斑马鱼快速骨质疏松模型方法的建立与优化  

Establishment and optimization of rapid model of osteoporosis in zebrafish

作　　者：毛洪运 刘雨彤 赵心月 姜德利 王晓仪 赵珂璇 华永庆 许惠琴[2,3] MAO Hongyun;LIU Yutong;ZHAO Xinyue;JIANG Deli;WANG Xiaoyi;ZHAO Kexuan;HUA Yongqing;XU Huiqin(Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization,Nanjing 210023,China;College of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China;Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica,Nanjing 210023,China)

机构地区：[1]江苏省中药资源产业化过程协同创新中心,南京210023 [2]南京中医药大学药学院,南京210023 [3]江苏省中药药效与安全性评价重点实验室,南京210023

出　　处：《中国实验动物学报》2024年第5期547-556,共10页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金(81473390);江苏省高等学校自然科学研究重大项目(20KJA360001);贵州省科技计划项目([2020]4Y090);贵阳市科技重大项目([2020]-18-4)。

摘　　要：目的本文通过比较常见斑马鱼骨质疏松(osteoporosis,OP)模型不同建立方法,优化建立一种快速、稳定、灵敏的造模方法。方法采用铁过载及泼尼松龙(prednisolone,Pred)诱导OP模型,观察骨形成情况及动物死亡率,分组为:空白对照组(Control组)、模型组(包括FAC组和Pred组)、阳性对照组(AC组)。铁过载诱导法采用枸橼酸铁铵(ammonium ferric citrate,FAC)为造模药物。Pred诱导法有Pred-3受精后天数(day post fertilization,dpf)法,造模时间为3~9 dpf;Pred-5 dpf法,造模时间为5~10 dpf;Pred撤药法,3 dpf开始造模,7~9 dpf每天撤去1/3造模药物。进而比较常用药物阿尔法骨化醇(alfacalcidol,AC)、骨化三醇(calcitriol,CA)、阿仑膦酸钠(alendronate,AL)抗OP效应,选择稳定、灵敏的阳性药,并进一步对不同染色方法及染色条件进行优化。结果FAC在500μg/mL剂量下未能观察到对骨形成的显著影响。骨节数及第一椎骨长度结果显示,与Control组比较,Pred-3 dpf诱导法Pred组均降低,具有显著性差异(P<0.01,P<0.05),但斑马鱼死亡率较高;Pred-5 dpf法无显著性差异;Pred撤药法Pred组均降低,具有显著性差异(P<0.01),且无死亡。给予AC、CA、AL后均具有抗OP作用,CA抗OP作用更为灵敏、稳定。茜素红(alizarin red,ARS)染色参数优化结果显示,染色浓度为0.02%,染色2 h,0.5%KOH和甘油分别以(3∶1,3 h)和(1∶1,14 h)洗涤条件为最佳。染色对比结果显示,钙黄绿素对骨节部位染色较为灵敏,ARS则对第一椎骨处染色较为灵敏。结论采用Pred撤药法可诱导建立快速、稳定、灵敏的斑马鱼OP模型,是研究OP的稳定可靠模型。Objective To establish a fast,stable,and sensitive zebrafish model of osteoporosis(OP)using different method.Methods OP models were induced by iron overload or prednisolone(Pred),and bone formation and mortality were observed.The groups were divided into:Control group,model group(include FAC group and Pred group),and positive control group(AC group).Ammonium ferric citrate was used as the model drug in the iron-overload induction method.For the Pred induction models,the modeling time for the Pred-3 days post-fertilization(dpf)method was 3~9 dpf,the modeling time for the Pred-5 dpf method was 5~10 dpf,and Pred was administered from 3 dpf and removed from 7~9 dpf for the Pred withdrawal method.To compare the anti-osteoporosis(OP)effects of commonly used drugs such as Alfacalcidol(AC),Calcitriol(CA),and Alendronate(AL),it’s important to select a stable and sensitive positive control drug and to further optimize different staining Methods and conditions.Results There was no significant effect of ammonium ferric citrate 500μg/mL on bone formation.Bone formation and the length of the first vertebra were significantly decreased in the Pred group induced by Pred-3 dpf compared with those in the control group(P<0.01,P<0.05),but zebrafish mortality was higher.There was no significant difference between the Pred-5 dpf method,but bone formation was significantly reduced in the Pred withdrawal group(P<0.01),with no mortality.Alfacalcidol,calcitriol,and alendronate all had anti-OP effects,with CA having the most sensitive and stable anti-OP effect.Alizarin red staining showed that the optimal dye parameters were 0.02%concentration for dyeing 2 h,with washing in 0.5%KOH and glycerol under the conditions of a 3∶1 ratio for 3 h followed by a 1∶1 ratio for 14 h.The result of staining showed that calcein was more sensitive for staining bone nodes and ARS staining was more sensitive for staining the first vertebra.Conclusions The Pred withdrawal method can be used to establish a rapid,stable,and sensitive OP model in zebrafis

关 键 词：斑马鱼 骨质疏松 泼尼松龙 茜素红 骨化三醇 

分 类 号：Q95-33[生物学—动物学]