AT1受体在尼古丁致血管内皮功能障碍中的作用研究

发布时间：2018-09-17 15:05

【摘要】：研究背景: 吸烟是心血管疾病的重要危险因素,可导致冠心病、脑血管疾病、主动脉瘤、外周血管等疾病。据报道2000年世界范围内约有160万心血管患者死亡直接由吸烟引起。研究表明吸烟通过促进内皮功能紊乱、增强炎症反应、增加氧化应激、促使心肌能量代谢障碍以及促进血栓形成等来影响心血管系统,而戒烟能降低冠心病、心脏骤停、卒中的风险,并改善外周血管症状。尼古丁作为烟草中的主要毒性物质,在由吸烟引起的各种心血管疾病的发展过程中起着极为重要的作用。然而尼古丁引起血管内皮功能损伤的机制仍不清楚。以往研究主要集中在烟碱乙酰胆碱受体(nicotinic acetylcholine receptor, nACHR)及其下游的机制。体外研究发现尼古丁通过nACHR介导内皮细胞增殖、迁移和血管新生,在体研究发现尼古丁可以介导血管新生,而这种生血管效应可导致粥样硬化斑块的不稳定性和斑块的进一步发展、肿瘤的生长及肿瘤血管的形成及其他吸烟相关的疾病。也有报道吸烟者体内肾素-血管紧张素-醛固酮系统过度激活,而血管紧张素转换酶抑制剂可抑制氧化应激反应调节内皮细胞中eNOS的表达,改善吸烟患者的内皮功能,从而减轻尼古丁对血管内皮细胞的损害。因此我们设想尼古丁可能通过除nACHR之外的受体,如血管紧张素II受体1(angiotensin II receptor 1, AT1R)引起内皮功能紊乱。因此本课题拟观察尼古丁对内皮细胞AT1R表达的影响,以及尼古丁通过AT1R对内皮功能的改变。目的: 1.探讨尼古丁对脐静脉内皮细胞AT1R表达的影响。 2.探讨尼古丁对脐静脉内皮细胞eNOS及ICAM-1表达的影响。 3.探讨运用氯沙坦抑制AT1受体及siRNA沉默AT1R,观察尼古丁对人脐静脉内皮细胞eNOS和ICAM-1表达的影响有无改变。方法: 培养HUVEC并进行传代,观察10~(-6)M尼古丁干预不同时间(0h、2h、6h、12h、18h、24h)细胞膜上AT1R和细胞内eNOS及ICAM-1的表达变化趋势;分别选取24h AT1R变化最明显的时间点以及12h eNOS和ICAM-1都有显著性差异的时间点再以0、10~(-8)、10~(-7)、10~(-6)M尼古丁干预HUVEC观察细胞膜上AT1R和细胞内eNOS和ICAM-1的表达变化。按上述实验得到尼古丁干预HUVEC引起AT1R、eNOS和ICAM-1变化最显著的时间和浓度点后又将实验随机分为对照组、尼古丁干预组、氯沙坦+尼古丁干预组、AT1R siRNA干扰+尼古丁干预组,用western blot方法检测eNOS及ICAM-1的表达情况。采用SPSS18.0软件分析,数据以均数±标准差(means±SD)表示,不同处理组间比较采用单因素方差分析,P0.05认为差异有统计学意义。结果: 1.用0、10~(-8)、10~(-7)、10~(-6)M尼古丁干预HUVEC 24小时可见细胞AT1R含量随尼古丁浓度增加而逐渐增多,10~(-8)和10~(-7)M尼古丁干预细胞后AT1含量与对照组比较无显著性差异,10~(-6)M尼古丁干预后AT1含量与对照组相比有显著性改变(p㩳0.05)。10~(-6)M尼古丁干预HUVEC 2小时即可见细胞膜上AT1R受体含量的增高,且在24小时内随着时间延长,AT1含量逐渐增多。 2.10~(-6)M尼古丁干预HUVEC 6小时出现eNOS的增加,在12小时达高峰,并持续至18小时,然后逐渐降低;2小时出现ICAM-1的增加,在6小时达高峰,然后逐渐降低,18小时后与对照组无显著性差异。同时用0、10~(-8)、10~(-7)、10~(-6)M尼古丁干预HUVEC 12小时可见随尼古丁浓度的增加,eNOS与ICAM-1含量均逐渐增加。 3.10~(-7)M氯沙坦或siRNA沉默HUVEC中的AT1R,,可减少10~(-6)M尼古丁引起的HUVEC eNOS的高表达,但对ICAM-1无影响。结论: AT1受体在尼古丁所致内皮功能障碍中发挥一定作用。
[Abstract]:Research background:
Smoking is an important risk factor for cardiovascular disease, which can lead to coronary heart disease, cerebrovascular disease, aortic aneurysm, peripheral blood vessels and other diseases. It is reported that about 1.6 million deaths of cardiovascular patients worldwide in 2000 were directly caused by smoking. Dysfunction of muscle energy metabolism and promotion of thrombosis affect the cardiovascular system, while smoking cessation reduces the risk of coronary heart disease, cardiac arrest, stroke, and improves peripheral vascular symptoms.
