【作者】 裴志忠；

【导师】 王顺祥；

【作者基本信息】 河北医科大学 ， 外科学， 2008， 硕士

【摘要】 目的:缺氧是实质性肿瘤微环境的基本特征之一,同时肿瘤细胞的缺氧也是肿瘤发生恶性转化甚至转移的启动因子。缺氧诱导因子-l （hypoxia inducible factor-1, HIF-1 ）是缺氧条件下广泛存在于哺乳动物和人体内的一种转录因子,由HIF-1α和HIF-1β两个亚单位组成,是调节细胞内氧代谢的关键因子之一,其中HIF-1α是主要的氧调节亚基。HIF-1作为一种核转录因子,在哺乳动物的生长发育、生理和病理反应过程中起重要作用。研究证实HIF-1α在肝癌等多种恶性肿瘤中普遍高表达,且与肿瘤的侵袭转移及预后等生物学特性有关。VEGF是HIF-1α的一个主要靶基因,通过调节VEGF的表达、增加肝细胞癌新生血管的生成,从而促进肝细胞癌的侵袭转移。HIF-1活性的调节,可能是治疗很多疾病的切入点。目前有关HIF-1α和VEGF关系的深入研究及以HIF-1α为治疗靶点的研究较少。本研究分别应用HIF-1α抑制剂YC-1[即3-（5’-羟甲基-2’-呋喃基）-1苯甲基引唑]及HIF-1α反义寡核苷酸作用于肝癌细胞株,检测药物干预前后肝癌细胞株SMMC-7721细胞、BEL-7402细胞HIF-1α、VEGF mRNA和蛋白表达的变化,明确HIF-1α对VEGF表达的影响,以及以HIF-1α为治疗靶点的可行性。方法:肝癌细胞株SMMC-7721细胞、BEL-7402细胞分对照组和试验组。对照组细胞常规培养,试验组:分别用HIF-1α抑制剂YC-1、HIF-1α反义寡核苷酸作用于人肝癌细胞,培养24小时后。采用逆转录-聚合酶链式反应（RT-PCR）技术,测定干预前后肝癌细胞株SMMC-7721细胞、BEL-7402细胞HIF-1α、VEGF mRNA表达的变化,以β-actin作为内参照标准,通过凝胶扫描仪对扩增产物进行DNA电泳条带的光密度值分析,将HIF-1α、VEGF分别与β-actin比值作为HIF-1α、VEGF mRNA表达水平的参数,对HIF-1α、VEGF mRNA产物相对定量;以Western blot方法检测SMMC-7721细胞、BEL-7402细胞HIF-1α、VEGF蛋白表达的变化,同样以β-actin作为内参照标准,对HIF-1α、VEGF蛋白进行相对定量分析。所有数据均以x±s表示,采用SPSS13.0统计分析软件进行单因素方差分析（ANOVA）和t检验。P<0.05为有显著差异。结果:1在肝癌细胞株SMMC-7721细胞、BEL-7402细胞中,对照组SMMC-7721细胞、BEL-7402细胞HIF-1α、VEGF mRNA的表达显著高于实验组（P<0.05）。2在肝癌细胞株SMMC-7721细胞、BEL-7402细胞中,对照组SMMC-7721细胞、BEL-7402细胞HIF-1α、VEGF蛋白的表达显著高于实验组（P<0.05）。。3在肝癌细胞株SMMC-7721细胞、BEL-7402细胞中,随着YC-1或HIF-1α反义寡核苷酸浓度的增加实验组细胞HIF-1αmRNA的表达量逐渐减少,组间比较差异具有显著性（P<0.05）;随着YC-1或HIF-1α反义寡核苷酸浓度的增加实验组细胞VEGF的表达产物VEGF₁₂₁（541bp）和VEGF₁₆₅ （408bp）的表达量亦呈减少趋势,组间比较差异具有显著性（P<0.05）,其中VEGF₁₆₅的表达要明显高于VEGF₁₂₁。4在肝癌细胞株SMMC-7721细胞、BEL-7402细胞中,随着YC-1或HIF-1α反义寡核苷酸浓度的增加实验组细胞HIF-1α蛋白的表达量逐渐减少,组间比较差异具有显著性（P<0.05）;随着YC-1或HIF-1α反义寡核苷酸浓度的增加实验组细胞VEGF蛋白的表达亦呈减少趋势,组间比较差异具有显著性（P<0.05）。结论:1 HIF-1α、VEGF在肝癌细胞株SMMC-7721细胞、BEL-7402细胞中高表达。说明HIF-1α和VEGF均与肝细胞癌的发生、发展有关。2 YC-1可以在基因和蛋白水平上抑制HIF-1α的表达,并下调VEGF的表达。同样HIF-1α反义寡核苷酸对肝癌细胞HIF-1α表达亦有抑制作用,同时VEGF的表达下降。说明HIF-1α可以促进肝癌细胞VEGF的表达。3在肝癌细胞株SMMC-7721细胞、BEL-7402细胞中,随着YC-1或HIF-1α反义寡核苷酸浓度的增加实验组细胞HIF-1α和VEGF的表达下降,说明针对HIF-1α为靶点的治疗是可行的。更多还原

