转基因斑马鱼模型吉非替尼抗血管生成作用研究

Effect of gefitinib on anti-angiogenesis activity of Tg(Flk1:EGFP)transgenic zebrafish

目的吉非替尼是第一个选择性作用于表皮生长因子受体的酪氨酸激酶,主要用于非小细胞肺癌。本研究主要探讨吉非替尼对Tg(Flk1:EGFP)转基因斑马鱼模型血管生成的影响。方法选择Tg(Flk1:EGFP)转基因斑马鱼为动物模型,分别给予10、50和100μmol/L浓度的吉非替尼干预处理。以0.025μmol/L的1-苯基-2-硫脲(1-phenyl-2-thiourea,PTU)处理组为空白对照组,以10μmol/L的SU5416处理组为阳性对照组,在受精后12h(12hours post-fertilization,12hpf)开始加药干预,用荧光显微镜观察并统计受精后30h(30hpf)的斑马鱼胚胎体节间血管的数量并计算血管抑制率。用多功能酶标仪检测受精后4d(4days post-fertilization,4dpf)的斑马鱼体内绿色荧光蛋白的表达量,并进行统计学分析。结果空白对照组斑马鱼体节间血管正常形成;10μmol/L的SU5416完全抑制了斑马鱼体节间血管的生成,血管生成抑制率为100%,10、50和100μmol/L浓度吉非替尼血管生成抑制率分别为10.4%、19.6%和26.4%,随着吉非替尼浓度的增加,血管抑制率增加(P<0.05),斑马鱼体内绿色荧光蛋白的表达量减少(P<0.05),并呈剂量依赖性。结论吉非替尼具有抑制斑马鱼血管生成的作用。吉非替尼抑制血管生成可能对其抗肿瘤治疗起一定的辅助作用。

OBJECTIVE Gefitinib is the first tyrosine kinase that selectively acts on epidermal growth factor receptor and is mainly used in non-small cell lung cancer.This study was to study the effects of gefitinib on angiogenesis in Tg(Flk1:EGFP)transgenic zebrafish model.METHODS Tg(Flk1:EGFP)transgenic zebrafish was selected as the animal model and treated with gefitinib at 10,50 and 100 μmol/L,respectively.Treated with 0.025 μmol/L of 1-phenyl-2-thiourea(PTU)was served as negative control group.Treated with 10 μmol/L of SU5416 was served as a positive control group.Dosing intervention began 12 hours after fertilization(12 hpf),the number of internode blood vessels of zebrafish embryos at 30 hpf was observed by fluorescence microscopy,and then the vascular inhibition rate was calculated.Four days after fertilization(4 dpf),the multi-functional microplate reader was used to detect the expression of green fluorescent protein in zebrafish and the statistical analysis was carried out.RESULTS Blank control group had completely formed normal angiogenesis.SU5416 at 10 μmol/L completely inhibited the angiogenesis of internode blood vessels in zebrafish.The inhibition rate of angiogenesis was 100%.The inhibition rate of angiogenesis of gefitinib at 10,50 and 100 μmol/L were 10.4%,19.6% and 26.4% respectively,the inhibition rate of blood vessel increased with the increase of gefitinib concentration(P<0.05),the expression of green fluorescent protein in zebrafish decreased(P<0.05),showing a dose dependency.CONCLUSION Gefitinib inhibited the angiogenesis of zebrafish,which may play a supporting role in antitumor therapy.