纳米氧化锌暴露对斑马鱼胚胎心脏发育的毒性效应和机制研究

发布时间：2018-06-04 17:53

本文选题：氧化锌 + 纳米颗粒　；参考：《中国药理学与毒理学杂志》2017年06期

【摘要】：目的观察纳米氧化锌暴露对斑马鱼胚胎心脏发育及大鼠心肌细胞(H9c2)的毒性作用及可能机制。方法对纳米氧化锌材料进行表征化处理。斑马鱼胚胎在受精后4 h(4 hpf)暴露纳米氧化锌颗粒0.5,2.5,5.0和10.0 mg·L~(-1)24~96 h,计算胚胎存活率,并于胚胎17 hpf时间点,收集并用实时定量PCR测定中胚层脊索同源(noto),T盒16(tbx16),过短同源a(ta)和tbx6 m RNA表达;观察斑马鱼72 hpf心率。将心肌细胞标记红色荧光蛋白Tg(cmlc2:nuc-ds Red)的斑马鱼交配取胚胎,染毒纳米氧化锌2.5和5.0 mg·L~(-1),计数72 hpf胚胎心肌细胞数目。大鼠心肌细胞(H9c2)与纳米氧化锌0,0.1,0.5,1,5.0和10.0 mg·L~(-1)共培养24 h,Alamar Blue方法检测细胞活力,透射电子显微镜观察线粒体超微结构,化学发光法和Seahorse仪分别检测细胞ATP和耗氧率水平。结果纳米氧化锌在双蒸水中的粒径分布为(331±3)nm。48 hpf的斑马鱼胚胎纳米氧化锌LC50为21.81 mg·L~(-1)。暴露纳米氧化锌2.5 mg·L~(-1)后,17 hpf斑马鱼胚胎的心脏发育关键基因noto,ta和tbx6表达降低(P0.05)。72 hpf斑马鱼暴露纳米氧化锌5.0 mg·L~(-1),心率为153 min-1,与正常对照组相比降低12.6%(P0.01),心肌细胞数目减少15.5%(P0.01)。纳米氧化锌0.5 mg·L~(-1)可引起大鼠心肌细胞(H9c2)细胞活力降低(P0.05)以及线粒体出现空泡化现象。与细胞对照组相比,细胞ATP减少25.7%(P0.05),耗氧率减少27.2%(P0.01)。结论低剂量纳米氧化锌暴露对斑马鱼心脏发育有一定影响,主要表现为心率降低和心肌细胞数目减少,这些变化可能与心脏发育相关基因表达减少以及细胞线粒体结构和功能损伤有关。
[Abstract]:Objective to observe the toxic effects of nano-zinc oxide exposure on zebrafish embryonic heart development and rat cardiomyocyte H9c2 and its possible mechanism. Methods the nanometer zinc oxide materials were characterized. Zebrafish embryos were exposed to 0.5 and 10.0 mg / L of zinc oxide particles at 4 h and 4 h after fertilization. The survival rate of the embryos was calculated and the expression of tbx6 m RNA was detected by real-time quantitative PCR at 17 hpf. The heart rate of zebrafish was observed at 72 hpf. Zebrafish (zebrafish) labeled with red fluorescent protein (Tg(cmlc2:nuc-ds Red) were mated to obtain embryos. The embryos were treated with 2.5 and 5.0 mg / L zinc oxide and the number of 72 hpf embryonic cardiomyocytes was counted. Rat cardiomyocytes (H9c2) were co-cultured with zinc oxide (0.1mg / L) for 24 h to detect the viability of the cells. The ultrastructure of mitochondria was observed by transmission electron microscopy (TEM), and the levels of ATP and oxygen consumption were detected by chemiluminescence and Seahorse. Results the nanometer zinc oxide (LC50) of zebrafish embryo was 21.81 mg / L ~ (-1), whose particle size distribution was 31 1 卤3)nm.48 hpf in double distilled water. After exposure to nano-zinc oxide (2.5 mg / L), the expression of Notota and tbx6 in 17 hpf zebrafish embryos was decreased, and the heart rate was 153 min ~ (-1). The heart rate of zebrafish exposed to nano-zinc oxide 5.0 mg / L ~ (-1) was 15.3 min ~ (-1). Nano-zinc oxide (0.5 mg / L) could reduce the viability of rat cardiomyocytes (H9c2) and induce vacuolation of mitochondria. Compared with the control group, the cell ATP was reduced by 25.7g / L and the oxygen consumption rate was decreased by 27.2% (P0.01g). Conclusion low dose nano-zinc oxide exposure has a certain effect on heart development of zebrafish, mainly manifested by the decrease of heart rate and the number of cardiac myocytes. These changes may be associated with decreased expression of cardiac development-related genes and damage of mitochondrial structure and function.
【作者单位】：南京医科大学公共卫生学院;
【基金】：国家自然科学基金(81573174) 江苏省杰出青年基金(SBK2014010296) 环境化学与生态毒理学国家重点实验室开放课题(KF2015-01)~~
【分类号】：R99

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