【Abstract】 Objective:Hypoxia is a common characteristic of locally advanced solid tumors that has been associated with diminished therapeutic response, and more recently, with malignant progression, that is, an increasing probability of recurrence, loco regional spread, and distant metastasis. The hypoxia inducible factor 1(HIF-1) is a heterodimer transcription factor that is an important regulator of the growing tumor’s response to hypoxia in mammal or human body. It consists of HIF-1αand HIF-1βsubunits, HIF-1 activity in tumors depends on the availability of the HIF-1αsubunit. As nuclear transcription factor, HIF-1 are important in mammal of regulating the growing development, physiology and pathology. The study indicated: HIF-1α, which has high expression in hepatocellular carcinoma and in other malignant tumors, is associated with the tumor aggressiveness, migration and mortality. VEGF, as a target gene of HIF-1α, stimulating angiogenesis, promoting the aggressiveness and migration in hepatocellular carcinoma, is regulated by HIF-1α. The activity of HIF-1 may be the key to cure the malignant tumors. At the present time, there was little deeply study about relation of HIF-1αand VEGF in clinical research. In this research, we explored whether VEGF can be up-regulated by HIF-1αin hepatocellular carcinoma. The cell lines of SMMC-7721 and BEL-7402 were treated with YC-1 [3-(5’- hydroxymethyl-2’-furyl)-1-benzylindazole] and ASODN of HIF-1αrespectively. By detecting the expression of HIF-1α、VEGFmRNA and protein in SMMC- 7721 cell and BEL-7402 cell culturing in normal and treated conditions by RT-PCR and Western blot, we investigate the relation of HIF-1αand VEGF, and the potential of HIF-1αas a valid therapeutic target.Methods: SMMC-7721 cells and BEL-7402 cells were divided as control group and experiment group. The control group were cultured with normal condition, the experiment group were treated by YC-1 or ASODN of HIF-1αrespectively with various concentration. All of the cells were collected on time (after cultured 24 hours). The expression of HIF-1α、VEGF mRNA levels was evaluated by RT-PCR. The HIF-1α、VEGF protein levels in cytoplasm was evaluated by western blot. The OD value of electrophoresis strip of DNA product or that of HIF-1α、VEGF protein was analyzed by gel scanner. Beta-actin was used as an internal standard. The relative expression level of HIF-1α、VEGF was represented with the ratio between produces of HIF-1α、VEGF and that of beta-actin. Data were analyzed with ANOVA and T test using SPSS13.0 statistical software. A level of P<0.05 was considered statistically significant.Results: 1 In SMMC-7721 cells and BEL-7402 cells, the expression of HIF-1αand VEGF mRNA in experiment group were obviously down-regulated compared with control group, the differences of the expression of HIF-1αand VEGF mRNA were statistically significant (P<0.05).2 In SMMC-7721 cells and BEL-7402 cells, the expression of HIF-1αand VEGF protein in experiment group were obviously down-regulated compared with control group, the differences of the expression of HIF-1αand VEGF protein were statistically significant (P<0.05).3 In SMMC-7721 cells and BEL-7402 cells, following the increasing of the concentration of YC-1 or ASODN of HIF-1α, the expression of HIF-1αmRNA were decreased gradually, and the differences of the expression of HIF-1αmRNA were statistically significant within the experiment group(P<0.05). The expression of HIF-1αmRNA showed a dose-dependent in cells cultured with YC-1 or ASODN of HIF-1α. The expression of VEGF mRNA (including VEGF121 and VEGF165) were decreased gradually in experiment group, the differences of the expression of VEGF mRNA were statistically significant (P<0.05). VEGF165 mRNA has high expression compared with VEGF121 mRNA.4 In experiment group of SMMC-7721 cells and BEL-7402 cells, following the increasing of the concentration of YC-1 or ASODN of HIF-1α, the expression of HIF-1αprotein showed decrease, it was statistically significant (P<0.05), and it showed a dose-dependent in cells cultured with YC-1 or ASODN of HIF-1α. The expression of VEGF protein were obviously down-regulated in cells cultured with YC-1 or ASODN of HIF-1α, it was statistically significant, too (P<0.05).Conclutions: 1 The high expression of HIF-1αand VEGF in SMMC-7721 cells and BEL-7402 cells indicated that HIF-1αand VEGF were associated with the occurrence and development of HCC.2 YC-1 inhibits the expression of HIF-1αat gene level and protein level. The expression of VEGF was down-regulated at gene level and protein level. ASODN of HIF-1αcould inhibited the expression of HIF-1αand VEGF at gene level and protein level, too. The result indicated that HIF-1αcan promote the expression of VEGF at gene level and protein level, in other word, that the expression of HIF-1αcan up-regulated the expression of VEGF in hepatocellular carcinoma.3 Following the increasing of the concentration of YC-1 or ASODN of HIF-1αin experiment group in SMMC-7721 cells and BEL-7402 cells, the expression of HIF-1αand VEGF were decreased gradually. The result indicated that it has high potential to treatment tumor of HIF-1αas a valid therapeutic target.更多还原