoxia inducible factor-1 alpha （HIF-1 α） are resemble, but they can not be replaced by each other. The expressions of them disaccord. This article is to examine the differential expression of hypoxia inducible factor-2 alpha （HIF-2α） and hypoxia inducible factor-1 alpha （HIF-1α） in SMMC-7721 hepatoma cells after prolonged hypoxia, and explore the possible regulatory mechanism.Methods: Using 1% mionectic incubator to simulate the anoxic circumstance of SMMC-7721 hepatoma cells, and measure the expressional quantum of mRNA and protein of HIF-1α and HIF-2α, and quantum of mRNA of natural antisense of HIF-1α lpha （aHIF）, and meantime measure the half life of HIF-1 and HIF-2α mRNA by CoCl₂ and transcription inhibitor 5,6 -dichlorobenzimidazole-1- beta-d-ribo furanoside （DRB） .Results: Actual hypoxia induces rapidly the accumulation of HIF-1 and HIF-2 α protein in hepatoma cells. With the prolonged hypoxia, the HIF-1 α mRNA drops due to its stability, whereas HIF-2α mRNA and protein and aHIF mRNA increase.Conclusions: In prolonged hypoxia, HIF-2 α may play a more important role than HIF-1α on the metabolic adaption of tumor cells, involving the progress, the invasion and metastasis, and the resistance of chems and radiotharepy of the hepatoma cells. aHIF may participate the regulation of HIF-1α mRNA .PART TWO The Clinical Significance of the HIF-2α Expression in HCCObjectives: To investigate the expressional and clinical meaning of hypoxia inducible factor-2 alpha （HIF-2α） in hepatocellular carcinoma （HCC）.Methods: Tumor tissues in 67 cases of HCC were studied by the semi-quantitative reverse transcriptase-polymerase chain reaction（RT-PCR） and immuno-histochemical technique.Results: HIF-2α could be detected in 27 cases （40.3%）. For the transcription of HIF-2α mRNA and the expression of HIF-2α protein, there are significant differences between large and small tumors, tumors of spreading type and non-spreading type, and tumors with necrosis and without necrosis. No apparent significant difference was observed between tumor histological grades.Concisions: HIF-2α is expressed in lots of HCC, and its expression is at significant high level in the HCC with large size, with necrosis and being spreading type （P<0.05）, which indicates the expression of HIF-2 α is not only responsible for the malignant growth of HCC, but a factor influencing the infiltration and metastasis of HCC （P>0.05）.PART THREEThe Effects of HIF-2α on The Proliferation and The Invasion of Hepatocellular Carcinoma CellsObjectives: To study the effects of hypoxia inducible factor-2 α （HIF-2α） on the proliferation , adhesion and the invasion of SMMC-7721 cells under anoxia culture.Methods: Lipofectamine-mediated HIF-2α ODN was transfected into SMMC-7721 cells, and cultured under 37°C、 1%O₂、5%CO₂. Then determine the expression of HIF-2α mRNA and protein to evaluate the effect of transfection. Using MTT to measure the proliferative and adhesive activity of SMMC-7721 cells, and transwell cell culture chambers toexplore the adhensive and invasive activity of SMMC-7721 cells.Results: The expressions of HIF-2α mRNA and protein were inhibited significantly by HIF-2 α ASODN （P<0.01）. The blockage of HIF-2 α expression significantly inhibited the adhesive activity （inhibition ratio 16. 99%—31.07%, P<0.01） and invasive activity （inhibition ratio 17.9%, P<0.01）, while had no significant influence on the proliferative activity of SMMC-7721 cells （P>0.05）.Conclusions: The blockage of HIF-2 α expression inhibits the adhesive and invasive activities of SMMC-7721cells, and has no impact on their proliferation.更多还原