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高糖对人脐静脉内皮细胞NO分泌及SIRT1表达的影响
The Effect of High Glucose on NO Secretion and SIRT1 Expression in Human Umbilical Vein Endothelial Cell
【作者】 王楠；
【作者基本信息】 天津医科大学 ， 内科学， 2009， 硕士
【摘要】 目的糖尿病大血管病变严重威胁着人类的健康。高血糖是2型糖尿病的基本特征,多项研究已证实高血糖可以经多种途径直接或间接损伤内皮细胞,促进动脉粥样硬化的发生。SIRT1(silence information regulator 1)是血管内皮中的保护性蛋白,SIRT1-eNOS轴在维持内皮稳态中发挥着重要作用,NO与SIRT1是该轴的重要组成部分,本研究旨在通过观察不同葡萄糖培养环境下HUVEC中NO分泌及SIRT1表达,观察高糖对SIRT1-eNOS轴的影响,为糖尿病大血管病变的预防和治疗寻找新靶点。内容取用无菌脐带,经胰酶消化后,原代培养脐静脉内皮细胞(HUVEC),达80%以上融合时予以传代培养并鉴定,采用第3～5代细胞用于下一步实验。然后将同源细胞随机分成4组,分别记为正常葡萄糖浓度组(5.5mmol/L)、高糖11.1mmol/L组、高糖22.2mmol/L组和高糖33.3mmol/L组,在相应浓度的培养基中培养48小时后,检测各组细胞上清液中NO含量,同时,检测各组细胞中SIRT1 mRNA和蛋白表达,观察葡萄糖浓度对HUVEC中NO分泌及SIRT1表达的影响。最后将同源细胞随机分为4组,记为0h、24h、48h、72h组,用高浓度葡萄糖33.3mmol/L培养细胞至上述时间后,检测各组细胞上清液中NO含量,同时,检测各组细胞中SIRT1 mRNA和蛋白的表达,观察高糖培养不同时间对HUVEC中NO分泌及SIRT1表达的影响。方法1、实验方法:原代及传代脐静脉内皮细胞培养;免疫组化法细胞鉴定;硝酸还原法检测NO分泌;细胞总RNA和总蛋白提取;RT-PCR和western blot技术。2、统计方法:所有数据通过SPSS11.5软件进行处理,经Levene检验验证方差齐性。多组间均数比较采用单因素方差分析,多组间均数两两比较采用SNK-q检验。P＜0.05被认为有统计学意义。结果1、HUVEC原代及传代培养成功,免疫细胞化学染色示培养细胞vonwillebrand因子(Ⅷ因子相关抗原)阳性。2、HUVEC培养48h,随葡萄糖培养浓度的增加,各组细胞NO分泌量逐渐下降(P＜0.05)。同时,HUVEC中SIRT1基因和蛋白表达均呈逐渐下降趋势,其中高糖22.2 mmol/L及33.3 mmol/L组与正常浓度组相比,SIRT1基因和蛋白表达差异有统计学意义(P＜0.05)。3、高糖(33.3mmol/L)培养HUVEC,与0h组相比,NO分泌量在24h组增加(P＜0.05),48h和72h组减少(P＜0.05)。同时,与0h组相比,48h和72h组SIRT1mRNA和蛋白表达下降有统计学意义(P＜0.05)。结论1、培养48h,HUVEC中NO分泌及SIRT1表达与葡萄糖浓度有关。葡萄糖浓度达22.2mmol/L以上,HUVEC中NO分泌水平及SIRT1表达均下降。2、高糖33.3mmol/L培养环境下,HUVEC中NO分泌及SIRT1表达水平与培养时间有关。随培养时间的延长HUVEC中NO分泌呈先升高后降低的变化。其中,培养48h以上HUVEC中NO分泌及SIRT1的表达均下降。以上结果表明高糖可以同时抑制HUVEC中的NO分泌和SIRT1表达,且抑制程度与葡萄糖浓度及高糖培养时间有关。由于NO和SIRT1是SIRT1-eNOS轴的重要信号分子,因此,抑制SIRT1-eNOS轴可能是高糖损伤内皮功能的又一途径。
【Abstract】 ObjectiveDiabetes is a major independent risk factor for cardiovascular disease and stroke. Previous studies showed that high glucose induced endothelial dysfunction by affecting NO secretion directly or indirectly. Recent studies point to silence information regulator 1 (SIRT1) as a key regulator of vascular endothelial homeostasis, controlling angiogenesis, vascular tone and endothelial dysfunction, and a positive feedback mechanism exists between SIRT1 and NO. The aim of this study is to investigate the effect of high glucose on the secretion of NO and the expression of SIRT1 in human umbilical vein endothelial cells (HUVEC). ContentsPrimary HUVECs were isolated and cultured, then they were identified by detecting human VIII factor associated antigen with immunohistochemical method. Subculture the cells when they are over 80% confluence. Experiments are done with cells between 2-4 passages. HUVEC were treated with glucose at different concentrations (5.5, 11.1, 22.2, 33.3mmol/L) for 48h. NO content in medium were detected by nitrate reductase test.SIRT1 mRNA expression levels were determined by reverse transcription-polymerase chain reaction (RT-PCR)and the protein expression levels were evaluated by Western blotting.Then HUVEC were treated with high glucose(33.3mmol/L) for different time(0h、24h、48h and 72h),NO content in medium and SIRT1 mRNA and protein level were detected by the times.Methods1、Experimental methods included: (1) Primary cultivation and serial subcultivation of HUVEC. (2) Immunocytochemistry. (3)NO detected by nitrogen oxide kit. (4) Extraction of total RNA and Protein,RT-PCR and western blotting.2、Statistic methods: All analysis were performed with SPSS11.5 software package.Normal distribution of the data was tested using kolmogorov-Smirnov nonparametric test.Groups comparions were performed with one way analysis of variance (post levene test).comparion of any two group among the groups were performed with SNK-q test. Difference were considered statistically significent at P<0.05.Results1、Primary HUVECs were isolated and cultured, immunocytochemical stain show human VIII factor related antigen positive.2、NO content in mediun significantly decreased by different glucose concentration (P<0.05) .In contrast to normal glucose, high glucose (22.2 mmol/L,33.3 mmol/L) significantly decreased SIRT1 expression in both protein and mRNA levels (P<0.05).3、In contrast to 0h , the NO content in medium increased after culturing 24h(P<0.05). The expression of SIRT1 in both RNA and protein level ,as well as the NO content in medium, significantly decreased after culturing 48h and 72h (P<0.05).ConclusionsHigh glucose may decrease NO secretion and SIRT1 expression in a dose-and time-dependent manner in HUVEC. SIRT1-eNOS axis may be a pathway that connecting high glucose and endothelial dysfunction.