WO2022193397A1 - 一种斑马鱼骨质疏松模型的构建方法 - Google Patents

一种斑马鱼骨质疏松模型的构建方法 Download PDF

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WO2022193397A1
WO2022193397A1 PCT/CN2021/088295 CN2021088295W WO2022193397A1 WO 2022193397 A1 WO2022193397 A1 WO 2022193397A1 CN 2021088295 W CN2021088295 W CN 2021088295W WO 2022193397 A1 WO2022193397 A1 WO 2022193397A1
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zebrafish
lead acetate
constant temperature
construction method
osteoporosis
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安艳
丁洁
严锐
杨乾磊
魏园杰
张晓云
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Suzhou University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/02—Breeding vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K61/00—Culture of aquatic animals
    • A01K61/10—Culture of aquatic animals of fish
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/40—Fish
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

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  • the invention belongs to the technical field of medicine, and in particular relates to a method for constructing a zebrafish osteoporosis model.
  • Osteoporosis is a metabolic bone disease characterized by decreased bone mass, abnormal microstructure of bone tissue, and increased risk of fracture. With the increasing aging of the population in my country, the incidence of osteoporosis and the fragility fractures caused by it is also increasing. At present, animal models of osteoporosis have been widely used in the study of the etiology, mechanism and prevention of osteoporosis.
  • An ideal experimental animal model for osteoporosis should have the following characteristics: (1) Convenience: it means that the modeling method is simple and the model is easy to use; (2) Relevance: it means that the model should be as close as possible to human conditions, and the pathological process, Pathological changes and outcomes should be as similar as possible to human osteoporosis. Therefore, the construction of an effective osteoporosis screening drug model is of great significance for the drug treatment of osteoporosis and the discovery of safe and effective new drugs.
  • the animals used to prepare osteoporosis models mainly include rats, mice, rabbits and sheep.
  • the existing animal osteoporosis models take a long time, use a large amount of samples, and have low activity screening efficiency; the existing cell models have a single function link, high cost, and difficulty in primary culture, which cannot be carried out by ordinary laboratories.
  • Zebrafish is an important model organism that has been widely used in the establishment of disease models and drug discovery in recent years. Its bone formation mechanism includes endochondral ossification and intramembranous ossification, and its process is affected by a series of complex mechanisms including transcription factors and hormones.
  • the strict regulation of zebrafish, the key genes involved in regulation, such as osteonectin, osteoprogenin, runx2, etc., are highly homologous to related mammalian genes, zebrafish are small, easy to raise, develop rapidly, have strong fertility, and are easy to observe and operate. The dose of zebrafish is small, the experiment time is short, and high-throughput screening of microplate formation can be carried out.
  • the main drugs used in the construction of zebrafish osteoporosis models are prednisolone or dexamethasone, but the above two drugs belong to glucocorticoid drugs, and it is very easy to use them due to inaccurate dose control during modeling. Excessive immunosuppression leads to teratogenicity and high lethality in zebrafish embryos.
  • some studies have found that high iron can also cause bone development defects in zebrafish, but whether the high iron-induced juvenile osteoporosis model can be used to screen other drugs for the treatment of osteoporosis other than iron chelators is unknown.
  • glucocorticoids and iron are essential substances for regulating physiology in the human body, and high concentrations of these two compounds have a greater impact on the physiology of organisms other than bone.
  • the present invention uses a zebrafish model that is easy to operate, and can quickly establish a zebrafish osteoporosis model in a short period of time after being treated with a lead acetate solution. It provides a simple, efficient and high-throughput biological model for high-speed screening of drugs for prevention and treatment of osteoporosis and drug mechanism research.
