VPO1对NO氧化作用及机制的初步研究

发布时间：2018-03-26 20:25

本文选题：血管过氧化物酶　切入点：一氧化氮　出处：《中南大学》2012年博士论文

【摘要】：目的：从生化实验和细胞实验水平,运用分子生物学技术,探讨VPO1对NO的氧化机制及VPO1在内皮细胞NO代谢中的作用。方法：实验分为三部分：(1)体外实验：用探针法实时监测VPO1与NO的反应,以及Cl-或Br-对该反应的影响,并用Griess试剂检测该反应的氧化产物。(2)用NO分析仪检测GJ-4细胞(稳定转染了VPO1的HEK293细胞)内的NO水平,并通过APF荧光检测法检测细胞内VPO1的活性。(3)通过Western Blot检测各种血管活性物质刺激后内皮细胞VPO1, Nox-4, iNOS的蛋白表达情况,以及内皮细胞中的NO含量。结果：(1)体外实验发现VPO1能与NO发生反应,氧化产物之一为硝酸盐；Cl-和Br-能竞争性抑制MPO对NO的消耗,而Cl-对VPO1与NO反应无明显影响。(2)GJ-4细胞中的NO含量明显低于对照组,而VPO1的活性明显高于对照组。(3)LPS(100ng/ml)和TNF-a(2ng/ml)刺激内皮细胞(HUVEC)24小时后,VPO1蛋白表达水平有不同程度升高。在HMEC-1细胞中,BK(1μM)与Ach(10μM)可呈时间依赖性的降低Nox-4和升高iNOS的蛋白表达。TNF-a(2ng/ml)刺激内皮细胞24小时后,细胞内NO含量明显低于对照组。结论：VPO1是NO的氧化酶。细胞内VPO1(?)消耗内源性NO,参与NO代谢。TNF-a能通过上调VPO1的表达参与调控内皮细胞的NO代谢。
[Abstract]:Aim: to investigate the oxidation mechanism of VPO1 on no and the role of VPO1 in the metabolism of no in endothelial cells by using molecular biology from the level of biochemical and cellular experiments. Methods: the experiment was divided into three parts: in vitro, the reaction of VPO1 with no and the effect of Cl- or Br- on the reaction were monitored in real time by probe method. The oxidation product of the reaction was detected by Griess reagent. The no level in GJ-4 cells (HEK293 cells stably transfected with VPO1) was detected by no analyzer. The expression of VPO1, Nox-4, iNOS protein and no content in endothelial cells were detected by Western Blot. Results (1) in vitro, VPO1 could react with no. One of the oxidation products was nitrate-Cl- and Br-can competitively inhibit the consumption of no by MPO, but Cl- had no effect on the reaction between VPO1 and no. The content of no in GJ-4 cells was significantly lower than that in control group. The activity of VPO1 was significantly higher than that of the control group (100 ng / ml) and TNF-a1 2ng / ml). The expression of VPO1 protein in endothelial cells was increased in varying degrees after 24 hours of stimulation. In HMEC-1 cells, BK1 渭 M) and Ach(10 渭 M) decreased the expression of Nox-4 and increased the expression of iNOS protein in a time-dependent manner. After 24 hours of stimulation of endothelial cells, The content of no in the cells was significantly lower than that in the control group. Conclusion VPO1 is the oxidase of no. ) consuming endogenous no and participating in no metabolism. TNF-a can regulate the no metabolism of endothelial cells by up-regulating the expression of VPO1.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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