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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。
Objective To investigate the role of nicotinamide adenine dinucleotide phosphate oxidase 4 in cardiomyocyte differentiation. Methods Nicotinamide adenine dinucleotide phosphate oxidase 4 was transfected into murine embryonic stem cell clones by ribozyme technology. The mouse embryonic stem cells were differentiated into embryonic bodies. The levels of reactive oxygen species (ROS) were measured by fluorochrome 2,7-dichlorofluorescein diester and quantitative nitroblue tetrazolium assay. The levels of nicotinamide adenine dinucleotide phosphate oxidase mRNA and ventricular Muscle myosin light chain 2 protein expression; DNA laddering detection of apoptosis; in situ hybridization nick nicotine adenine dinucleotide phosphate oxidase 4 expression in the embryonic heart. Results Active oxygen scavengers N-acetylcysteine, catalase and nicotinamide adenine dinucleotide phosphate oxidase inhibitor xylenyl iodide inhibited the differentiation of embryonic stem cells into cardiomyocytes, whereas lower concentrations (1 nmol / L ~ 100nmol / L) could obviously promote the cardiomyocyte differentiation, but the higher concentration (1μmol / L) hydrogen peroxide showed inhibition (P <0.001). The experiment found that the production of endogenous reactive oxygen species during cardiomyocyte differentiation mainly came from nicotinamide adenine dinucleotide phosphate oxidase 4. Ribonuclease inhibition of nicotinamide adenine dinucleotide phosphate oxidase 4 expression in embryonic stem cells resulted in a decrease in reactive oxygen species (P <0.001), a significant decrease in myosin light chain 2, and an increase in cardiomyocyte differentiation Was significantly inhibited (P <0.001). Nicotinamide adenine dinucleotide phosphate oxidase 4 at high levels of expression of the stem cells produce a large number of reactive oxygen species, but also significantly inhibited the differentiation of cardiomyocytes (P <0.05). Conclusion Nicotinamide adenine dinucleotide phosphate oxidase 4 plays a key role in cardiomyocyte differentiation by generating reactive oxygen species.