【摘要】： 一氧化氮(nitric oxide, NO)信號(hào)轉(zhuǎn)導(dǎo)途徑不僅維系血管自穩(wěn),而且影響血管性疾病的發(fā)生與發(fā)展。在病理?xiàng)l件下,NO過(guò)量產(chǎn)生導(dǎo)致的細(xì)胞毒作用,主要?dú)w咎于自由基ONOO-的形成。ONOO-是NO和超氧陰離子相互作用的產(chǎn)物,是一種具有普遍性的強(qiáng)氧化劑。以往對(duì)ONOO-作用于心血管系統(tǒng)的研究大多偏重于其對(duì)血管平滑肌收縮或舒張狀態(tài)的影響。有關(guān)ONOO-對(duì)HCVSMCs生物學(xué)特性的影響,尤其是凋亡的研究報(bào)道較少。本實(shí)驗(yàn)以兩種新型分子—凋亡基因PDCD4和盤狀結(jié)構(gòu)域受體(discoidin domain receptor2, DDR2)的表達(dá)為切入點(diǎn),探討ONOO-作用于HCVSMCs分子調(diào)節(jié)機(jī)制。 PDCD4是近年來(lái)備受關(guān)注的一種程序性細(xì)胞死亡因子,也是一種抑癌基因。它位于染色體10q24上,其編碼蛋白具有多個(gè)磷酸化位點(diǎn),可與蛋白激酶C、脯氨酸激酶、酪氨酸激酶等相結(jié)合。它的退變對(duì)于體內(nèi)高效的蛋白翻譯過(guò)程來(lái)說(shuō)是必需的。DDRs是最近發(fā)現(xiàn)的一類帶有盤狀結(jié)構(gòu)域的跨膜受體型酪氨酸激酶,作為膠原受體和膠原傳感器,對(duì)膠原質(zhì)和量的變化進(jìn)行監(jiān)測(cè),并把信息傳遞給成纖維細(xì)胞,調(diào)節(jié)成纖維細(xì)胞的增殖、凋亡、分化、遷移、黏附和膠原蛋白的分泌、降解。哺乳動(dòng)物體內(nèi)DDRs以兩種亞型存在,其中DDR2主要表達(dá)于心、骨骼肌、肺、腦和腎的間質(zhì)細(xì)胞,與相關(guān)細(xì)胞分泌MMP2相關(guān)。血管平滑肌細(xì)胞的結(jié)構(gòu)、功能異常是血管性疾病發(fā)生和發(fā)展的病理基礎(chǔ)之一。血管平滑肌細(xì)胞的異常增殖、積聚、遷移及凋亡在心腦血管疾病的發(fā)生、發(fā)展過(guò)程中起到關(guān)鍵性作用。 在本實(shí)驗(yàn)中,首先以終濃度分別為10、50、100μmol／L的ONOO-作用于體外培養(yǎng)的HCVSMCs,采用噻唑藍(lán)(MTT)比色法測(cè)定細(xì)胞生存率,采用流式細(xì)胞儀測(cè)定細(xì)胞凋亡率,分別以吖啶橙染色、Ho33342／PI熒光雙染進(jìn)行形態(tài)學(xué)觀察。采用RT-PCR檢測(cè)PDCD4、DDR2 mRNA的表達(dá)。采用Western-blot檢測(cè)PDCD4、DDR2蛋白的表達(dá)。 結(jié)果顯示：(1)以O(shè)NOO-作用于HCVSMCs 24 h,顯著抑制該系細(xì)胞增殖。(2)以O(shè)NOO-作用于HCVSMCs24 h,明顯誘導(dǎo)該系細(xì)胞凋亡。(3)以O(shè)NOO-作用于HCVSMCs 24 h,可引起DDR2mRNA及蛋白水平總體上呈現(xiàn)下降趨勢(shì)。(4)以O(shè)NOO-作用于HCVSMCs 24 h,引起PDCD4 mRNA及蛋白水平顯著增加。 結(jié)論ONOO-是通過(guò)誘導(dǎo)凋亡途徑抑制HCVSMCs的增殖。PDCD4及DDR2基因在ONOO-誘導(dǎo)HCVSMCs凋亡的信號(hào)轉(zhuǎn)導(dǎo)途徑中起到關(guān)鍵作用。
[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-.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R363

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