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目的以致胚胎毒性阳性药物全反式维甲酸(ATRA)及丙戊酸钠进行斑马鱼胚胎发育毒性试验,建立有效的斑马鱼胚胎发育毒性评价方法。方法采用水浴染毒法,将受精后2 h(2 hpf)的斑马鱼胚胎暴露于不同浓度梯度的阳性药物。分别在24、48、72和144 hpf观察并记录畸形及死亡的胚胎数目。统计阳性药物的EC50和LC50,计算致畸指数(TI=LC50/EC50)。结果两种阳性药物所致斑马鱼胚胎发育早期(24~48 hpf)与发育后期(72~144 hpf)的畸形表现不同。在72hpf,两种阳性药物对胚胎孵化率均有明显抑制作用;144 hpf可见ATRA(≥1.6×10-3mg/L)和丙戊酸钠(≥1.25×102mg/L)严重致畸作用,TI分别为10.35和5.72。两种阳性药物胚胎致畸率及死亡率均呈明显的浓度依赖关系,且与已有动物试验及体外试验结果相符。结论以两种阳性药物建立有效的斑马鱼胚胎发育毒性评价方法,可进行进一步的验证。
OBJECTIVE: To establish a zebrafish embryonic developmental toxicity test by using embryonic toxicity-positive all-trans retinoic acid (ATRA) and sodium valproate. Methods The zebrafish embryos at 2 hpf after fertilization were exposed to positive drug with different concentration gradient by water bath method. At 24, 48, 72 and 144 hpf were observed and recorded the number of deformed and dead embryos. The EC50 and LC50 of the positive drugs were counted and the teratogenic index (TI = LC50 / EC50) was calculated. Results The abnormalities of zebrafish embryo development (24 ~ 48 hpf) and late development (72 ~ 144 hpf) caused by two kinds of positive drugs were different. At 72 hpf, both positive drugs significantly inhibited embryo hatching rate; 144 hpf showed severe teratogenic effects of ATRA (≥1.6 × 10-3mg / L) and sodium valproate (≥1.25 × 102mg / L), TI Respectively 10.35 and 5.72. The embryo teratogenicity and mortality of the two kinds of positive drugs showed a significant concentration-dependent relationship, and with the existing animal experiments and in vitro test results. Conclusions The establishment of an effective zebrafish embryo developmental toxicity evaluation method with two positive drugs can be further verified.