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目的:通过观察缺氧反应元件(hypoxia-response element,HRE)调控下,绿色荧光蛋白质(green fluorescent protein,GFP)构建的肝癌细胞Bel-7402/5HRE-EGFP在缺氧条件下,缺氧诱导因子1α(hypoxia-inducible factor 1α,HIF-1α)、血管内皮生长因子(vascular endothelial growth factor,VEGF)等表达水平的变化,研究HRE对微环境的反应特性。方法:以5个串连的HRE和巨细胞病毒(cytomegalovirus,CMV)的微小启动子为转录调控元件、GFP为报告基因构建表达载体,应用FCM和细胞免疫染色法观察缺氧、一氧化氮、过氧化物和酸性pH等微环境的变化对人肝癌细胞Bel-7402中HRE活性的影响,以及缺氧条件下HIF-1α、VEGF表达水平的变化。观察缺氧探针哌莫硝唑的染色强度和分布与HIF-1α、VEGF和GFP表达强度和分布之间的关系,探求裸鼠体内肿瘤组织缺氧对HRE活性及其相关基因表达的影响。结果:肝癌细胞中HRE对缺氧非常敏感,肿瘤细胞和组织缺氧时可上调HIF-1α和VEGF的表达,两者的分布也基本一致。结论:缺氧在调控肝癌血管生成因子表达方面可能起着重要的作用。
OBJECTIVE: To observe the effect of hypoxia-inducible factor (HIF-1α) on the expression of Bel-7402 / 5HRE-EGFP in hepatocarcinoma cells under the control of hypoxia-response element (HRE) Hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) were measured to investigate the response of HRE to microenvironment. METHODS: Five small promoters of HRE and cytomegalovirus (CMV) in series were used as transcriptional regulatory elements. GFP was used as a reporter gene to construct expression vector. FCM and cell immunostaining were used to observe the effects of hypoxia, nitric oxide, Peroxide and acidic pH on the activity of HRE in human hepatocellular carcinoma cell line Bel-7402, and the changes of HIF-1α and VEGF expression under hypoxic conditions. To observe the relationship between the staining intensity and distribution of hypoxia probe pimientanzole and the expression intensity and distribution of HIF-1α, VEGF and GFP and explore the effect of hypoxia on HRE activity and related gene expression in nude mice. Results: HRE was highly sensitive to hypoxia in HCC cells. HIF-1α and VEGF were up-regulated in tumor cells and tissues when hypoxia, and their distributions were also consistent. Conclusion: Hypoxia may play an important role in regulating the expression of angiogenic factors in hepatocellular carcinoma.