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目的:在心脏特异的骨形态形成蛋白受体IA(the bone morphogenetic protein receptor IA,ALK3)基因敲除小鼠模型中,纯合子的心脏特异ALK3基因敲除的小鼠死于胚胎中期,同时伴有室间隔缺损。用基因芯片(microarray)法筛选了ALK3下游基因,并获得下游候选基因(转录因子Pax-8)。为了进一步明确Pax-8基因在胚胎心脏发育中的角色,Pax-8基因在小鼠被系统敲除。方法:运用实时荧光定量RT-PCR法检测Pax-8基因在小鼠胚胎中的表达情况。运用原位杂交法显示Pax-8基因在小鼠胚胎心脏中的表达部位。TUNEL法检测心肌细胞的凋亡情况,电镜下观察Pax-8基因敲除的小鼠心脏组织发育的病理学异常。结果:正常胚胎小鼠9.5、10.5、11.5、12.5以及13.5d的Pax-8基因的表达水平以10.5d表达最高。胚胎切片的原位杂交显示:转录因子Pax-8表达在13.5d的α-MHCCre+/-,ALK3F/+小鼠整个心脏中。Pax-8基因敲除的小鼠心脏表现出形态改变(心脏呈球形,左心室壁肥厚,室间隔增厚,左室乳头肌肥大),左心室壁和室间隔心肌组织细胞凋亡率明显增高,同时线粒体体积较对照组明显缩小并且数量增多。结论:Pax-8基因是心脏较特异的参与心脏正常发育的ALK3下游基因,并与心肌细胞凋亡有关。
OBJECTIVE: In a mouse model of heart-specific bone morphogenetic protein receptor IA (ALK3) knockout mice homozygous for heart-specific ALK3 knockout mice died of metaphase, accompanied by Ventricular septal defect. The downstream gene of ALK3 was screened by microarray and the downstream candidate gene (transcription factor Pax-8) was obtained. To further clarify the role of Pax-8 in embryonic heart development, the Pax-8 gene was knocked out systematically in mice. Methods: The expression of Pax-8 gene in mouse embryos was detected by real-time fluorescence quantitative RT-PCR. In situ hybridization was used to show the expression site of Pax-8 gene in mouse embryonic heart. TUNEL method was used to detect the apoptosis of cardiomyocytes. The pathological abnormalities of heart tissues of Pax-8 knockout mice were observed under electron microscope. Results: The expression levels of Pax-8 gene in normal embryonic mice at 9.5, 10.5, 11.5, 12.5 and 13.5 d were the highest at 10.5 d. In situ hybridization of embryo sections showed that the transcription factor Pax-8 was expressed throughout 13.5 days of α-MHCCre +/-, ALK3F / + mice. Pax-8 knockout mouse heart showed morphological changes (spherical heart, left ventricular wall hypertrophy, ventricular septal thickening, left ventricular papillary hypertrophy), left ventricular wall and interventricular septal myocardial tissue apoptosis rate was significantly increased, At the same time mitochondrial volume was significantly reduced and increased compared with the control group. CONCLUSION: Pax-8 gene is a specific downstream gene of ALK3 involved in normal heart development and is associated with cardiomyocyte apoptosis.