斑马鱼皮肤损伤模型的建立及分子机制

Model establishment of skin damage and molecular mechanism on transgenic zebrafish embryos

目的以皮肤荧光转基因斑马鱼(ktr4:NTR-h Kik GR)为实验动物,研究长春新碱对皮肤细胞的损伤作用及其分子机制,建立皮肤损伤模型。方法取受精后24 h皮肤荧光斑马鱼胚胎,将脱膜后的胚胎分别置于0.01~0.04 mmol·L^(-1)不同浓度的长春新碱中,24 h后采用荧光显像的方法对斑马鱼皮肤荧光斑点进行计数和统计处理;同时对鱼体细胞死亡情况进行TUNEL试剂盒检测,确定其皮肤细胞死亡的水平;利用蛋白免疫印迹的方法对各组斑马鱼胚胎的caspase-3、KRT4及p53表达水平进行检测。结果在长春新碱与斑马鱼胚胎共同孵育24 h后,0.02、0.04 mmol·L^(-1)长春新碱明显引起斑马鱼荧光皮肤细胞减少(P<0.01);TUNEL检测表明,长春新碱与对照组比较能明显引起斑马鱼皮肤细胞凋亡;Western bolt分析显示,0.02、0.04 mmol·L^(-1)长春新碱能诱导斑马鱼胚胎caspase-3和p53表达增加(P<0.01),同时高浓度的长春新碱可使皮肤角蛋白KRT4表达明显减少(P<0.01)。结论利用长春新碱引起斑马鱼皮肤损伤,该局部毒性作用可作为皮肤损伤的造模新方法。

Aim To establish a new model of skin damage by using vincristine to transgenic zebrafish（ krt4： NTR-h Kik GR）.Methods Skin fluorescent transgenic zebrafish embryos after24 h fertilization were treated with the 0. 01 ~ 0. 04 mmol·L^（-1）vincristine,and zebrafish skin cell ablation was investigated under fluorescence microscope after 24 h,at same time skin death cells were detected with TUNEL assay and image processed,then the protein expressions of KRT4,caspase-3 and p53 were assessed with Western blot. Results 0. 02 mmol · L^（-1） and0. 04 mmol · L^（-1） vincristine could obviously induce zebrafish skin cell apoptosis（ P〈 0. 01） with statistically significant difference compared with the control,and the same result could beaccomplished in TUNEL assay. Results of Western bolt showed that vincristine could increase the embryos caspase-3 and p53 expression（ P 〈0. 01）,while vincristine in high concentration might decrease KRT4 markedly（ P 〈0. 01）. Conclusion Vincristine can induce damage on zebrafish skin with suppression KTR4,which can be used as a new skin damage model.