本文選題：pyridoxine + 血小板��； 參考：《南京醫(yī)科大學(xué)》2010年博士論文

【摘要】： 動脈粥樣硬化(Atherosclerosis, AS)是一種以內(nèi)皮細(xì)胞受損、平滑肌細(xì)胞侵入血管內(nèi)膜、血管平滑肌細(xì)胞增殖為主的血管重塑的病理過程,其發(fā)病過程十分復(fù)雜,其確切病因尚未完全闡明。大量的研究證實(shí)血小板的激活和聚集在AS的發(fā)生發(fā)展中發(fā)揮了重要作用。 一氧化氮(nitric oxide, NO)是內(nèi)皮產(chǎn)生的最主要的舒張因子,其參與了體內(nèi)多種生理性活動,例如舒張血管、抑制血管平滑肌細(xì)胞的增殖、降低血管的滲透性。同時越來越多的研究證實(shí)NO除以上作用外,還能夠抑制血小板的聚集,維持血管內(nèi)環(huán)境的平衡,從而預(yù)防多種心血管疾病的發(fā)生與發(fā)展。NO是L-精氨酸在一氧化氮合酶(NOS)催化下的產(chǎn)物,NOS有三種亞型:內(nèi)皮型(eNOS)、神經(jīng)型(nNOS)、誘導(dǎo)型(iNOS)。目前認(rèn)為,血小板上主要存在eNOS和iNOS,其中發(fā)揮主要作用的是eNOS。已知PI3K/Akt途徑是影響eNOS活性的經(jīng)典的信號轉(zhuǎn)導(dǎo)通路,但是目前對于血小板上eNOS的活性調(diào)控研究并不多。 Pyridoxine是維生素B6的有效的生物成分,參與人體多種代謝過程并發(fā)揮了重要的作用,尤其是已經(jīng)有研究報道,維生素B6能夠增加內(nèi)皮NO的生物合成,并在心血管疾病中發(fā)揮治療作用�，F(xiàn)階段對Pyridoxine的研究發(fā)現(xiàn),其具有降低血液中高同型半胱氨酸濃度的作用,以及抑制血小板聚集等多種功效,對于其機(jī)制的研究最初認(rèn)為Pyridoxine在體內(nèi)作為一種輔酶參與多種代謝活動,但其在血小板上的作用機(jī)制目前尚未有報道。 本研究的目的首先是檢測維生素B6在體外是否能增加血小板NO的合成,其次研究其發(fā)揮這一作用的機(jī)制。提取正常人的血小板,觀察Pyridoxine是否能抑制聚誘劑誘導(dǎo)的血小板的聚集,及其對血小板上cGMP(NO生物活性檢測指標(biāo))的水平的影響。用Western blot檢測eNOS Ser1177磷酸化水平和蛋白激酶Akt磷酸化水平。HTRF分析血小板上磷脂酰肌醇-3-激酶的活性。研究結(jié)果顯示,Pyridoxine呈濃度依賴性的抑制由ADP或者凝血酶引起的血小板聚集,當(dāng)濃度達(dá)到2mmol/L時,其抑制血小板聚集的能力不再隨著濃度的增加而增加。放射免疫法試驗(yàn)結(jié)果顯示Pyridoxine能增加NO的生物活性。我們進(jìn)一步研究發(fā)現(xiàn),Pyridoxine能增加eNOS Ser1177位磷酸化,增加Akt絲氨酸磷酸化,增加PI3K的活性,并且這一作用能夠被PI3K的抑制劑LY294002抑制。本研究結(jié)果表明,Pyridoxine能夠有效抑制ADP或凝血酶誘導(dǎo)的血小板的激活,并增加血小板上NO的生物合成。該作用是通過改善PI3K的活性,增加下游Akt磷酸化,進(jìn)一步增加eNOS Ser 1177位點(diǎn)磷酸化來實(shí)現(xiàn)的。本研究為pyridoxine對于血栓性疾病相關(guān)的疾病的治療和/或預(yù)防提供了理論基礎(chǔ)。
[Abstract]:Atherosclerotic atherosclerosis (ASS) is a pathological process of vascular remodeling with endothelial cells damaged smooth muscle cells invading the intima and vascular smooth muscle cells proliferating. The pathogenesis of ASS is very complicated and its exact etiology has not been fully elucidated. Numerous studies have confirmed that platelet activation and aggregation play an important role in the development of as. Nitric oxide (no) is the most important relaxation factor produced by endothelium, which is involved in many physiological activities, such as vasodilating, inhibiting the proliferation of vascular smooth muscle cells and reducing the permeability of blood vessels. At the same time, more and more studies have proved that no can inhibit platelet aggregation and maintain the balance of the intravascular environment in addition to the above effects. Therefore, to prevent the occurrence and development of many cardiovascular diseases. No is a product of L-arginine catalyzed by nitric oxide synthase (NOS). There are three subtypes of nitric oxide synthase (NOS): endothelial-type eNOSN, nerve-type nNOSN, and inducible iNOS. At present, eNOS and iNOS exist mainly in platelets, among which eNOSs play a major role. It is known that PI3K/Akt pathway is a classical signal transduction pathway affecting the activity of eNOS, but there are few studies on the regulation of eNOS activity in platelets. Pyridoxine is an effective biological component of vitamin B6, which is involved in many metabolic processes and plays an important role in human body. Especially, it has been reported that vitamin B6 can increase the biosynthesis of no in endothelial cells. And play a role in the treatment of cardiovascular disease. Recent studies on Pyridoxine have shown that it can reduce the concentration of homocysteine in blood and inhibit platelet aggregation. As a coenzyme, Pyridoxine is thought to be involved in many metabolic activities in vivo, but the mechanism of its action on platelets has not been reported. The aim of this study was to investigate whether vitamin B _ 6 could increase platelet no synthesis in vitro and to study the mechanism by which vitamin B _ 6 could play this role. To observe whether Pyridoxine can inhibit platelet aggregation induced by polymeric attractant and its effect on the detection of cGMP(NO bioactivity on platelets. The activity of phosphatidylinositol-3-kinase on platelets was determined by Western blot, eNOS Ser1177 phosphorylation level and protein kinase Akt phosphorylation level. The results showed that Pyridoxine inhibited platelet aggregation induced by ADP or thrombin in a concentration-dependent manner. When the concentration reached 2mmol/L, its ability to inhibit platelet aggregation was no longer increased with the increase of concentration. The results of radioimmunoassay showed that Pyridoxine could increase the biological activity of no. We further found that Pyridoxine could increase the phosphorylation of eNOS Ser1177 site, increase Akt serine phosphorylation, and increase the activity of PI3K, which could be inhibited by LY294002, an inhibitor of PI3K. These results suggest that Pyridoxine can effectively inhibit the activation of platelets induced by ADP or thrombin and increase the biosynthesis of no on platelets. This effect was achieved by improving the activity of PI3K, increasing the phosphorylation of downstream Akt and further increasing the phosphorylation of eNOS Ser 1177 site. This study provides a theoretical basis for the treatment and / or prevention of thrombotic disease-related diseases by pyridoxine.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R363

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