斑马鱼Tbx2基因阻抑先天性心脏病模型的建立与研究

目的建立斑马鱼Tbx2基因阻抑先天性心脏病动物模型,并研究Tbx2基因对心脏发育的影响。方法通过吗啉环寡聚核苷酸显微注射介导的翻译抑制,观察Tbx2基因阻抑胚胎的心脏发育,并通过心脏特异性分子标记整胚原位杂交技术进一步阐明Tbx2在心脏发育中的作用。结果600枚Tbx2基因阻抑的斑马鱼胚胎受精后8h27．2％（163／600）胚胎死亡,24～96h出现轻、中、重度不同程度的心脏发育异常,比例分别为21．3％（128／600）、32．8％（197／600）、18．7％（112／600）,心脏缺陷包括心室发育不良、心房扩张、房室区异常、心率缓慢、心律不齐、血液反流等,整胚原位杂交结果显示了房室区特异性分子标记多能聚糖（versican）基因表达的异常。结论斑马鱼是研究心脏发育的理想模式生物,吗啉环寡聚核苷酸基因阻抑是研究基因功能的良好方法。Tbx2基因在房室特异性分化和房室管形成方面起了重要的作用。

Establishment of model of congenital heart disease with Tbx2 gene knockdown: experiment with zebrafish

OBJECTIVE: To establish a model of congenital heart disease with Tbx2 gene knockdown. METHODS: 1200 fertilized eggs of zebrafish (Danio rerio) were divided into 3 groups: wild type group (n = 100), Tbx2-morpholino (MO) group (n = 600, to be injected with Tbx2-MO so as to knock down the Tbx2 gene), and control MO group (n = 500, to be injected with control MO). 12, 24, 36, 48, 72, and 96 hours after fertilization the heart structure, heart rate, rhythm, contractibility, and blood flow of the embryos were observed under anatomic microscope and digital camera was used to record the findings. Whole-mount in situ hybridization was conducted to observe the expression of versican in the heart. RESULTS: Eight hours after fertilization 27.2% of the fertilized eggs of the Tbx2-MO group died, and 21.3%, 32.8%, and 18.7% of them showed mild, moderate, or severe developmental abnormality of heart, including hypogenetic ventricle, dilatation of atria, abnormal atrioventricular canal, slow heartbeat, arrhythmia, and blood regurgitation. Whole-mount in situ hybridization showed that versican did not decrease with the passage of time but was expressed abnormally in the atria and ventricles, and did not disappear 72 hours after fertilization; however, it was expressed normally in the statolith. CONCLUSION: MO antisense oligonucleotide knockdown is a good method for the study of heart development. Tbx2 gene plays an important role in the differentiation of atria and ventricles and formation of atrioventricular canal.