Peroxynitrite诱导下咽癌细胞凋亡与PDCD4，DDR1及DDR2基因活性相关性的研究

发布时间：2018-11-17 12:21

【摘要】：Peroxynitrite (ONOO-)是一氧化氮和超氧阴离子的反应产物。ONOO-作为强氧化剂,参与体内一系列病理生理过程。在病理条件下,NO过量产生的细胞毒作用主要归咎于ONOO的形成。ONOO-可通过不同的信号途径影响肿瘤的发生发展过程。事实上,ONOO-可以穿透双层磷脂膜,诱导脂质过氧化,干扰线粒体的功能,影响细胞的代谢,进而损伤DNA,引起包括肿瘤细胞在内的各种细胞的坏死和/或凋亡。下咽癌,是头颈部最常见的恶性肿瘤之一,近年来其发生率呈显著上升趋势。尽管治疗手段的进步在一定程度上改善了患者的生命质量,但多年来患者总体生存率未有明显提高。因此,探讨新的肿瘤相关基因,揭示下咽癌发生的分子机制,有助于产生新的有效治疗策略。 PDCD4是近年来备受关注的一种程序性细胞死亡调控基因,也是一种抑癌基因。PDCD4位于染色体10q24上,其表达产物与真核细胞翻译起始因子e IF4A结合,从而抑制核糖体复合物的形成和蛋白质的合成。PDCD4在正常组织中普遍表达,而在恶性肿瘤中表达下调甚至缺失。PDCD4在肿瘤组织中的低表达状态与肿瘤的浸润、转移及预后密切相关。值得关注的是,在一些细胞的凋亡过程中,PDCD4表达增高。迄今为止,ONOO-诱干预条件下,PDCD4在FaDu细胞(下咽癌细胞系)表达状况的研究鲜有报道。盘状结构域受体(Discoidin Domain receptors, DDRs)是最近发现的一类带有盘状结构域的跨膜受体型酪氨酸激酶,包括DDR1和DDR2两种亚型存在。DDR1主要在一些肿瘤细胞中高表达,而DDR2主要表达于心、骨骼肌、肺、脑和肾的间质细胞。本课题从PDCD4和盘状结构域受体(Discoidin domain receptor,DDRs)的表达为切入点,探讨ONOO对FaDu细胞的作用及机制。在本实验中,以不同浓度的ONOO-作用于体外培养的FaDu细胞,采用流式细胞仪测定细胞凋亡率,采用噻唑蓝(MTT)比色法测定细胞生存率。以吖啶橙和Ho33342荧光染色观察细胞的形态学改变。采用Western-blot技术检测PDCD4、DDR1、DDR2蛋白的表达。采用RT-PCR技术检测PDCD4、DDR1、DDR2 mRNA的表达。结果显示： (1) ONOO显著抑制FaDu细胞细胞增殖。 (2) ONOO主要通过诱导细胞凋亡抑制FaDu细胞增殖。 (3) ONOO-作用下,FaDu细胞PDCD4 mRNA及蛋白水平增加显著。 (4) ONOO作用下,FaDu细胞DDR1mRNA及蛋白水平总体上呈现下降趋势。 (5)在ONOO作用下,FaDu细胞中的DDR2 mRNA及蛋白水平总体上呈现下降趋势。结论： ONOO-主要通过启动凋亡途径有效抑制(?)FaDu细胞增殖。在ONOO-诱导FaDu细胞凋亡的信号转导途径中,PDCD4及DDR1、DDR2基因可能起到关键性作用。本研究从一个侧面,为揭示下咽癌与氧化胁迫的关系提供了实验依据。
[Abstract]:Peroxynitrite (ONOO-) is the product of the reaction of nitric oxide and superoxide anion. As a strong oxidant, ONOO- is involved in a series of pathophysiological processes in vivo. Under pathological conditions, the excessive cytotoxicity of NO is mainly attributed to the formation of ONOO. ONOO- can influence the development of tumor through different signal pathways. In fact, ONOO- can penetrate bilayer phospholipid membrane, induce lipid peroxidation, interfere with mitochondrial function, affect cell metabolism, and then damage DNA, to induce necrosis and / or apoptosis of various cells, including tumor cells. Hypopharyngeal carcinoma is one of the most common malignant tumors in head and neck. Although the improvement of treatment has improved the quality of life to some extent, the overall survival rate of patients has not improved significantly over the years. Therefore, exploring new tumor-related genes and revealing the molecular mechanism of hypopharyngeal carcinogenesis may contribute to the development of new effective therapeutic strategies. PDCD4 is a kind of programmed cell death regulating gene and a tumor suppressor gene. PDCD4 is located on chromosome 10q24 and its expression product binds to eukaryotic translation initiation factor e IF4A. Thus inhibiting the formation of ribosomal complexes and the synthesis of proteins. PDCD4 is generally expressed in normal tissues, but down-regulated or even absent in malignant tumors. Metastasis and prognosis are closely related. It is noteworthy that the expression of PDCD4 is increased during the apoptosis of some cells. Up to now, the expression of PDCD4 in FaDu cells (hypopharyngeal cancer cells) has been seldom reported under the condition of ONOO- induced intervention. Discoid domain receptor (Discoidin Domain receptors, DDRs) is a recently discovered transmembrane receptor tyrosine kinase with discoid domain, including DDR1 and DDR2 subtypes. DDR1 is highly expressed in some tumor cells, while DDR2 is mainly expressed in the heart. Interstitial cells in skeletal muscle, lung, brain and kidney. In this study, the effect and mechanism of ONOO on FaDu cells were studied from the expression of PDCD4 and disc domain receptor (Discoidin domain receptor,DDRs). In this experiment, FaDu cells were treated with different concentrations of ONOO-, the apoptosis rate was measured by flow cytometry, and the cell survival rate was determined by thiazolyl (MTT) colorimetry. The morphological changes of the cells were observed by acridine orange and Ho33342 fluorescence staining. Western-blot technique was used to detect the expression of PDCD4,DDR1,DDR2 protein. RT-PCR technique was used to detect the expression of PDCD4,DDR1,DDR2 mRNA. The results showed that: (1) ONOO significantly inhibited the proliferation of FaDu cells. (2) ONOO inhibits the proliferation of FaDu cells by inducing apoptosis. (3) the level of PDCD4 mRNA and protein in FaDu cells increased significantly under the action of ONOO-. (4) the level of DDR1mRNA and protein in FaDu cells decreased under the action of ONOO. (5) under the action of ONOO, the level of DDR2 mRNA and protein in FaDu cells decreased. Conclusion: ONOO- can effectively inhibit the proliferation of FaDu cells by initiating apoptosis pathway. PDCD4 and DDR1,DDR2 genes may play a key role in the signal transduction pathway of FaDu cell apoptosis induced by ONOO-. This study provides an experimental basis for revealing the relationship between hypopharyngeal carcinoma and oxidative stress.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63

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