汉防己甲素联合化疗药物在斑马鱼体内逆转多药耐药的研究

Tetrandrine combined with chemotherapeutic drugs induces reversal of multidrug resistance in zebrafish

目的探讨汉防己甲素对斑马鱼胚胎早期发育及多药耐药基因abcb4表达的影响及其机制。方法通过药物毒性实验分析汉防己甲素药液处理后斑马鱼胚胎的死亡率、孵化率、心率和畸形率情况。取斑马鱼胚胎设置分组:空白对照组、阿霉素药液处理组、长春碱药液处理组、汉防己甲素药液处理组、汉防己甲素和阿霉素混合药液处理组及汉防己甲素和长春碱混合药液处理组,每组胚胎用药液处理至第5天后取50尾用于实验。通过实时荧光定量PCR检测abcb4基因的表达情况,罗丹明123实验检测胚胎的外排功能,分析耐药程度。结果低剂量汉防己甲素对斑马鱼胚胎毒性较低,高剂量汉防己甲素对胚胎孵化有抑制作用,并有致畸致死作用;实时荧光定量PCR结果显示,与对照组相比abcb4基因的mRNA在阿霉素和长春碱药液单独使用时表达增高,而在汉防己甲素联合阿霉素、长春碱使用时表达降低;罗丹明123实验结果显示,阿霉素和长春碱单独使用时斑马鱼体内产生耐药,而汉防己甲素联合阿霉素、长春碱使用时耐药程度降低。结论汉防己甲素对斑马鱼胚胎毒性较低,且汉防己甲素能逆转物阿霉素和长春碱的耐药作用,对提高治疗效果有重要意义。

Objective To investigate the effects of tetrandrine on early development in zebrafish embryos and the expression of multidrug resistance gene abcb4 and its mechanisms.Methods We used a drug toxicity test to analyze the mortality,hatching rate,heart rate,and deformity rate of zebrafish embryos treated with tetrandrine.Wild-type zebrafish embryos were divided into a blank control group,doxorubicin treatment group,vinblastine treatment group,tetrandrine treatment group,tetrandrine combined with doxorubicin treatment group,and tetrandrine combined with vinblastine treatment group.Drugs were administered to each group of embryos until the fifth day,and the embryos were then used for the experiment.Expression of the abcb4 gene was detected using real-time fluorescent quantitative PCR,and drug resistance was detected using the Rhodamine 123 test.Results Compared with the control group,low-dose tetrandrine had no teratogenic lethal effects on zebrafish embryos.High-dose tetrandrine significantly inhibited embryonic hatching rate and had clear lethal and teratogenic effects.Compared with the control group,the mRNA of the abcb4 gene increased when treated with doxorubicin and vinblastine alone,but decreased when exposed to tetrandrine combined with doxorubicin and vinblastine.The Rhodamine 123 experimental result showed evidence of drug resistance in zebrafish treated with doxorubicin and vinblastine alone,and indicated that tetrandrine combined with doxorubicin and vinblastine could reverse drug resistance.Conclusions Tetrandrine has low toxicity for zebrafish embryos early in development,and can reverse drug resistance to doxorubicin and vinblastine.These data suggest that tetrandrine may improve sensitivity to chemotherapy drugs and thus may enhance the therapeutic effect.