斑马鱼眼部细胞凋亡模型的建立

Establishment of ocular apoptosis model in zebrafish

目的建立邻苯二甲酸二丁酯(DBP)诱导的斑马鱼眼部细胞凋亡模型,并用于抗眼部细胞凋亡药物的活性评价。方法斑马鱼受精后30 h(hpf),加入DBP(0.1、10、50、100μmol·L^-1)18 h和42 h,观察斑马鱼的存活率;将30或54 hpf的斑马鱼在直射光或非直射光条件下,加入DBP(0.1、1、5、10、50μmol·L^-1)造模18 h,吖啶橙染色(AO),采用体视荧光显微镜观察斑马鱼眼部细胞凋亡情况;造模同时,给予不同浓度N-乙酰-L-半胱氨酸(NAC)或人参皂苷Rg1,观察药物对斑马鱼眼部细胞凋亡的影响。结果DBP造模18、42 h,10μmol·L^-1及以下浓度对鱼卵存活率无明显影响,50μmol·L^-1及以上浓度会致使斑马鱼存活率明显降低(P<0.01);30 hpf的斑马鱼在10μmol·L^-1 DBP造模18 h后显示明显的眼部细胞凋亡(P<0.01)。斑马鱼在72 h可见眼部细胞自发性凋亡,且与光照条件无关。在该造模条件下,NAC或人参皂苷Rg1均能抑制斑马鱼眼部细胞凋亡(P<0.05)。结论利用DBP可快速高效地建立斑马鱼眼部细胞凋亡模型,该模型可用于药物活性的快速评价。

Aim To establish a model of ocular apoptosis in zebrafish by using dibutyl phthalate(DBP), and accordingly to evaluate the activity of anti-ocular apoptosis drugs. Methods Zebrafish embryos(30 hpf) were treated with DBP(0.1, 10, 50, 100 μmol·L^-1 ) for 18 h and 42 h, and the survival rate was calculated. Then zebrafish embryos(30 or 54 hpf) were incubated with DBP(0.1, 1, 5, 10, 50 μmol·L ^-1 ) for 18 h under direct or indirect light conditions, and visualized under microscope, then the ocular apoptosis was assessed by AO staining. Furthermore, modeled zebrafish was treated with N-acetyl-L-cysteine(NAC) or ginsenoside Rg1, and the effects on ocular apoptosis were observed. Results There was no mortality in zebrafish at concentrations below 10 μmol·L ^-1 after modeling with DBP for 18 h and 42 h. The survival rate of zebrafish was significantly reduced with a concentration above 50 μmol·L^1 ( P <0.01). Furthermore, obvious ocular apoptosis was found at 30 hpf after 18 h with DBP(10 μmol·L -1 )( P <0.01). The zebrafish showed spontaneous apoptosis of the ocular cells at 72 h, which was not associated with light conditions. Effect of NAC or ginsenoside Rg1 was observed on decreasing ocular apoptosis in zebrafish( P <0.05). Conclusions DBP can be used to rapidly and efficiently establish a model of ocular apoptosis in zebrafish, which can be used for rapid evaluation of drug activity.