【作者】 赵玉玲；

【导师】 龚跃法； 高中洪；

【作者基本信息】 华中科技大学 ， 无机化学， 2008， 博士

【摘要】 糖尿病对身体的危害主要来自高糖导致的多种并发症,在糖尿病并发症的发生发展中,氧化应激发挥着重要的作用。伴随着高糖所导致的氧化应激的发生,过量产生的O2.-降低NO的生物活性,同时生成更强的氧化剂,如过氧亚硝基阴离子(ONOO-),进而损伤蛋白质生成3-硝基酪氨酸(3-NT)。目前已在许多糖尿病并发症中发现3-NT的存在,但并没有对其机理进行系统的研究。本论文主要对高糖和外源性硝化试剂(hemin-nitrite-H2O2和SIN-1)作用人脐静脉内皮细胞(ECV304)以及大鼠血管平滑肌细胞(VSMC)后所致细胞氧化损伤进行了研究,观察蛋白质硝化反应是否进一步加重了细胞损伤的程度。同时我们还观察了高糖和外源性硝化导致的细胞硝化蛋白之间的异同。所取得的主要结果如下:1、高糖导致人脐静脉内皮细胞(ECV304)氧化损伤以及引起蛋白质硝化内皮功能丧失以及进一步的损害在糖尿病并发症的发生发展过程中发挥着重要的作用。在高糖导致的内皮细胞损伤过程中,蛋白质酪氨酸硝化参与其中。有关高糖导致人脐静脉内皮细胞(ECV304)发生蛋白质硝化反应的研究还很少。用终浓度分别为30 mM和40 mM的D-葡萄糖作用ECV304细胞72小时,通过检测细胞存活率、测定脂质过氧化水平、荧光检测胞内谷胱甘肽、荧光观察胞内活性氧、毛细管电泳检测胞内和胞外活性氮含量、凝胶电泳以及蛋白印迹分析细胞蛋白质硝化等观察高糖所导致的氧化损伤情况。经过高糖作用72小时后,发现高糖可以导致ECV304发生严重的氧化损伤,且有剂量依赖性。高糖所导致的氧化损伤表现在细胞存活率降低、丙二醛(MDA)的含量增加、谷胱甘肽(GSH)的含量降低、细胞内活性氧(ROS)的含量增加以及一氧化氮(NO)及其代谢产物的含量(包括增加细胞内亚硝酸钠和培养基中硝酸钠含量)增加。实验结果还表明高糖可以导致ECV304细胞产生硝化蛋白,主要发生在分子量为66 kDa、50 kDa、43 kDa、34 kDa、30 kDa和23 kDa的蛋白上。通过对高糖作用ECV304进行研究后发现,体内高糖可以导致内皮细胞发生严重的氧化损伤,并且推断蛋白质硝化反应参与了氧化损伤的过程。2、高糖导致大鼠血管平滑肌细胞(VSMC)氧化损伤以及引起蛋白质硝化高糖可以导致血管平滑肌细胞产生大量活性氧,进而在氧化应激状态下可能会引起细胞内发生蛋白质酪氨酸硝化。目前尚无高糖与平滑肌细胞蛋白质硝化的相关性研究。用终浓度分别为30 mM和40 mM的D-葡萄糖作用大鼠血管平滑肌细胞(VSMC)3d和7d,通过检测细胞存活率、荧光检测胞内谷胱甘肽、荧光观察胞内活性氧、毛细管电泳检测胞内活性氮含量、凝胶电泳以及蛋白印迹分析细胞蛋白质硝化等观察高糖所导致的氧化损伤情况。结果表明,高糖以终浓度为30 mM和40 mM分别作用VSMC细胞3d和7d后,可以显著增加细胞的增殖、诱发细胞产生氧化损伤(包括降低GSH含量以及增加细胞内活性氧ROS的含量)、导致VSMC中活性氮生成量增加,并证实高糖可以导致细胞内产生蛋白质的酪氨酸硝化,硝化主要发生在分子量为55 kDa、30 kDa、26 kDa和20 kDa的蛋白上。在高糖导致的细胞氧化损伤过程中,氧化损伤的程度随着高糖浓度和作用时间的增加而加重。3、高糖和外源性硝化试剂(hemin-nitrite-H2O2和SIN-1)所致细胞蛋白质硝化有一定差别糖尿病并发症的病理过程是复杂的,而导致蛋白质发生硝化反应的途径又是多样的。铁卟啉或者某些含铁卟啉过氧化酶催化NO2-—H2O2蛋白质硝化反应在病理条件下是比ONOO-更容易发生的。有关高糖所导致的细胞蛋白质硝化和外源性硝化试剂所导致的细胞蛋白质硝化之间的差别研究的很少。分别用高糖和外源性硝化试剂(hemin-nitrite-H2O2和SIN-1)作用ECV304和VSMC细胞72小时,通过检测细胞存活率、凝胶电泳以及蛋白印迹分析细胞蛋白质硝化等观察高糖和外源性硝化试剂所导致的细胞氧化损伤。研究结果表明:(1)蛋白质硝化可以加重细胞损伤的程度;(2)对某些蛋白质来说,高糖所导致的蛋白质硝化现象同其它外源性硝化试剂(如hemin-nitrite-H2O2体系和SIN-1)所导致的蛋白质硝化相类似;(3)外源性硝化试剂(如hemin-nitrite-H2O2体系和SIN-1)所导致的蛋白质硝化主要发生在小分子量蛋白上(～11、17和20 kDa),高糖引起的蛋白质硝化对不同的蛋白质有一定的选择性。更多还原

