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本研究目的是在斑马鱼体内建立符合高通量药物筛选条件的胰岛β细胞破坏模型,用于筛选出针对糖尿病胰岛β细胞再生的药物。利用胰岛素-硝基还原酶-绿色荧光蛋白(insulin-nfs B-green fluorescent protein;INS-nfs B-GFP)转基因系F1/F2代胚胎分为A组(正常对照组),根据胚胎时相大小分为3个大组,B组受精后24 h(24 hours postfertilization,24 hpf)、C组受精后36 h(36 hours postfertilization,36 hpf)、D组受精后48 h(48 hours postfertilization,48 hpf),分别在不同毫摩尔(mmol/L)浓度(5 mmol/L,7.5 mmol/L,10 mmol/L,15 mmol/L)的甲硝唑中孵育24~48 h,通过体式倒置荧光显微镜和共焦显微镜观察斑马鱼胰岛β细胞破坏及荧光表达的情况,同时用原位杂交方法进一步在分子水平证实。结果显示,斑马鱼胚胎36 hpf加10 mmol/L浓度的甲硝唑并孵育36 h,荧光明显消失,并且畸形率和死亡率较低,是创造胰岛条件β细胞破坏的转基因斑马鱼模型的理想条件,在此基础上去甲硝唑10~86 h后继续观察胰岛β细胞再生,结果发现在去除甲硝唑24 h(即斑马鱼胚胎96 hpf)时胰岛β细胞开始再生。本模型的建立对于糖尿病的临床价值和应用前景有着非常重要的意义,利用这一条件使高通量筛选具有胰岛功能恢复作用药物成为可能,将为糖尿病的治疗开辟新的道路。
The purpose of this study was to establish a model of islet β-cell destruction in high-throughput drug screening in zebrafish, which was used to screen the drug for the islet β-cell regeneration of diabetes mellitus. The F1 / F2 generation of INS-nfs B-green fluorescent protein (INS-nfs B-GFP) transgenic embryos were divided into A group (normal control group), embryo size The rats were divided into three groups: group B, 24 hours postfertilization, 24 hours post-fertilization, 36 hours postfertilization, 36 hours post-fertilization, 48 hours post-fertilization, 48 h post- ) Were incubated in metronidazole with different concentrations (5 mmol / L, 7.5 mmol / L, 10 mmol / L, 15 mmol / L) for 24-48 h. And confocal microscopy observation of zebrafish pancreatic β-cell destruction and fluorescence expression, while using in situ hybridization method further confirmed at the molecular level. The results showed that 36 hpf plus 10 mmol / L metronidazole in 36-h zebrafish embryos incubated for 36 h, the fluorescence disappeared obviously and the rate of deformity and mortality was low, which was ideal for creating a transgenic zebrafish model of pancreatic β-cell disruption On the basis of which metronidazole was continuously observed after 10-86 hrs. It was found that pancreatic beta cells began to regenerate 24 h after metronidazole removal (ie, 96 hpf in zebrafish embryos). The establishment of this model is of great significance for the clinical value and application prospect of diabetes. Using this condition makes it possible to screen high-throughput drugs with pancreatic islet function recovery, which will open a new path for the treatment of diabetes.