尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4在心肌细胞分化中的作用  

Nicotinamide Adenine Dinucleotide Phosphate Oxidase 4 Regulates Cardiomyocyte Differentiation

作　　者：张小勇[1] 唐蔚青[1] 国汉邦[1] Michael Stouffs Marisa Jaconi 黎健[1] 

机构地区：[1]卫生部北京老年医学研究所/卫生部北京医院,卫生部老年医学重点实验室,北京100730 [2]Department of Pathology and Immunology,Faculty of Medicine,Geneva University

出　　处：《中国动脉硬化杂志》2006年第9期741-746,共6页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金(30370582)

摘　　要：目的探讨尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4在心肌细胞分化中的作用。方法用核糖酶技术获得尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4低表达小鼠胚胎干细胞克隆,将小鼠胚胎干细胞分化为胚小体。应用荧光染料2,7二氯氢化荧光素二酯和定量氮蓝四唑试验检测活性氧水平;逆转录聚合酶链反应和Western blot测定尼克酰胺腺嘌呤二核苷酸磷酸氧化酶mRNA水平和心室肌肌球蛋白轻链2蛋白表达水平;DNA laddering检测细胞凋亡;原位杂交分析尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4在胚胎心脏中的表达。结果活性氧清除剂N乙酰基半胱氨酸、过氧化氢酶和尼克酰胺腺嘌呤二核苷酸磷酸氧化酶抑制剂二甲苯基碘可抑制胚胎干细胞分化成心肌细胞,而较低浓度(1nmol/L^100nmol/L)的过氧化氢可明显促进心肌细胞分化,但较高浓度(1μmol/L)的过氧化氢显示出抑制作用(P<0.001)。实验发现,心肌细胞分化过程中内源性活性氧的产生主要来自尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4。应用核糖酶技术抑制胚胎干细胞中尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4的表达,可引起活性氧水平下降(P<0.001),肌球蛋白轻链2含量显著降低,使心肌细胞的分化受到明显的抑制(P<0.001)。尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4的高水平表达使干细胞产生大量活性氧,也显著抑制心肌细胞的分化(P<0.05)。结论尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4通过产生活性氧在心肌细胞分化中起关键作用。Aim To investigate the role of nicotinamide adenine dinucleofide phosphate oxidase 4 （NOX4） in cardiomyocyte differentiation. Methods Anti-NOX4 ribozyme clones of mouse embryonic stem （ES） cells were differentiated into embryoid bodies （EBs）. To measure reactive oxygenspecies （ROS） generation, H2DCF-DA and quantitative nitro blue tetrazolitan （NBT） test was used. The mRNA levels of NOX were assayed by RT-PCR while the protein level of ventricular myosin light chain 2 （MLC2v） was detected by westem blotting. Andibeapoptosis of ES cell wasdetected by DNA leddering. Moreover, NOX4 mRNA in the heart of mouse embryo sect.ices was analyzed by in situ hybridization. Results The exposure of embryoid bodies to 1-100 nmol/L H2O2 led to an enhanced beating activity, whereas 1/nnol/L H2O2 depressed cardiomyocyte differentiation as compared with eontrol conditions （P〈0.001）. In contrast , ROS scavengers, snchasN-acetylcysteine and catalase, and NOX inhibitor diphenyleneiedonium chloride impaired H2O2-stimulated cardiogenesis. The results revealed NOX4 as source of ROS responsible for KS cell differentiation into cardiomyocytes. Down-regulation of NOX4 expression in KS cells by ribozyme severely reduced ROS generation （ P〈 0.001） and MLC2v expression, resulting in the suppression of cardiomyocyte differentiation （ P 〈 0.001 ）. Moreover, NOX4 overexpression increased the production of ROS （ P 〈 0.05） and induced KS cell apo ptosis. Conclusion These results suggested a key role of NOX4 in cardiomyocyte differentiation through the generation of ROS.

关 键 词：病理学与病理生理学 尼克酰胺腺嘌呤二核苷酸磷酸氧化酶 活性氧 胚胎干细胞 心肌细胞 

分 类 号：R363[医药卫生—病理学]