【作者】 张霞；

【导师】 张友忠；

【作者基本信息】 山东大学 ， 妇产科学， 2005， 硕士

【摘要】 研究背景: 肿瘤的生长具有血管依赖性,肿瘤组织中新生血管的形成不仅为肿瘤细胞的生长、增殖提供了氧和养料以及排除代谢产物的途径,同时也为肿瘤细胞的转移提供了有利条件。而肿瘤血管生成过程受到诸多因素的影响,其中血管内皮生长因子（VEGF）直接作用于血管内皮细胞,能够特异性地刺激血管内皮细胞的增殖和血管生成,增加血管通透性,在肿瘤血管生成中发挥中心调控作用。而VEGF家族中有多个成员,其中VEGF-A通过与血管内皮细胞表面特异性受体Flt-1及Flk-1结合发挥其生物学功能。乏氧诱导因子1（HIF-1）是细胞在缺氧条件下产生的一种核转录因子,它可作用于多种靶基因,使其转录活性发生改变,从而介导组织细胞产生一系列的缺氧适应性反应。 目前,研究者们已在多种实体肿瘤中检测到HIF-1α及VEGF mRNA及其蛋白的过表达,然而,关于HIF-1α与VEGF在卵巢癌中的分析国内外均比较分散,而且大部分仅对HIF-1α或VEGF在卵巢癌中的表达进行了检测。 目的: 本研究旨在通过检测SKOV₃人卵巢癌细胞株在乏氧条件下及化疗药物（紫杉醇）调节下HIF-1α及VEGF-A基因mRNA水平表达的变化及其相互关系,为探索卵巢癌的早期诊断及其新的治疗靶点提供重要的研究依据。 方法:更多还原

【Abstract】 Backgroud:Angiogenesis is the development of new blood vessels from a preexisting vasculature. Several basic and clinical studies have indicated that tumor growth is dependent on angiogenesis. Vascularization not only provides oxygen and nutrient for proliferating cells, but also promotes it’s extension. Angiogenesis in tumors is influenced by many factors. Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor that stimulates cell division,and migration of vascular endothelial cells, increases the permeability of blood vessels,and play a magor role in angiogenisis. VEGFs are a family of secreted polypeptides with a highly conserved receptor-binding cystine-knot structure similar to that of the platelet-derived growth factors, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placenta growth factor. VEGF-A, the founding member of the family, has important roles in mammalian vascular development and in diseases involving abnormal growth of blood vessels combined with its special receptors Flt-1 and Flk-1 which located on the surface of vascular endothelial cells; other VEGFs are also involved in the development of lymphatic vessels and disease-related angiogenesis. Hypoxia-inducible factor la (HIF-1α) is a heterodimeric transcription factor inresponse to hypoxia that plays a regulatory role in the transcriptional activity of target genes to adapt cells to hypoxia.Currently, overexpression of HIF-la and VEGF mRNA and protein has been demonstrated in a variety of human solid carcinomas, while the data regarding HIF-la and VEGF-A in human ovarian carcinoma are scant and most reports measure either HIF-1 a or VEGF-A in ovarian cancer.Objective:The aim of this study was to investigate expression of both HIF-la and VEGF-A mRNA in SKOV3 cell line in response to hypoxia and regulated by chemotherapeutic drugs (Paclitaxel) and to describe the correlation between HIF-la and VEGF-A in SKOV3 cell line, to provide evidences for the early diagnosis and new therapeutic targets of ovarian cancer.Methods:1. SKOV3 cell line was cultivated to exponential phase of growth, then counting the cell and adjusting the concentration.2. Twenty-five-mililiter culture flasks were seeded with 2 * 106 human ovarian carcinoma cells, then were supplied with total culture fluid to make the bulk volume to lml. Meanwhile, SKOV3 cells were treated with paclitaxel at concentrations of 3.4ng/ml (low), 4.5ug/ml (middle) and 0.45mg/ml (high), respectively. Moreover, a blank control group (no paclitaxel) was set up. Then, cells were incubated in humidified multi-gas incubators containing either air (21% oxygen; normoxia), or 1% oxygen (hypoxia). Five percent CO2 was used in all normoxic and hypoxic incubators, with the balance being nitrogen in hypoxic incubators.3. After different times (30 minutes, 1 hour, 4 hours, 8 hours and 24hours), investigating cells’ changes in morphology and collecting cells.4. Total RNA from SKOV3 cells was prepared by Trizol. Expression levels of HIF-la and VEGF-A mRNA were measured using reverse transcription-polymerase chain reation (RT-PCR) in SKOV3 cells.5. Doing the column figures according to the relative expression value of HIF-la and VEGF-A mRNA to investigate the correlation of the two genes. The experimental5data were analyzed with SPSS. Results:1. The rate of HIF-la mRNA expression in SKOV3 cells is 85% (34/40), so is the rate of VEGF-A mRNA expression.2. According to the column figures of HIF-la and VEGF-A mRNA expression, we can discover the changed tendency of the two genes expression is so similar to each other. The result analyzed with Spearman rank correlation illustrated that there was a strong correlation between HIF-la and VEGF-A mRNA expression (Rs— 0.9227, PO.01).3. Not considering the influence of middle and high concentration paclitaxel on cells, the result indicated there was a significant increase in expression of HIF-la in SKOV3 cells cultured in the environment of hypoxia over that in normoxia (P=0.0059); there was also a significant increase in expression of VEGF-A in SKOV3 cells cultured in the environment of hypoxia over that in normoxia (P=0.0137).4. Analyzing the effect of paclitaxel in different concentrations on expression of HIF-1 a and VEGF-A mRNA with two-way AN OVA and making multiple comparisions with Student - Newman - Keuls and Tukey HSD - test, respectively, the results did not prove HIF-la (F=1.460, P=0.248; P multiple comparisions =0.273, respectively) and VEGF-A (F=1.239, P=0.3l5; P multiple comParisions=0.253, respectively) mRNA expression was affected by the concentration of paclitaxel.5. Analyzing the effect of the action time on expression of HIF-1 a and VEGF-A mRNA with the same method as 4, the results indicated that paclitaxel could not influence the expression of HIF-la (F=0.894, P=0.480; P multiple comparisions =0.481, respectively) and VEGF-A (F=1.627, P=0.195; P multiple comparisions=0.174, respectively) mRNA in 24 hours.6. Morphological change in SKOV3 cells indicated that paclitaxel could affect their proliferation in short times.Conclusions:1. Hypoxia is general phenomenon in solid tumors, while HIF-la mediates tumor cells to adapt hypoxia.2. VEGF-A is a target gene of HIF-la. HIF-la plays a regulatory role in the expression of VEGF-A to promote angiogenesis in human solid carcinomas and tumor cell proliferation , invasion and metastasis.3. Expression and activity of HIF-la is regulated by O2 homeostasis.4. HIF-la and VEGF-A could act as new therapeutic targets of human carcinomas. Their inhibitors have widely applied perspective in tumor therapy.更多还原