PDCD4及DDR2在Peroxynitrite诱导人中枢血管平滑肌细胞凋亡过程中表达的变化

发布时间：2018-11-24 11:09

【摘要】： 一氧化氮(nitric oxide, NO)信号转导途径不仅维系血管自稳,而且影响血管性疾病的发生与发展。在病理条件下,NO过量产生导致的细胞毒作用,主要归咎于自由基ONOO-的形成。ONOO-是NO和超氧阴离子相互作用的产物,是一种具有普遍性的强氧化剂。以往对ONOO-作用于心血管系统的研究大多偏重于其对血管平滑肌收缩或舒张状态的影响。有关ONOO-对HCVSMCs生物学特性的影响,尤其是凋亡的研究报道较少。本实验以两种新型分子—凋亡基因PDCD4和盘状结构域受体(discoidin domain receptor2, DDR2)的表达为切入点,探讨ONOO-作用于HCVSMCs分子调节机制。 PDCD4是近年来备受关注的一种程序性细胞死亡因子,也是一种抑癌基因。它位于染色体10q24上,其编码蛋白具有多个磷酸化位点,可与蛋白激酶C、脯氨酸激酶、酪氨酸激酶等相结合。它的退变对于体内高效的蛋白翻译过程来说是必需的。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,作为胶原受体和胶原传感器,对胶原质和量的变化进行监测,并把信息传递给成纤维细胞,调节成纤维细胞的增殖、凋亡、分化、迁移、黏附和胶原蛋白的分泌、降解。哺乳动物体内DDRs以两种亚型存在,其中DDR2主要表达于心、骨骼肌、肺、脑和肾的间质细胞,与相关细胞分泌MMP2相关。血管平滑肌细胞的结构、功能异常是血管性疾病发生和发展的病理基础之一。血管平滑肌细胞的异常增殖、积聚、迁移及凋亡在心脑血管疾病的发生、发展过程中起到关键性作用。在本实验中,首先以终浓度分别为10、50、100μmol／L的ONOO-作用于体外培养的HCVSMCs,采用噻唑蓝(MTT)比色法测定细胞生存率,采用流式细胞仪测定细胞凋亡率,分别以吖啶橙染色、Ho33342／PI荧光双染进行形态学观察。采用RT-PCR检测PDCD4、DDR2 mRNA的表达。采用Western-blot检测PDCD4、DDR2蛋白的表达。结果显示：(1)以ONOO-作用于HCVSMCs 24 h,显著抑制该系细胞增殖。(2)以ONOO-作用于HCVSMCs24 h,明显诱导该系细胞凋亡。(3)以ONOO-作用于HCVSMCs 24 h,可引起DDR2mRNA及蛋白水平总体上呈现下降趋势。(4)以ONOO-作用于HCVSMCs 24 h,引起PDCD4 mRNA及蛋白水平显著增加。结论ONOO-是通过诱导凋亡途径抑制HCVSMCs的增殖。PDCD4及DDR2基因在ONOO-诱导HCVSMCs凋亡的信号转导途径中起到关键作用。
[Abstract]:The signal transduction pathway of nitric oxide (nitric oxide, NO) not only maintains self-stabilization of blood vessels, but also affects the occurrence and development of vascular diseases. Under pathological conditions, the cytotoxicity caused by excessive production of NO is mainly attributed to the formation of free radical ONOO-. ONOO- is the product of the interaction between NO and superoxide anion and is a universal strong oxidant. Previous studies on the effects of ONOO- on cardiovascular system have focused on the effects of ONOO- on the contractile or diastolic state of vascular smooth muscle. There are few reports on the effects of ONOO- on the biological characteristics of HCVSMCs, especially on apoptosis. The aim of this study was to explore the regulatory mechanism of ONOO- on HCVSMCs molecules by using the expression of two novel molecules, the apoptotic gene PDCD4 and the discoid domain receptor (discoidin domain receptor2, DDR2). PDCD4 is a kind of programmed cell death factor and a tumor suppressor gene. It is located on chromosome 10q24 and has many phosphorylation sites, which can bind to protein kinase C, proline kinase, tyrosine kinase and so on. Its degeneration is essential for efficient protein translation in vivo. DDRs is a recently discovered transmembrane receptor tyrosine kinase with a disk-like domain that acts as a collagen receptor and collagen sensor. The changes of collagen quality and quantity were monitored, and the information was transmitted to fibroblasts to regulate the proliferation, apoptosis, differentiation, migration, adhesion and collagen secretion and degradation of fibroblasts. There are two subtypes of DDRs in mammalian, in which DDR2 is mainly expressed in the interstitial cells of heart, skeletal muscle, lung, brain and kidney, which is related to the secretion of MMP2 by related cells. Abnormal structure and function of vascular smooth muscle cells are one of the pathological bases for the occurrence and development of vascular diseases. Abnormal proliferation, accumulation, migration and apoptosis of vascular smooth muscle cells play a key role in the occurrence and development of cardiovascular and cerebrovascular diseases. In this experiment, the survival rate of HCVSMCs, cultured in vitro was determined by thiazolyl blue (MTT) colorimetry, apoptosis rate was measured by flow cytometry and acridine orange staining was used. Ho33342/PI fluorescence double staining was used to observe the morphology. The expression of PDCD4,DDR2 mRNA was detected by RT-PCR. The expression of PDCD4,DDR2 protein was detected by Western-blot. The results showed that: (1) ONOO- significantly inhibited the proliferation of HCVSMCs for 24 h, (2) ONOO- significantly induced the apoptosis of the cell line HCVSMCs24 h, and (3) ONOO- induced HCVSMCs 24 h. (4) the level of PDCD4 mRNA and protein increased significantly when HCVSMCs was treated with ONOO- for 24 h. Conclusion ONOO- inhibits the proliferation of HCVSMCs by inducing apoptosis. PDCD4 and DDR2 genes play a key role in the signal transduction pathway of HCVSMCs apoptosis induced by ONOO-.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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