甲硝唑诱导斑马鱼骨丢失模型的建立

目的通过构建甲硝唑诱导胰岛β细胞破坏的转基因斑马鱼骨丢失的动物模型,为寻找可保护胰岛β细胞的促骨形成药物提供经济便捷的平台。方法用酶切鉴定正确的重组质粒p-IsceI-INS:nfsB进行显微注射,建立胰岛β细胞转基因斑马鱼系。选用受精后30 h的转基因斑马鱼胚胎分别暴露于5,10,15μmol·L^-1甲硝唑溶液中,作为甲硝唑组;同时设1%二甲亚砜溶媒作为阴性对照组,nfsB蛋白将无毒性的前体药物转变为细胞毒素,在活体直接破坏胰岛β细胞。48 h后及时更换胚胎养殖水终止甲硝唑的破坏作用,常规28.5℃下在24孔板中培养至斑马鱼颅骨形成。测定斑马鱼血糖水平,采用酶联免疫吸附测定(ELISA)试剂盒定量测定糖基化终产物;采用茜素红对斑马鱼骨骼进行染色,以显微检测、数码成像方法定量分析骨骼染色区域。结果与阴性对照组比较,5,10,15μmol·L^-1甲硝唑组斑马鱼骨骼染色面积和累计吸光度显著减小,提示利用甲硝唑破坏胰岛β细胞构建转基因斑马鱼的骨骼矿化量和骨密度都显著降低,成功诱导斑马鱼骨丢失模型。并用该模型成功验证依替膦酸二钠防治骨质疏松的作用。结论成功构建破坏胰岛β细胞诱导转基因斑马鱼骨丢失模型,为在整体动物水平筛选可保护胰岛β细胞的促骨形成的小分子化合物和中草药提供一个新型的动物模型。

Establishment of A Zebrafish Model of Bone Loss Induced by Metronidazole

Objective To construct a transgenic zebrafish model of bone loss induced by metronidazole,and to provide a convenient in vivo model for screening drugs protected the islet of pancreas and promoted bone formation.Methods Enzyme digestion was used to identify the correct recombinant plasmid(p-Iscei-INS:nfsB)for micro-injection to establish the islet mesenchymal transgenic zebrafish line.Thirty hours after fertilization,transgenic zebrafish embryos were exposed to metronidazole solution of different concentrations,and 1%DMSO was set as the negative control group.NfsB protein transformed the non-toxic precursor drug into a cytotoxin and directly destroyed the pancreatic islet cells in vivo.After 48 hours,the destruction of metronidazole was stopped by replacing the embryo culture water.The zebrafish was cultured in 24-well plate at 28.5℃till the skull formation.After the experiment,blood glucose level of zebrafish was measured.Alizarin red was used to stain zebrafish bones,and the bone staining areas were quantitatively analyzed by means of microscopic examination and digital imaging.Results Compared with the negative control group,the staining area and optical density of zebrafish bones in the 5,10 and 15μmol·L^-1 metronidazole groups were significantly reduced,suggesting that the bone mineralization and bone mineral density of transgenic zebrafish were significantly reduced when metronidazole was used,and the zebrafish bone loss model was successfully established.In this model,the effect of disodium etiphosphonate on osteoporosis was verified.Conclusion The transgenic zebrafish model of bone loss induced by the destruction of islet cells was successfully established and it is a new animal model for screening small molecular compounds and the Chinese herbal medicines that can protect the islet formation at the whole animal level.