  • the first object of the present invention is to provide a method for constructing a zebrafish osteoporosis model, comprising the following steps:
  • the fish eggs are randomly divided into blank control group and lead acetate group, the blank control group is added with a culture solution containing bacteriocin, and the lead acetate group is added with a bacteriostat-containing culture solution and lead acetate solution to carry out constant temperature cultivation;
  • the final concentration of lead acetate in the lead acetate group was 5-20 mg/L;
  • step 4) Take the zebrafish cultured in step 3) for cartilage staining. Compared with the blank control group, if the staining area of the lead acetate group is reduced, a zebrafish osteoporosis model is obtained.
  • step 3 the temperature of constant temperature cultivation is 28-29°C.
  • step 3 the time of constant temperature cultivation is 8-10 days, and the culture medium is replaced every day.
  • step 3 during the culturing process, 13 to 15 hours of light/9 to 11 hours of darkness is used every day.
  • step 2) the temperature of constant temperature incubation is 28-29°C.
  • step 2) the time of constant temperature incubation is 1-3 hours.
  • the culture medium is fish culture water.
  • the culture medium in the present invention is a culture medium capable of normal growth and development of fertilized eggs.
  • the parameter conditions of the fish culture water are: pH: 7.0-7.3; temperature: 27-29°C; conductivity: 450-550 ⁇ s; salinity: 0.25-0.75 ⁇ ; dissolved oxygen: ⁇ 6mg/L ; Photoperiod: 14/10 hours; Hardness: 100 ⁇ 200mg/L; Chlorine: 0mg/L; Ammonia nitrogen concentration: ⁇ 0.02mg/L; Nitrite: ⁇ 1mg/L; Nitrate: ⁇ 50mg/L; Carbon dioxide : ⁇ 50mg/L.
  • the concentration of bacteriocin is 0.5 ⁇ 10-5 ⁇ g/L.
  • the second object of the present invention is to provide a zebrafish osteoporosis model constructed by the method.
  • the present invention has at least the following advantages:
  • the invention uses a zebrafish model that is easy to operate, and can quickly establish a zebrafish osteoporosis model in a short period of time after being treated with a lead acetate solution. It provides a simple, efficient and high-throughput biological model for high-speed screening of drugs for prevention and treatment of osteoporosis and drug mechanism research.
  • Figure 1 is a comparison of pictures of alizarin red staining of juveniles in each concentration group 9 days after fertilization;
  • the construction method of lead-induced zebrafish osteoporosis model including the following steps:
  • the fish eggs were randomly inhaled into a 6-well plate, with 30 embryos per well, 3 replicate groups; 3 ml of fish culture water, 3 ⁇ l of bacteriostatin and 3 ⁇ l of lead acetate solutions of different concentrations were added to each well to make the final concentrations 0 mg respectively.
  • /L, 5mg/L, 10mg/L, 20mg/L, namely a blank control group and three lead acetate concentration groups and then placed in a 28.5°C constant temperature incubator, under the conditions of 14 hours of light/10 hours of darkness every day. Incubate at constant temperature, and change the medium every 24h until 9 days after fertilization;
  • step (3) Take the cultured samples in step (3) for cartilage staining. Compared with the blank control group, if the staining area of the PbAc solution group is reduced, a zebrafish osteoporosis model is obtained.
  • Alizarin red staining to quantify the bone mineralization area including the following steps:
  • 50% absolute ethanol solution (50 ml): Take 26.3 ml of 95% absolute ethanol, 5 ml of 1M Tris (pH 7.5), add ddH2O to 50 ml.
  • Alizarin Red 50 ml: Weigh 0.25 g of Alizarin red (Alizarin) powder and dissolve it in 50 ml of ddH 2 O.
  • Alizarin Red 50ml, pH7.5: Take 1ml 0.5% Alizarin Red, 12.5ml 100% glycerol, 5ml 1M Tris (pH7.5), add ddH2O to 50ml.
  • Tris pH7.5/10mM MgCl 2 Take 9-day-old zebrafish larvae, soak them in 1ml 2% PFA/1 X PBS and fix them overnight, rinse with 100mM Tris pH7.5/10mM MgCl 2 for 10mins, add 1ml of 80%ETOH/100mM 80% ETOH/100mM respectively after aspirating the solution Tris pH7.5/10mM MgCl2 , 50% ETOH/100 mM Tris Ph7.5, 25% ETOH/100 mM Tris Ph7.5 were rinsed for 5 mins for decolorization.

