胡秀英;方琴;王季石;马丹;蒋丽;
HU Xiuying;FANG Qin;WANG Jishi;MA Dan;JIANG Li;Department of Hematology,the Affiliated Hospital of Guizhou Medical University;Department of Pharmacy,the Affiliated Baiyun Hospital of Guizhou Medical University;

• 1:贵州医科大学附院血液内科
• 2:贵州医科大学附属白云医院药剂科

摘要(Abstract)：

目的:探讨乙醛脱氢酶2(ALDH2)基因导入人脐静脉内皮细胞(HUVECs)后对其增殖和抗氧化损伤的影响。方法:将培养的HUVECs分为转染目的基因组(转基因组)、转染空载体组(空质粒对照组)和未转染质粒组(阴性对照组),转基因组采用脂质体介导的方法将含人ALDH2基因的真核表达载体导入HUVECs中,采用RT-PCR和Western blot检测各组ALDH2基因和蛋白的表达;采用不同浓度过氧化氢(H_2O_2)(0、50、75、100和125μmol/L)孵育HUVECs 48 h后,检测各组细胞增殖水平及氧化损伤程度,检测各组细胞中血红素氧合酶(HO-1)、ROS及一氧化氮(NO)含量的变化。结果:转基因组荧光强度和数量比阴性对照组显著提高,提示导入的ALDH2基因在HUVECs细胞得到高表达;与空质粒对照组和阴性对照组比较,转染72 h后,转基因组细胞ALDH2 mRNA和蛋白表达增加(P<0.01);不同浓度H_2O_2孵育48 h后,转基因组HUVECs存活率明显升高(P<0.05);转基因组可抑制一定浓度H_2O_2诱导的ROS水平的升高、降低HO-1表达及增加NO的产生,差异有统计学意义(P<0.05)。结论:ALDH2基因高表达可降低低氧自由基引起的HUVECs细胞的损伤,保护内皮细胞的生物学功能。
Objective: To investigate the effect of aldehyde dehydrogenase 2(ALDH2) gene on the proliferation and anti- oxidative damage of human umbilical vein endothelial cells(HUVECs). Methods: HUVECs were divided into 3 groups: transfected target gene group(transgenic group) transfected empty vector group(empty plasmid control group) and non transfected plasmid group(negative control group). In the transgenic group,the eukaryotic expression vector containing human ALDH2 gene was introduced into HUVECs by liposome mediated method. Western blot RT-PCR were used to detect the expression of ALDH2 gene and protein in each group. Different concentration of H_2O_2(0,50,75,100 and 125 μmol / L) were incubated with HUVECs. 48 hours later,the level of cell proliferation and the degree of oxidative damage were detected in the cells of each group and the changes of HO-1,ROS and NO level were detected in the cells of each group. Results: The fluorescence intensity and number of transgenic group were significantly higher than that of negative control group,suggesting that the ALDH2 gene was highly expressed in HUVECs cells. Compared with empty plasmid control group and negative control group,the expression of mRNA and protein of ALDH2 gene in the transgenic group were increased(P < 0. 01) 72 h after transfection. The survival rate of HUVECs cells in transgenic group was significantly increased(P < 0. 05) after 48 h incubation with different concentrations of H_2O_2. The transgenic group could inhibit the increase of ROS induced by a certain concentration of H_2O_2,reduce the expression of HO-1 and increase the production of NO,and the difference was statistically significant(P < 0. 05). Conclusion: High expression of ALDH2 Gene can reduce the damage of HUVECs cells induced by low oxygen free radicals,and protect the biological function of endothelial cells.

关键词(KeyWords)： 乙醛脱氢酶2;转染;氧化损伤;一氧化氮;内皮细胞
aldehyde dehydrogenase 2;transfection;oxidation damage;nitric oxide;endothelial cell

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金资助项目(No.81360501);; 贵州省科学技术基金资助项目[黔科合SY字(2012)3138];贵州省科学技术基金资助项目[黔科合LH字(2014)7130];; 贵州省省长基金临床应用课题专项研究资助项目[黔省专合字(2012)97]

作者(Author): 胡秀英;方琴;王季石;马丹;蒋丽;
HU Xiuying;FANG Qin;WANG Jishi;MA Dan;JIANG Li;Department of Hematology,the Affiliated Hospital of Guizhou Medical University;Department of Pharmacy,the Affiliated Baiyun Hospital of Guizhou Medical University;

Email:

DOI: 10.19367/j.cnki.1000-2707.2016.11.006

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