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目的选择斑马鱼作为实验对象,研究阿霉素(doxorubicin,DOX)对其abcb4基因表达的影响,进一步了解abcb4基因在斑马鱼多药耐药机制中可能的作用。方法分别以2mL/L二甲基亚砜(DMSO),10μmol/L DOX及含2mL/L DMSO的10μmol/L DOX对斑马鱼胚胎进行药物处理,另以Eggwater处理的胚胎作为对照组。将正常发育的4~16个细胞期斑马鱼胚胎,随机分入以上各组中,药物处理至120hpf。收集药物处理下的斑马鱼不同时期胚胎运用实时荧光定量PCR和胚胎原位杂交技术,检测abcb4基因在斑马鱼中的表达变化情况。结果相较于对照组,药物处理的斑马鱼胚胎abcb4基因mRNA表达水平升高(P<0.05),而abcb5基因mRNA表达情况则无明显变化。通过斑马鱼胚胎原位杂交,均在斑马鱼120hpf胚胎小肠部位发现有abcb4基因阳性杂交信号,且药物处理的斑马鱼胚胎在脑及心脏部位发现abcb4基因阳性杂交信号。结论 DOX能诱导斑马鱼胚胎abcb4基因表达水平增高,对阐明abcb4基因在斑马鱼多药耐药产生机制中的作用具有重要意义。
Objective To study the effect of doxorubicin (DOX) on the gene expression of abcb4 in zebrafish as an experimental object to further understand the possible role of abcb4 in multidrug resistance of zebrafish. Methods The zebrafish embryos were treated with 2mL / L dimethylsulfoxide (DMSO), 10μmol / L DOX and 10μmol / L DOX containing 2mL / L DMSO respectively. The other Eggwater-treated embryos were used as control. The normal 4 to 16 cell-stage zebrafish embryos were randomly assigned to each of the above groups and the drug was treated to 120 hpf. Collect zebrafish embryos under drug treatment using real-time fluorescence quantitative PCR and in situ hybridization to detect the expression of abcb4 in zebrafish. Results Compared with the control group, the mRNA expression of abcb4 mRNA in zebrafish embryos treated with drug treatment increased (P <0.05), while the expression of abcb5 mRNA had no significant change. The zebrafish embryo in situ hybridization, were found in the zebrafish 120hpf embryonic small intestine site abcb4 gene positive hybridization signal, and drug-treated zebrafish embryos in the brain and heart found abcb4 gene positive hybridization signal. Conclusions DOX can induce the increase of abcb4 gene expression in zebrafish embryos, which is of great significance for clarifying the role of abcb4 in the mechanism of multidrug resistance in zebrafish.