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  • Life Sciences & Earth Sciences (AREA)
  • Environmental Sciences (AREA)
  • Zoology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

一种斑马鱼骨质疏松模型的构建方法，将性成熟的斑马鱼按雌雄比1:1.5～2.5的比例放入交配鱼缸内，插上隔板，雌雄斑马鱼分开；隔夜取出隔板，待90～150分钟后收集鱼卵，置于含有抑菌素的培养液中，恒温孵育；孵育后将鱼卵随机分为空白对照组和醋酸铅组，空白对照组中加入含有抑菌素的培养液，醋酸铅组中还加入醋酸铅溶液，进行恒温培养；其中，醋酸铅的终浓度为5～20mg/L；培养后的斑马鱼进行软骨染色，与空白对照组相比，若醋酸铅组的染色区域减小，则得到斑马鱼骨质疏松模型。本构建方法使用操作简便的斑马鱼模型，在醋酸铅溶液处理后可短期快速建立斑马鱼骨质疏松模型，为高速筛选防治骨质疏松的药物及药物机制研究提供简单、高效、高通量的生物模型。

Description

一种斑马鱼骨质疏松模型的构建方法 技术领域
本发明属于医药技术领域，尤其涉及一种斑马鱼骨质疏松模型的构建方法。
背景技术
骨质疏松是一种以骨量减少、骨组织显微结构异常和骨折危险性增加为特征的一种代谢性骨病。随着我国人口老龄化现象的不断增加，骨质疏松症及其所导致的脆性骨折的发生率也越来越高。目前，骨质疏松动物模型在骨质疏松病因、机制及防治等方面的研究中已经得到广泛的应用。理想的骨质疏松试验动物模型应具有以下特点：(1)方便性：指造模方法简单、模型使用方便；(2)关联性：指模型应最大限度的与人体条件相接近，病变过程、病理改变及转归都应与人类骨质疏松尽可能相似。因此，构建有效的骨质疏松筛药模型，对骨质疏松症的药物治疗和发现安全有效的新药具有重要意义。
目前，用于制备骨质疏松模型的动物主要有大鼠、小鼠、兔和羊等。现有动物骨质疏松模型耗时长，用样量大，活性筛选效率低；现有的细胞模型作用环节单一，成本高，原代培养难度大，一般的实验室不能进行。
斑马鱼是近年来被广泛应用于建立疾病模型和药物发现的重要模式生物，其骨骼形成机制包括软骨内骨化和膜内骨化，其过程受到一系列包括转录因子和激素在内的复杂机制的严格调控，参与调控的关键基因，如osteonectin,osteoprogenin,runx2等与哺乳动物的相关基因具有高度同源性，斑马鱼个体小、易饲养、发育快速、繁殖力强、易于观察和操作，且斑马鱼给药量少，实验耗时短，可进行微孔板形成的高通量筛选。
现阶段，构建斑马鱼骨质疏松模型时主要使用的药物是泼尼松龙或地塞米松，但上述两种药物属于糖皮质激素类药物，在造模时极易因为剂量把握不准而使免疫抑制过强导致斑马鱼胚胎致畸、致死率高。目前还有研究发现，高铁也能够造成斑马鱼骨骼发育缺陷，但高铁诱导的幼鱼骨质疏松模型是否能够用于筛选铁螯合剂以外的其他治疗骨质疏松的药物尚未可知。另外，糖皮质激素和铁是人体内调节生理的必需物质，高浓度的这两种化合物对生物体骨之外的生理影响较大。
发明内容
为解决上述技术问题，本发明使用操作简便的斑马鱼模型，在醋酸铅溶液处理后可短期 快速建立斑马鱼骨质疏松模型。为高速筛选防治骨质疏松的药物及药物机制研究提供简单、高效、高通量的生物模型。
本发明的第一个目的是提供一种斑马鱼骨质疏松模型的构建方法，包括如下步骤：
1)将性成熟的斑马鱼按雌雄比＝1:1.5～2.5的比例放入交配鱼缸内，插上隔板，雌雄斑马鱼分开；培养10～20小时后，取出隔板，待90～150分钟后收集鱼卵；
2)收集到的鱼卵置于含有抑菌素的培养液中，恒温孵育；
3)恒温孵育后将鱼卵随机分为空白对照组和醋酸铅组，空白对照组中加入含有抑菌素的培养液，醋酸铅组中加入含有抑菌素的培养液和醋酸铅溶液，进行恒温培养；其中，醋酸铅组中醋酸铅的终浓度为5～20mg/L；
4)取步骤3)培养后的斑马鱼进行软骨染色，与空白对照组相比，若醋酸铅组的染色区域减小，则得到斑马鱼骨质疏松模型。
进一步地，在步骤3)中，恒温培养的温度为28～29℃。
进一步地，在步骤3)中，恒温培养的时间为8～10天，每天更换培养液。
进一步地，在步骤3)中，在培养过程中，每天以13～15小时光照/9～11小时黑暗处理。
进一步地，在步骤2)中，恒温孵育的温度为28～29℃。
进一步地，在步骤2)中，恒温孵育的时间为1～3小时。
进一步地，所述的培养液为养鱼水。本发明中的培养液为能够使受精卵正常生长发育的培养液。
进一步地，所述的养鱼水的参数条件为：酸碱度：7.0～7.3；温度：27～29℃；电导率：450～550μs；盐度：0.25～0.75‰；溶氧度：≧6mg/L；光周期：14/10小时；硬度：100～200mg/L；氯：0mg/L；氨氮浓度：<0.02mg/L；亚硝酸盐：<1mg/L；硝酸盐：<50mg/L；二氧化碳：<50mg/L。
进一步地，在含有抑菌素的培养液中，抑菌素的浓度为0.5×10-5μg/L。
本发明的第二个目的是提供一种所述方法构建得到的斑马鱼骨质疏松模型。
借由上述方案，本发明至少具有以下优点：
本发明使用操作简便的斑马鱼模型，在醋酸铅溶液处理后可短期快速建立斑马鱼骨质疏松模型。为高速筛选防治骨质疏松的药物及药物机制研究提供简单、高效、高通量的生物模型。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细说明如后。