As a major toxic substance in tobacco, nicotine plays an important role in the development of various cardiovascular diseases caused by smoking. However, the mechanism of nicotine-induced vascular endothelial dysfunction remains unclear.
Previous studies have focused on nicotinic acetylcholine receptor (nACHR) and its downstream mechanism. In vitro studies have shown that nicotine mediates endothelial cell proliferation, migration and angiogenesis through nACHR. In vivo studies have shown that nicotine can mediate angiogenesis, and this angiogenic effect can lead to atherosclerotic plaque. There are also reports of over-activation of the renin-angiotensin-aldosterone system in smokers. Angiotensin-converting enzyme inhibitors can inhibit oxidative stress response and modulate the expression of eNOS in endothelial cells. Therefore, we hypothesized that nicotine may cause endothelial dysfunction through receptors other than nACHR, such as angiotensin II receptor 1 (AT1R). Therefore, we intend to observe the effect of nicotine on the expression of AT1R in endothelial cells. And nicotine through AT1R on endothelial function changes.
Objective:
1. to investigate the effect of nicotine on AT1R expression in human umbilical vein endothelial cells.
2. to investigate the effect of nicotine on the expression of eNOS and ICAM-1 in human umbilical vein endothelial cells.
3. To observe the effect of nicotine on the expression of eNOS and ICAM-1 in human umbilical vein endothelial cells by inhibiting AT1 receptor and siRNA silencing of AT1R with losartan.
Method:
The expression of AT1R and intracellular eNOS and ICAM-1 in HUVEC were observed at different intervals (0h, 2h, 6h, 12h, 18h, 24h) after 10~(-6) M nicotine treatment. The expression of AT1R and intracellular eNOS and ICAM-1 in HUVEC were observed by intervention with nicotine. According to the above experiment, the time and concentration of the most significant changes of AT1R, eNOS and ICAM-1 induced by nicotine intervention in HUVEC were randomly divided into control group, nicotine intervention group, losartan + Nicotine intervention group, AT1R siRNA interference + Nicotine intervention group. Western blot was used to detect the expression of eNOS and ICAM-1. SPSS18.0 software was used to analyze the data. Mean (+ SD) was used to express the data. One-way ANOVA was used to analyze the difference between different treatment groups. P 0.05 showed that the difference was statistically significant.
Result:
1. The AT1R content of HUVEC treated with 0,10-8,10-7,10-6 M nicotine increased with the increase of nicotine concentration in 24 hours. There was no significant difference in AT1 content between the two groups after 10-8 and 10-7 M nicotine treatment. The AT1 content of HUVEC treated with 10-6 M nicotine had significant change compared with the control group (p?0.05). The content of AT1R receptor on HUVEC membrane increased after 2 hours of nicotine treatment, and the content of AT1 increased gradually with the prolongation of 24 hours.
2.10-6 M nicotine increased eNOS at 6 hours, peaked at 12 hours, lasted to 18 hours, and then gradually decreased; 2 hours increased ICAM-1 at 6 hours, then decreased gradually, after 18 hours, there was no significant difference between the two groups. At the same time, 0,10-8, 10-7, 10-6 M nicotine intervened HUVEC for 12 hours. With the increase of nicotine concentration, the contents of eNOS and ICAM-1 gradually increased.
3.10~(-7) M losartan or siRNA silencing AT1R in HUVEC decreased the high expression of HUVEC eNOS induced by 10~(-6) M nicotine, but had no effect on ICAM-1.
Conclusion:
AT1 receptors play a role in nicotine induced endothelial dysfunction.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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