dhfr基因过表达对乙醇致斑马鱼胚胎心血管发育异常的效应研究

Effect of dhfr gene overexpression on ethanol-induced abnormal cardiovascular development in zebrafish embryos

摘要:

目的 观察二氢叶酸还原酶基因（dhfr）过表达对乙醇致斑马鱼胚胎心脏和血管发育异常的作用，并初步探讨其机制。方法 体外转录dhfr mRNA并将其显微注射入斑马鱼受精卵内使dhfr基因过表达，并进行过表达的效率验证。将野生型斑马鱼分为正常对照组、乙醇处理组（400?mmol/L乙醇溶液）、乙醇+dhfr mRNA组（显微注射6?nL dhfr mRNA），观察各组胚胎的整体发育情况。按照上述分组，利用红色荧光蛋白标记心脏的转基因斑马鱼系Tg（cmlc2：mcherry）观察胚胎心房和心室的发育状况；利用荧光显微造影观察野生型斑马鱼胚胎心脏流出道及血管发育情况；通过心率和心室收缩指数评估心功能情况。构建基因探针并通过胚胎整体原位杂交及Real-time PCR法，检测胚胎nkx2.5、tbx1、flk-1的基因表达情况。结果 与乙醇处理组相比，乙醇+dhfr mRNA组斑马鱼胚胎发育异常百分率明显下降，存活百分率显著上升（P < 0.05）；心房、心室、心脏流出道和血管发育的异常表型及心功能情况明显改善。乙醇处理组nkx2.5、tbx1、flk-1 mRNA的表达较对照组下降（P < 0.05）；乙醇+dhfr mRNA组nkx2.5、tbx1、flk-1 mRNA的表达较乙醇处理组上调，但仍未达到正常对照组水平（P < 0.05）。结论 dhfr基因过表达可部分挽救乙醇所致胚胎心脏和血管发育异常，其机制可能与dhfr基因过表达后上调乙醇导致的nkx2.5、tbx1、flk-1水平降低有关。

Abstract:

Objective To study the effect of dhfr gene overexpression on ethanol-induced abnormal cardiac and vascular development in zebrafish embryos and underlying mechanisms. Methods dhfr mRNA was transcribed in vitro and microinjected into zebrafish fertilized eggs to induce the overexpression of dhfr gene, and the efficiency of overexpression was verified. Wild-type zebrafish were divided into a control group, an ethanol group, and an ethanol+dhfr overexpression group (microinjection of 6 nL dhfr mRNA). The embryonic development was observed for each group. The transgenic zebrafish Tg (cmlc2:mcherry) with heart-specific red fluorescence was used to observe atrial and ventricular development. Fluorescence microscopy was performed to observe the development of cardiac outflow tract and blood vessels. Heart rate and ventricular shortening fraction were used to assess cardiac function. Gene probes were constructed, and embryo in situ hybridization and real-time PCR were used to measure the expression of nkx2.5, tbx1, and flk-1 in the embryo. Results Compared with the ethanol group, the ethanol+dhfr overexpression group had a significant reduction in the percentage of abnormal embryonic development and a significant increase in the percentage of embryonic survival (P < 0.05), with significant improvements in the abnormalities of the atrium, ventricle, outflow tract, and blood vessels and cardiac function. Compared with the control group, the ethanol group had significant reductions in the expression of nkx2.5, tbx1, and flk-1 (P < 0.05), and compared with the ethanol group, the ethanol+dhfr overexpression group had significant increases in the expression of nkx2.5, tbx1, and flk-1 (P < 0.05), which were still lower than their expression in the control group. Conclusions The overexpression of the dhfr gene can partially improve the abnormal development of embryonic heart and blood vessels induced by ethanol, possibly by upregulating the decreased expression of nkx2.5, tbx1, and flk-1 caused by ethanol.