附图说明
图1为各浓度组幼鱼在受精后9天的茜素红染色图片对比；
图2为染色区域的定量分析。
具体实施方式
实施例1：
1、铅致斑马鱼骨质疏松模型的构建方法，包括以下步骤：
(1)配置母液：称取5g醋酸铅(Lead Acetate,PbAc)粉末溶于50ml双蒸水，即得到100mg/ml的醋酸铅溶液；
(2)配置工作液：取所述母液200μl于1.5ml离心管中加入800μl双蒸水，得20mg/ml PbAc溶液；取所述母液100μl于1.5ml离心管中加入900μl双蒸水，得10mg/ml PbAc溶液；取所述母液50μl于1.5ml离心管中加入950μl双蒸水，得5mg/ml PbAc溶液；
(3)将性成熟的斑马鱼按雌雄比＝1:2的比例放入交配鱼缸内，插上隔板，雌雄斑马鱼分开；隔夜，取出隔板，待2小时后收集鱼卵；
(4)将收集到的鱼卵放入加有抑菌素的养鱼水中，恒温孵育2小时；
(5)将鱼卵随机吸入6孔板中，每孔30个胚胎，3个重复组；每孔加3ml养鱼水，3μl抑菌素和3μl不同浓度的醋酸铅溶液使之终浓度分别为0mg/L、5mg/L、10mg/L、20mg/L，即为一个空白对照组和三个醋酸铅浓度组，随后放入28.5℃恒温培养箱中，每天以14小时光照/10小时黑暗的条件进行恒温孵育，每隔24h换液一次至受精后9天；
(6)取步骤(3)培养后的样品进行软骨染色，与空白对照组相比，若PbAc溶液组的染色区域减小，即得到斑马鱼骨质疏松模型。
2、茜素红染色量化骨矿化区，包括以下步骤：
(1)溶液配制：
2％多聚甲醛(PFA)与1×PBS(pH7.5)混合液(50ml)：取2.5ml 4％PFA，5ml 10×PBS,调节pH至7.5，加双蒸水补齐至50ml。
100mM Tris(pH7.5)与10mM MgCl 2混合液(50ml)：取5ml 1M Tris(pH7.5)，0.5ml 1M MgCl 2，加双蒸水至50ml。
80％的无水乙醇与10mM MgCl 2混合液(50ml)：取42.1ml 95％的无水乙醇，5ml 1M Tris(pH7.5)，0.5ml 1M MgCl 2，加ddH 2O至50ml。
80％的无水乙醇与25mM MgCl 2混合液(50ml)：取42.1ml 95％的无水乙醇(ETOH)，5ml 1M Tris(pH7.5)，1.25ml 1M MgCl 2，加ddH 2O至50ml。
50％的无水乙醇溶液(50ml)：取26.3ml 95％的无水乙醇，5ml 1M Tris(pH 7.5)，加ddH 2O 至50ml。
25％的无水乙醇溶液(50ml)：取13.2ml 95％的无水乙醇，5ml 1M Tris(pH 7.5)，加ddH 2O至50ml。
0.5％茜素红(50ml)：称取0.25g茜素红(Alizarin)粉末溶于50ml ddH 2O中。
0.01％茜素红(50ml，pH7.5)：取1ml 0.5％茜素红，12.5ml 100％的甘油，5ml 1M Tris(pH7.5)，加ddH 2O至50ml。
3％H 2O 2和0.5％KOH混合液：等量的6％H 2O 2和0.1％KOH，在使用前混合。
25％甘油和0.1％KOH混合液(50ml)：取12.5ml 100％甘油，0.25ml 20％KOH，加ddH 2O至50ml。
10％甘油和0.5％KOH混合液(50ml)：取5ml 100％甘油，1.25ml 20％KOH，加ddH 2O至50ml。
50％甘油和0.1％KOH混合液(50ml)：取25ml 100％甘油，0.25ml 20％KOH，加ddH 2O至50ml。
50％甘油和0.5％KOH混合液(50ml)：取25ml 100％甘油，1.25ml 20％KOH，加ddH 2O至50ml。
(2)染色具体步骤：
取9天的斑马鱼幼鱼，用1ml 2％PFA/1 X PBS浸泡并固定过夜，用100mM Tris pH7.5/10mM MgCl 2漂洗10mins，吸干净溶液后加入1ml依次分别用80％ETOH/100mM Tris pH7.5/10mM MgCl 2、50％ETOH/100mM Tris Ph7.5、25％ETOH/100mM Tris Ph7.5漂洗5mins进行脱色处理。
加1ml 3％H 2O 2和0.5％KOH漂白去色素，每10mins观察一次，直到色素漂干净为止，用25％甘油/0.1％KOH漂洗数次，每次10mins，直到没有气泡产生为止。
加入0.01％alizarin浸泡50mins进行硬骨染色，染色完后的斑马鱼幼鱼用50％甘油/0.1KOH浸泡10mins，换新的50％甘油/0.1％KOH溶液浸泡，显微镜下观察拍照，通过图像软件Image-proplus 6.0对图像进行分析，定量骨染色区域。
结果如图1～2所示，与空白对照组相比，随着醋酸铅处理浓度的增加，斑马鱼头部的染色区域逐渐减小，尤其在20mg/L PbAc处理组，只可见耳石骨轻微着色，且染色面积及程度都大大减弱，此外，通过图像软件Image-proplus 6.0对图像进行分析，计算斑马鱼的骨染色区域。其骨染色区域变化是逐渐减少的趋势，浓度越高差异性越明显，呈浓度梯度依赖性。
以上仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的 普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。