【Abstract】 The hazards of diabetes to body mostly come from complications induced by hyperglycemia. Oxidative stress plays an important role in the development and progression of diabetic complications. When oxidative stress induced by high glucose happens, overproduction of superoxide anion (O2.-) would decrease the bioactivity of nitric oxide (NO), thus leading to the generation of more potent oxidant, such as peroxynitrite (ONOO-), which will damage protein and produce 3-NO2Tyr (3-NT). Though 3-NT has been found in many diabetic complications, the mechanism has been not studied systematically. In this study, human umbilical vein endothelial cells (ECV304) and rat vascular smooth muscle cell (VSMC) were cultured in high glucose medium or incubated with exogenous nitrating agents (hemin-nitrite-H2O2 and SIN-1), then the cell oxidative and nitrative injury was studied and the role of protein tyrosine nitration in cell injury, as well as the difference between protein tyrosine nitration induced by high glucose and exogenous nitrating agents were discussed. The main results in this work are as follows:1. Protein tyrosine nitration presents in high glucose induced human umbilical vein endothelial cell injuryThe dysfunction and further damage of endothelium play an important role in the development and progression of diabetic vascular complications. Protein tyrosine nitration is involved in endothelial cell injury induced by high glucose. Little is known about protein nitration in human umbilical vein endothelial cells (ECV304) induced by high glucose. In the present article, ECV304 was cultured in 30 mM glucose and 40 mM glucose for 72h, then the cell viability, lipid peroxidation, intracellular glutathione, intracellular reactive oxygen species, nitrite and nitrate contents and protein tyrosine nitration were assayed. After 72h treatment, it was found that high glucose stimulated ECV304 injury in a dose-dependent manner, including reducing cell viability, increasing malondialdehyde (MDA) content, decreasing glutathione (GSH) content, increasing intracellular reactive oxygen species (ROS), increasing the production of nitric oxygen (NO) (increased nitrite content in cell and nitrate content in medium) and generating protein tyrosine nitration ( protein nitration with molecular masses ~ 66, 50, 43, 34, 30 and 23 kDa), which demonstrated that high glucose could induce cell oxidative injury to ECV304.2. Protein tyrosine nitration presents in high glucose induced rat vascular smooth muscle cell injuryThe dysfunction of vascular smooth muscle cell involves in the development and progression of diabetic vascular complications. When oxidative stress induced by high glucose happens, reactive oxygen species involve in vascular smooth muscle cell, then generates protein tyrosine nitration. No investigation on protein tyrosine nitration in vascular smooth muscle cell has been found. In the present study, exposure of rat vascular smooth muscle cell (VSMC) to 30 mM glucose and 40 mM glucose for 3d and 7d, then the cell viability, intracellular glutathione, intracellular reactive oxygen species, nitrite content, protein tyrosine nitration were assayed. After 3d and 7d treatment, it was found that high glucose stimulated VSMC injury in a dose-dependent and time-dependent manner, including increasing cell viability, inducing cell oxidative injury (decreased GSH content and increased intracellular ROS content), increasing the production of NO (increased nitrite content in cell). It was demonstrated that protein tyrosine nitration with molecular masses ~ 55, 30, 26 and 20 kDa was generated in cell. The cell oxidative injury induced by high glucose would aggravate with the dose and time.3. Protein tyrosine nitration induced by high glucose and exogenous nitrating agents (hemin-nitrite-H2O2 and SIN-1) are differentThe development and progression of diabetic vascular complications is complicated and there are multiple pathways leading to protein tyrosine nitration. It is well accepted that heme or heme containing proteins could catalyze NO2--H2O2 to nitrate tyrosine residue of protein. This is a more possible mechanism under physiological and pathological conditions to produce tyrosine nitration than peroxynitrite. There is no comparison between protein tyrosine nitration induced by high glucose and exogenous nitrating agents. In the present study, exposure of ECV304 and VSMC to high glucose and exogenous nitrating agents (hemin-nitrite-H2O2 and SIN-1) for 72h, the cell oxidative injury in ECV304 and VSMC induced by high glucose and exogenous nitrating agents was studied by cell viability and protein tyrosine nitration. After 72h treatment, it was found that with the addition of nitrite, the cytotoxicities of hemin-H2O2 on ECV304 and VSMC were significantly augmented, at the same time, protein nitration in cellular protein of both cell lines was also significantly increased. Secondly, with regard to some proteins, protein nitration induced by high glucose was similar to those induced by extrinsic factors (hemin-nitrite-H2O2 system and SIN-1). Furthermore, the difference between protein tyrosine nitration induced by high glucose condition and extrinsic factors (hemin-nitrite-H2O2 system and SIN-1) was that the later could also generate protein nitration with low molecular masses (~11, 17, and 20 kDa). By comparison protein tyrosine nitration induced by high glucose condition with those induced by extrinsic factors (hemin-nitrite-H2O2 system and SIN-1), it may be speculated that protein tyrosine nitration is selective in diabetic vascular complications.更多还原