雌二醇暴露中国青鳉原代肝细胞转录组分析

Transcriptome Analysis of Oryzias sinensis Primary Hepatocytes under the Exposure of Estradiol

鱼类雌激素响应基因的发掘及其作为生物标记物基因的应用一直是生态毒理学的研究热点。近年来的相关研究普遍采用活体实验,易受到动物个体差异影响,存在一定不确定性。通过建立鱼类原代肝细胞培养方法,利用实验室多代繁育的中国青鳉(Oryzias sinensis)作为实验动物,基于新一代测序技术(NGS),开展了不同浓度雌二醇(0.01、0.1、1、10和100 nmol·L-1)暴露下的中国青鳉肝细胞转录组分析,获得并注释中国青鳉表达序列标签(ESTs)65765条,发现雌激素暴露后具有良好剂量效应关系的显著差异表达基因(DEG)105个(37个上调,68个下调),其中有9个未知基因。GO和KEGG Pathway分析发现,DEG显著富集于雌激素的细胞响应、脂质运输、骨骼肌组织发育、钙离子运输、蛋白磷酸化等生物学过程以及钙离子信号通路和MAPK信号通路等。通过RT-qPCR对部分雌激素响应基因进行了分析验证,结果表明NGS数据可靠。卵黄原蛋白(VTG)和卵壳前体蛋白(CHG)基因家族在0.1 nmol·L-1雌二醇浓度时均能够显著升高,vtg2响应最明显。该研究提供了一个可靠的中国青鳉肝脏雌激素响应基因列表,以及一种利用本土物种细胞离体实验开展化学物质雌激素活性测定的方法,该方法比一些常用环境雌激素生物测试方法灵敏度更高。

The identification of estrogen-responsive genes in fishes and their uses as biomarkers in monitoring environmental estrogens in the water are of concern in ecotoxicological studies.Recently,many estrogen-responsive genes have been investigated in model fishes such as zebrafish using in vivo exposure experiments.Because of the individual difference,there are always non-negligible errors and uncertainties during the in vivo studies.In this paper,we developed a method for fish primary hepatocyte culture,and studied the gene expressions in response to estradiol exposure at different concentrations(0.01,0.1,1,10 and 100 nmol·L-1)in primary hepatocytes of Chinese medaka(Oryzias sinensis)using next-generation sequencing technology(NGS).A total of 65765 expression sequence tags(ESTs)were obtained and annotated by NGS,and 105 differentially expressed genes(DEGs)(37 up-regulated,68 down-regulated,9 unknown genes)were detected and show good dose-response relationship after estradiol exposure.GO and KEGG Pathway analyses showed that the DEGs were significantly enriched in the biological process of“response to estradiol”,“lipid transport”,“skeletal muscle tissue development”,“calcium ion transport”,“protein phosphorylation”,etc.,and KEGG pathways of“ubiquitin mediated proteolysis”,“MAPK signaling pathway”,etc.Analyses of some DEGs by RT-qPCR confirmed the expression results of NGS.The expressions of vitellogenin(VTG)genes and choriogenin(CHG)genes were found to be significantly increased at concentration of 0.1 nmol·L-1 estradiol and higher,and the expression of vtg2 was the largest increased.This study provides a reliable list of estrogen-responsive genes in liver of Chinese medaka and a method for measuring the estrogenic activity of chemicals in vitro using native species.The method is more sensitive than some traditional bioassays.