Wnt/β-catenin信号通路参与化疗药物诱导的转基因斑马鱼abcb4基因的表达和耐药

Wnt/β-catenin signaling pathway involved in the expression and drug resistance of abcb4 gene in transgenic zebrafish induced by chemotherapeutic drugs

目的探讨斑马鱼耐药基因ATP结合盒亚家族B成员4(ABCB4)与Wnt/β联蛋白(β-catenin)信号通路的关系。方法取野生型斑马鱼和转基因斑马鱼均设置空白对照组、阿霉素处理组、长春碱处理组、吉非替尼处理组、阿霉素联合吉非替尼处理组、长春碱联合吉非替尼处理组。每组100枚胚胎分别进行药物处理至第5天,每组取第5天斑马鱼幼鱼50枚进行实验。采用罗丹明123实验检测野生型斑马鱼耐药程度,酶标仪检测转基因的斑马鱼荧光强度,活细胞工作站检测荧光分布位置。Western blot法检测转基因的斑马鱼β-catenin、糖原合成酶激酶-3β(GSK-3β)、 ATP结合盒亚家族B成员4(ABCB4)、增强型绿色荧光蛋白(EGFP)的蛋白水平。结果罗丹明123实验证实经阿霉素和长春碱药物暴露后的斑马鱼体内产生耐药, EGFP荧光强度检测结果表明经阿霉素和长春碱药物暴露后的转基因斑马鱼荧光强度高,经阿霉素和长春碱药物暴露后的转基因斑马鱼体内β-catenin的累积升高伴随着EGFP和ABCB4蛋白的增加。结论斑马鱼体内Wnt/β-catenin信号通路参与斑马鱼abcb4基因的激活与耐药。

Objective To explore the relationship between the drug resistance gene ATP binding cassette sub-family B member 4(abcb4) in zebrafish and Wnt/β-catenin signaling pathway. Methods Wild-type zebrafish and transgenic zebrafish were set in the blank control group, doxorubicin treatment group, vinblastine treatment group, gefitinib treatment group, doxorubicin combined with gefitinib treatment group, and vinblastine combined with gefitinib treatment group. The 100 embryos of each group were treated with drugs until the 5th day, and 50 zebrafish juveniles were tested on the 5th day in each group. The drug resistance of wild-type zebrafish was tested by rhodamine 123 experiment. The fluorescence intensity of transgenic zebrafish was tested by microplate reader, and the fluorescence distribution was tested by living cell workstation. The protein levels of transgenic zebrafish β-catenin, GSK-3β, ABCB4 and enhanced green fluorescent protein(EGFP) were tested by Western blot analysis. Results Rhodamine 123 experiments proved that there was drug resistance in zebrafish when treated with doxorubicin and vinblastine. Compared with the blank group, the EGFP fluorescence intensity increased in the transgenic zebrafish when treated with doxorubicin and vinblastine. Western blot assay showed the accumulation of β-catenin accompanied by the increase of EGFP and ABCB4 proteins in the transgenic zebrafish exposed to adriamycin and vincristine. Conclusion The Wnt/β-catenin signaling pathway in zebrafish is involved in the activation and drug resistance of zebrafish abcb4 gene.