尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4源性活性氧过量产生抑制胚胎干细胞向心肌细胞的分化(英文)  

Excessive production of nicotinamide adenine dinucleotide phosphate oxidase 4-dependent reactive oxygen species suppresses cardiomyocyte differentiation from embryonic stem cells

作　　者：张小勇[1] 国汉邦[1] 黎健[1] 

机构地区：[1]卫生部北京医院老年医学研究所,北京市100730

出　　处：《中国组织工程研究与临床康复》2007年第7期1386-1390,F0003,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金(30370582,30440065,30572082);北京自然科学基金(7052059)~~

摘　　要：背景:作为信号传导通路中的第二信使,活性氧(主要指超氧离子和过氧化氢)对细胞的生长、分化起重要作用。过量的活性氧在心脏疾病中启动了心肌细胞的凋亡,尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4源性活性氧的过量产生诱导凋亡是否抑制了胚胎干细胞向心肌细胞的分化。目的:探讨尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4活性氧对心肌细胞分化的抑制作用。设计:随机对照实验观察。单位:卫生部北京医院老年医学研究所。材料:胚胎干细胞CGR8为本实验室自存,其他主要试剂除特别注明均购自Sigma公司。方法:实验于2003-10/2005-08在卫生部北京医院老年医学研究所进行。①将小鼠胚胎干细胞分化成胚小体。在分化4d后以不同浓度的过氧化氢(1,10,100,1000nmol/L)处理胚小体1h,在分化8d后观察心肌细胞的生成,分析活性氧对心肌细胞分化的影响。②将pcDNA3.1和pcDNA3.1-NOX4质粒分别转染CGR8细胞,同时以未进行转染的CGR8细胞为对照,以RT-PCR和WesternBlotting测定尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4mRNA和MLC2v蛋白水平,应用定量四唑氮蓝高水平表达尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4干细胞中活性氧水平。③采用活性氧清除剂N-乙酰半胱氨酸(5mmol/L)和过氧化氢酶(200U/mL)转染前处理2h作为对照,用Hoechst染色、TUNEL和DNAladder分析尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4过表达后CGR8细胞凋亡情况,同时检测转染尼克酰胺腺嘌呤二核苷酸磷酸氧化酶氧化酶4后CGR8细胞中p21、p53及Bcl-2的表达。主要观察指标:①活性氧对心肌细胞分化的影响。②胚胎干细胞中尼克酰胺腺嘌呤二核苷酸磷酸氧化酶的表达。③尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4过表达后活性氧产生量、心肌细胞的分化和胚胎干细胞凋亡的观察。④p53,p21和Bcl-2是否参与了尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4过表达诱导的细胞�BACKGROUND： Reactive oxygen species （ROS）, including superoxide anion （O2） and hydrogen peroxide （H2O2）, have been recognized as specific second messengers in signaling cascades involved in the growth and differentiation of cells. The generation of excessive ROS initiates cardiomyocyte apoptosis. This paper is aimed to corroborate the hypothesis that excessive amounts of nicotinamide adenine dinucleotide phosphate （NADPH） oxidase （NOX） 4-dependent ROS can suppress cardiomyocyte differentiation from embryonic stem （ES） cells by induction of apoptosis. OBJECTIVE： To investigate the role of NOX4 ROS on cardiomyocyte differentiation DESIGN ： Randomized and controlled trial observation SETTING ： Beijing Institute of Geriatrics, Beijing Hospital, Key Laboratory of Geriatrics, Ministry of Health MATERIALS： ES cells were preserved by the laboratory of Beijing Institute of Geriatrics, Beijing Hospital, Key Laboratory of Geriatncs, Ministry of Health, while other reagents were purchased from Sigma Company without specific notification. METHODS ： The experiment was carried out in the Beijing Institute of Genatrics, Beijing Hospital, Key Laboratory of Geriatncs, Ministry of Health from October 2003 to August 2005. （1）Mouse ES cells were differentiated into embryoid bodies （EBs）. At day 4, EBs were managed for one hour with different concentrations （1, 10, 100, 1 000 nmol/L） of H2O2, and generation of cardiomyocytes was observed at day 8 to analyze the effect of ROS on cardiomyocyte differentiation.（2） CGB8 cells transfected with pcDNA3.1 and pcDNA3.1-NOX4 respectively were adopted, while those untransfected CGB8 cells were taken as controls. A quantitative NBT （nitro blue tetrazolium） test was used to measure NOX4 ROS genera- tion. The levels of NOX4 mRNA and MLC2v protein were assayed by RT-PCR and western blotting, respectively.（3）ROS scavenger N-acetylcysteine （5 mmol/L） and catalase （200 U/mL） managed for 2 hours before transcnption were taken as control

关 键 词：活性氧 NADPH氧化酶 胚胎 干细胞 心肌 细胞分化 细胞凋亡 

分 类 号：R394.2[医药卫生—医学遗传学]