胚胎期铅暴露对斑马鱼胚胎及幼鱼NMDA受体mRNA表达的影响

Effect of lead exposure on mRNA expression of NMDA receptors in zebrafish embryos and larvae

目的:了解胚胎期铅暴露对斑马鱼胚胎及幼鱼N-甲基-D-天冬氨酸(NMDA)受体mRNA表达的影响。方法:野生型AB品系斑马鱼胚胎醋酸铅暴露浓度分别为0、0.1、0.5、2.5和12.5μmol/L,提取各组受精后24、48、72、96和120 h(hpf)斑马鱼胚胎或幼鱼总RNA,实时定量PCR检测NMDA受体亚基NR1.1、NR1.2和NR2B的mRNA表达量。结果:(1)对照组NR1.1和NR1.2及NR2B表达量在胚胎发育过程中逐渐升高,在孵化期(72 hpf)表达量增加明显,在幼鱼早期(96 hpf)时达到高峰(与24 hpf时比较,P<0.01),在120 hpf时仍处于较高水平。(2)随着铅暴露浓度增高,NR1.1表达量增加并有高峰前移的趋势,2.5μmol/L和12.5μmol/L铅暴露组NR1.1表达高峰期在72 hpf,并且显著高于对照组(P<0.05);铅暴露组NR1.2和NR2B动态表达也呈类似规律,但NR1.2表达高峰期呈平台化趋势,横跨72 hpf至120 hpf阶段,NR2B表达高峰期出现在72 hpf和120 hpf阶段。(3)NR1.1、NR1.2及NR2B mRNA表达量之间Pearson相关系数值分别为rNR1.1-1.2=0.681、rNR1.1-2B=0.637和rNR1.2-2B=0.514,均有统计学意义(P<0.01)。结论:在斑马鱼胚胎发育过程NR1.1、NR1.2及NR2B mRNA表达水平逐渐升高,在幼鱼早期达到高峰;胚胎和幼鱼阶段NR1.1、NR1.2及NR2B之间mRNA表达水平存在关联;铅有上调NR1.1、NR1.2和NR2B mRNA表达作用并使表达峰期前移,改变了正常的NMDA受体表达规律。

AIM ： To investigate the effect of lead exposure on mRNA expression of N - methyl - D - aspartate （NMDA） receptors in zebrafish embryos and larvae. METHODS： Zebrafish embryos （wild type; AB line） were exposed to lead acetate （PbAc） at concentrations of 0, 0.1 1 0.5, 2.5 and 12.5 μmol/L, respectively. Total RNA was extracted from zebrafish embryos or larvae at the time points of 24, 48, 72, 96 and 120 hours post fertilization （hpf）. The mRNA levels of NR1.1, NR1.2 and NR2B were determined by real - time quantitative PCR. RESULTS ： The mRNA expression of NR1.1, NR1.2 and NR2B gradually increased during embryonic development, raised rapidly at 72 hpf, peaked at 96 hpf （vs that at 24 hpf, P 〈0.01 ）, and still kept in high level at 120 hpf in control group. The impact of lead exposure on the mRNA expression of NR1.1, NR1.2 and NR2B varied with lead concentrations. With the increasing concentrations of PbAc, the mRNA expression of NR1.1 generally increased and reached the highest level ahead of 96 hpf. The peak mRNA level of NR1.1 was observed at 72 hpf under the condition of PbAe exposure at the concentrations of 2.5 and 12.5 μmol/ L, and were higher than that in control group （ P 〈 0.05 ）. Similarly, the mRNA levels of NR1.2 and NR2B showed an in- creasing trend with PbAe exposure. However, the peaking time of NR1.2 and NR2B in mRNA expression spanned from 72 to 120 hpf. The significant correlations between the expression levels of NR1.1, NR1.2 and NR2B were observed （ P 〈 0. O1 ） and the Pearson＇ s correlation coefficient values of ^rNR1, 1 -1. 2, ^rNR1.1-2B and ^rNR1.2-2B were 0.681, 0.637 and 0.514,respectively. CONCLUSION： The mRNA expression of NRI. 1, NR1.2 and NR2B gradually increases throughout the embryonic development of zebrafish and reaches the highest levels at early stage of larva. Close correlations between the mRNA expression of NRI. 1, NR1.2 and NR2B are present during the period of embryo and larva. Lead exposure induces up - regulation and forward shift of NR1.1, NR1.2 and NR2B at mRNA level, indicating that lead induces abnormal ex- pression of NMDA receptors.