Claims (10)

  1. 一种斑马鱼骨质疏松模型的构建方法，其特征在于，包括如下步骤：
    1)将性成熟的斑马鱼按雌雄比＝1:1.5～2.5的比例放入交配鱼缸内，插上隔板，雌雄斑马鱼分开；培养10～20小时后，取出隔板，待90～150分钟后收集鱼卵；
    2)收集到的鱼卵置于含有抑菌素的培养液中，恒温孵育；
    3)恒温孵育后将鱼卵随机分为空白对照组和醋酸铅组，空白对照组中加入含有抑菌素的培养液，醋酸铅组中加入含有抑菌素的培养液和醋酸铅溶液，进行恒温培养；其中，醋酸铅组中醋酸铅的终浓度为5～20mg/L；
    4)取步骤3)培养后的斑马鱼进行软骨染色，与空白对照组相比，若醋酸铅组的染色区域减小，则得到斑马鱼骨质疏松模型。
  2. 根据权利要求1所述的构建方法，其特征在于，在步骤3)中，恒温培养的温度为28～29℃。
  3. 根据权利要求1所述的构建方法，其特征在于，在步骤3)中，恒温培养的时间为8～10天，每天更换培养液。
  4. 根据权利要求1所述的构建方法，其特征在于，在步骤3)中，在培养过程中，每天以13～15小时光照/9～11小时黑暗处理。
  5. 根据权利要求1所述的构建方法，其特征在于，在步骤2)中，恒温孵育的温度为28～29℃。
  6. 根据权利要求1所述的构建方法，其特征在于，在步骤2)中，恒温孵育的时间为1～3小时。
  7. 根据权利要求1所述的构建方法，其特征在于，所述的培养液为养鱼水。
  8. 根据权利要求7所述的构建方法，其特征在于，所述的养鱼水的参数条件为：酸碱度：7.0～7.3；温度：27～29℃；电导率：450～550μs；盐度：0.25～0.75‰；溶氧度：≧6mg/L；光周期：14/10小时；硬度：100～200mg/L；氯：0mg/L；氨氮浓度：<0.02mg/L；亚硝酸盐：<1mg/L；硝酸盐：<50mg/L；二氧化碳：<50mg/L。
  9. 根据权利要求1所述的构建方法，其特征在于，在含有抑菌素的培养液中，抑菌素的浓度为0.5×10-5μg/L。
  10. 一种权利要求1～9所述方法构建得到的斑马鱼骨质疏松模型。
PCT/CN2021/088295 2021-03-19 2021-04-20 一种斑马鱼骨质疏松模型的构建方法 Ceased WO2022193397A1 (zh)

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