阿霉素致斑马鱼心血管毒性模型的建立

EstabiUshment of Cardiotoxicity Model in Zebrafish Induced by Doxorubicin

目的采用阿霉素（doxorubicin，DOX）作为阳性药物建立斑马鱼的心血管毒性模型。方法确定DOX的10％致死浓度（LC10）与最大非致死浓度（MNLC）,设置4个不同浓度的DOx溶液组（浓度：MNLC／10、MNLC／3、MNLC和LC10）、空白对照组（养鱼水处理组）和溶剂对照组（0．9％NaCl注射液）处理一定阶段的野生型AB系斑马鱼幼鱼至实验终点受精后725（72hpf）。在72hpf处收集药物处理的斑马鱼，Westem blotting分析心肌肌钙蛋白T（cTnT）和肌红蛋白（Mb）的表达，在立体解剖显微镜下观察心血管系统的形态学改变，荧光定量RT—PCR检测斑马鱼组织中miRNA146a的表达。结果在浓度≤MNLC／10时，DOX不诱发斑马鱼心血管毒性；当浓度≥MNLC／3时，可诱发斑马鱼心血管毒性，表现为血流变慢、血流缺失、静脉窦处瘀血和静脉窦处水肿，且具有剂量相关性。荧光定量RT—PCR结果显示，DOX的三个剂量MNLC／3、MNLC、LC10均显著提高miRNA146a的表达（与空白对照组比较，P〈0．01），且存在一定的剂量关系。Western blotting结果显示，DOX模型组的cTnT和Mb蛋白表达与空白对照组比较均未见明显变化（P〉0．05）。结论DOx对斑马鱼的心血管毒性作用明显，可以作为药物评价的心血管毒性模型。

Objective To establish zebrafish model of cardiotoxicity induced by doxorubicin （DOX）. Methods The wild type zebrafish was divided into 6 groups： 4 different concentrations of DOX solution, control group and 0.9% NaCl groups. To assay the expression of cardiac troponin T（cTnT） and myoglobin （Mb） by Western blot at 72 hpf （72 hours post fertilization）. Morphologic changes in the cardiovascular system were observed under stereomicroscope. The expression of miRNA 146a in zebrafish was tested by RT-PCR. Results DOX had no cardiotoxicity in zebrafish within 0-MNLC/10; when the concentration of DOX ≥ MNLC/3, it can be induced cardiovascular toxicty in the zebrafish, manifested as blood flow slows down, the lack of blood flow, venous sinus congestion, venous sinus edema and it is dose-dependent. Tissue expression of miRNA146a on zebadfishes of 3 concentrations were all increased compared with control group （P〈0.01）, and it is dose-dependent. There was no significant differences in the expression of cTnT and Mb between DOX groups and control group （P〉0.05）. Conclusion The cardiotoxicity induced by DOX was significant and ithad a similar toxic effect to mammal. So the zebrafish model of cardiotoxicity induced by DOX was successfully established.