nr1h4调控转基因斑马鱼模型建立及相关研究

Establishment of transgenic zebrafish model regulated by nr1h4 and related studies

目的建立法尼醇X受体(FXR,基因名称nr1h4)组织特异性荧光标记转基因斑马鱼模型,为胆汁酸代谢相关药物筛选及安全预警提供可视化检测动物模型。方法在UCSC网站查找斑马鱼nr1h4基因调控序列,并利用Promoter 2.0软件对其进行优化,设计引物,利用PCR方法调取斑马鱼nr1h4调控序列,构建于pT2AL200R150G转座载体克隆位点,将pTol2-nr1h4-EGFP质粒与pCS-TP转座酶mRNA共同注入斑马鱼受精卵单细胞,筛选出在肝脏和肠道中特异性表达的荧光个体,通过遗传学筛选,建立Tg(-1.6nr1h4-EGFP)斑马鱼品系。将受精后第4天的Tg(-1.6nr1h4-EGFP)斑马鱼幼鱼随机分为空白对照组、溶媒对照组、甘氨酸-β-鼠胆酸组、奥贝胆酸组以及低、中、高浓度(10、20、40μg·mL)熊去氧胆酸组、胆宁片组、大黄酸组和芦荟大黄素组,连续给药4 d,观察各组转基因斑马鱼不同时间的荧光强度及发育情况,并对荧光强度进行分析。结果成功建立了Tg(-1.6nr1h4-EGFP)荧光标记转基因斑马鱼品系,绿色荧光于体节期开始在斑马鱼腹部表达,受精后第4天(4dpf)主要集中在肝脏和肠道表达。与空白对照组相比,甘氨酸-β-鼠胆酸组转基因斑马鱼在各给药时间的荧光表达水平均显著降低(4dpf:P<0.01;5~7dpf:P<0.0001),奥贝胆酸组在各给药时间荧光均显著增强(P<0.0001),溶媒组荧光表达水平与对照组无显著差异,表明了该模型的有效建立。与空白对照组相比,低浓度的熊去氧胆酸组和芦荟大黄素组转基因斑马鱼在给药第2~4天荧光表达水平逐渐增强(P<0.0001),中浓度的熊去氧胆酸组和芦荟大黄素组在各给药时间荧光均显著增强(P<0.0001),高浓度的芦荟大黄素组在给药第2~4天荧光逐渐增强(5~6dpf:P<0.001;7dpf:P<0.05),高浓度的熊去氧胆酸组荧光表达水平与空白组无显著差异;低浓度的胆宁片组和大黄酸组转基因斑马鱼在各给药时间的荧光表达水平与空白组无显著差异,中浓度的胆宁片组在各给药时间荧光均显著增强(4dpf和7dpf:P<0.0001;5dpf和6dpf:P<0.001),高浓度的胆宁片组在各给药时间荧光依旧显著增强(P<0.0001),中浓度的大黄酸组在给药第2~4天荧光逐渐增强(5dpf:P<0.0001;6dpf:P<0.001;7dpf:P<0.01),而高浓度的大黄酸组在给药第2~4天荧光开始减弱(P<0.0001)。结论Tg(-1.6nr1h4-EGFP)斑马鱼模型为胆汁酸药物机制研究、药物筛选和安全性评价提供了新的动物模型;低、中浓度的熊去氧胆酸,中、高浓度的胆宁片,中浓度的大黄酸和各浓度的芦荟大黄素均能够通过上调斑马鱼肝脏和肠道FXR促进胆汁酸转运,而高浓度的熊去氧胆酸对FXR无明显作用,高浓度的大黄酸抑制FXR表达。

Objective A tissue-specific fluorescent labeled transgenic zebrafish model of farnesol X receptor(FXR,gene name nr1h4)was established to provide a visual detection animal model for the screening and safety warning of drugs related to bile acid metabolism.Methods The regulatory sequence of nr1h4 in zebrafish was found on UCSC website and optimized by promoter 2.0 software.Primers were designed and the nr1h4 regulatory sequence of zebrafish was obtained by PCR and constructed at the cloning site of pT2AL200R150G transposable vector.Then pTol2-nr1h4-EGFP plasmid and pCS-TP transposase mRNA were co-injected into zebrafish fertilized egg single cells to screen fluorescent individuals specifically expressed in liver and intestine,and Tg(-1.6nr1h4-EGFP)zebrafish strain was established through genetic screening.Tg(-1.6nr1h4:EGFP)zebrafish larvaes at the fourth day post fertilization were randomly divided into blank control group,solvent control group,glycine-β-muricholic acid group,obeticholic acid group,and low,medium and high concentration(10,20 and 40μg·mL)ursodeoxycholic acid group,danning tablet group,rhein group and aloe-emodin group,and were administered continuously for 4 days.And the fluorescence intensity and development of transgenic zebrafish in each group at different times were observed for 4 days,and the fluorescence intensity was analyzed.Results Tg(-1.6nr1h4:EGFP)fluorescent labeled transgenic zebrafish strain was successfully established.Green fluorescence was expressed in the abdomen of zebrafish from the somite stage and mainly concentrated in the liver and intestine at the fourth day post fertilization.Compared with the blank control group,the fluorescence expression level of transgenic zebrafish in glycine-β-muricholic acid group decreased significantly at each administration time(4dpf:P<0.01;5~7dpf:P<0.0001),the fluorescence in obecholic acid group increased significantly at each administration time(P<0.0001),and there was no significant difference between solvent control group and blank control group,which indicate the effective establishment of the model.Compared with the blank control group,the fluorescence expression level of transgenic zebrafish in low concentration ursodeoxycholic acid group and aloe-emodin group increased gradually from 2 to 4 days after administration(P<0.0001),and in medium concentration ursodeoxycholic acid group and aloe-emodin group inecreased significantly at each administration time(P<0.0001),and the fluorescence in high concentration aloe-emodin group increased gradually from 2 to 4 days after administration(5~6dpf:P<0.001;7dpf:P<0.05),but the fluorescences were almost same between high concentration ursodeoxycholic acid group and blank control group.The fluorescence expression level of transgenic zebrafish in low concentration danning tablet group and rhein group were not different from that of blank control group at each administration time,and the fluorescence in medium concentration danning tablet group increased significantly at each administration time(4 and 7dpf:P<0.0001;5 and 6dpf:P<0.001)and in high danning tablet group still increased significantly at each administration time(P<0.0001),and the fluorescence in medium concentration rhein group increased gradually from 2 to 4 days after administration(5dpf:P<0.0001;6dpf:P<0.001;7dpf:P<0.01)while in high concentration rhein group began to decrease from 2 to 4 days after administration(P<0.0001).Conclusion Tg(-1.6nr1h4-EGFP)zebrafish model provides a new animal model for the study of bile acid drug mechanism,drug screening and safety evaluation.Low and medium concentration ursodeoxycholic acid,medium and high concentrationcan danning tablet,medium concentration rhein and all concentration aloe-emodin can promote bile acid transport by upregulating FXR expression in zebrafish liver and intestine,while high concentration ursodeoxycholic acid have no significant effect on FXR,and high concentration rhein inhibit FXR expression.