WO2022165860A1 - 一种斑马鱼黑色素沉着模型的构建方法 - Google Patents

一种斑马鱼黑色素沉着模型的构建方法 Download PDF

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WO2022165860A1
WO2022165860A1 PCT/CN2021/077087 CN2021077087W WO2022165860A1 WO 2022165860 A1 WO2022165860 A1 WO 2022165860A1 CN 2021077087 W CN2021077087 W CN 2021077087W WO 2022165860 A1 WO2022165860 A1 WO 2022165860A1
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zebrafish
sodium arsenite
construction method
constant temperature
model
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安艳
魏园杰
严锐
景楠
王鲁娜
杨乾磊
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苏州大学
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  • the invention belongs to the technical field of medicine, and in particular relates to a method for constructing a zebrafish melanosis model.
  • Arsenic is a naturally occurring environmental poison. Long-term arsenic exposure can cause endemic arsenic poisoning, referred to as arsenic disease.
  • Arsenic disease is a biogeochemical disease, which is caused by depigmentation or/and hyperpigmentation of the skin caused by excessive intake of inorganic arsenic through drinking water, air or food for a long time by residents living under specific geographical conditions. , palmoplantar keratosis and carcinogenesis of the main systemic chronic poisoning. Symptoms of arsenic disease first appear on the skin, so skin lesions are considered an early sign of arsenic poisoning. At present, animal models of pigmentation have been widely used in the study of arsenic-induced skin hyperpigmentation.
  • An ideal animal model for pigment production 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 hyperpigmentation.
  • the animals used to study hyperpigmentation models mainly include guinea pigs, mice, Smyth line chickens and zebrafish.
  • Guinea pigs, mice and chickens are time-consuming, cost-intensive, sample volumes are large, and require high laboratory
  • Zebrafish is one of the classic model organisms in modern scientific research. The majority of melanin masses in the stripes on both sides of its body are mainly produced by melanocytes, which can better simulate human pigment spots. Compared with mammals, its reproductive cost is low; and its organ system and gene sequence are similar to those of humans; in addition, zebrafish has melanin deposition on the body surface, which can change rapidly with environmental changes, easy to monitor and can be analyzed and quantified by microscopy. In addition, zebrafish, as a model organism, is similar to human in terms of genome structure and many molecular mechanisms, and various mutant and transgenic zebrafish strains are also beneficial to study the cellular and molecular mechanisms of pathological pigmentation. In conclusion, the visibility of zebrafish melanocytes and their shared conserved features with human melanocytes make zebrafish an ideal model for studying melanogenesis.
  • the present invention uses a zebrafish model that is easy to operate, and can quickly establish a zebrafish model of increased melanin production in a short period of time after being treated with a sodium arsenite solution. To provide a simple, efficient and inexpensive biological model for the mechanism study of arsenic-induced skin pigmentation.
  • the first object of the present invention is to provide a method for constructing a zebrafish melanosis model, comprising the following steps:
  • the collected fish eggs are placed in a petri dish containing methylene blue, the fertilized fish eggs are selected to be divided into plates, an embryo culture medium is added, and incubated at a constant temperature;
  • step 4) Take the zebrafish cultured in step 3) and place them on a glass slide for microscope observation, take pictures and save them. Compared with the control group, if the melanin production in the sodium arsenite solution group increases, a zebrafish melanosis model is obtained.
  • step 3 the temperature of constant temperature cultivation is 28-29°C.
  • step 3 the time of constant temperature cultivation is 4-6 days.
  • 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 4-6 hours.
  • step 2) the disks are divided according to the density of 80-120 pieces/disk.
  • sodium arsenite solution is prepared by the following method:
  • Preparation of working solution take mother liquor, add water to prepare a sodium arsenite solution with a concentration of 8-12 mmol/L, and then add fish farming water to prepare a sodium arsenite solution of the required concentration.
  • the embryo culture medium in the present invention is a culture medium capable of normal growth and development of fertilized eggs, and fish culture water can also be used.
  • 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 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 melanin production increase model in a short period of time after being treated with a sodium arsenite solution. And because zebrafish has a short growth cycle, easy reproduction, large egg production, small individuals, and easy feeding, the experimental requirements are greatly reduced, providing a simple, efficient and inexpensive biological model for the study of the mechanism of arsenic-induced skin pigmentation.
  • Figure 1 shows the pictures and quantitative results of the zebrafish melanosis model constructed using different concentrations of sodium arsenite.
  • the solution preparation method is as follows:
  • Preparation of working solution take 5 ⁇ l of the mother liquor, add water to 50 ml, and obtain 10 mmol/l sodium arsenite solution;
  • Nitrate ⁇ 50mg/L
  • a method for constructing a zebrafish melanosis model comprising the following steps:
  • step 4) Take the cultured samples in step 3) for microscope observation. Compared with the blank control group, if the melanin production in the sodium arsenite solution group increases, a zebrafish melanin production model is obtained.
  • step 3 Take the zebrafish cultured on the 5th day of the fertilized eggs in step 3), place them on a glass slide and observe them under a microscope;
  • Picture A is a representative picture of the four concentration groups, and it can be seen that the size of the melanin spots has increased significantly;
  • picture B is the result of quantification of the captured zebrafish pictures using ImageJ, which can be seen compared with the control group.
  • 0.2mM had no statistical difference, while the 0.4 and 0.8 concentration groups showed a concentration-dependent increase, and there was a statistical difference, so the model of arsenic-induced increase in zebrafish melanin production was completed.

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Abstract

一种斑马鱼黑色素沉着模型的构建方法，将性成熟的斑马鱼按雌雄放入交配鱼缸内，插隔板分开雌雄斑马鱼；隔夜后开灯换水，取隔板，30-60分钟后收集鱼卵；置于含有亚甲基蓝的培养皿中，挑选受精鱼卵分盘，加入胚胎培养液恒温孵育；后挑出发育优良的受精卵随机分为对照组和亚砷酸钠溶液组进行恒温培养；取培养后的斑马鱼进行显微观察，与对照相比，若亚砷酸钠溶液组的黑色素生成增加，得到斑马鱼黑色素沉着模型。在亚砷酸钠溶液处理后可短期快速建立斑马鱼黑色素生成增加模型。且由于斑马鱼生长周期短，容易繁殖，产卵量大，个体小，饲养容易，使得实验要求大大降低，为砷致皮肤色素沉着的机制研究提供简单、高效，低廉的生物模型。

Description

一种斑马鱼黑色素沉着模型的构建方法 技术领域
本发明属于医药技术领域，尤其涉及一种斑马鱼黑色素沉着模型的构建方法。
背景技术
砷是一种天然存在的环境毒物，长期砷暴露会引起地方性砷中毒，简称地砷病。地砷病，是一种生物地球化学性疾病，是居住在特定地理环境条件下的居民，长期通过饮水、空气或食物摄入过量的无机砷而引起的以皮肤色素脱失或/和过度沉着、掌跖角化及癌变为主的全身性的慢性中毒。地砷病患者症状最早出现于皮肤，因此皮肤病变被视为砷中毒的早期标志。目前，色素沉着动物模型在砷致皮肤色素沉着过度的研究中已经得到广泛的应用。理想的色素生成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色素过度沉着尽可能相似。
目前，用于研究色素过度沉着的模型的动物主要有豚鼠、小鼠、Smyth line鸡和斑马鱼等。豚鼠，小鼠和鸡耗时长，成本大，样品量大，对实验室要求高，
斑马鱼作为现代科学研究的经典模式生物之一,其身体两侧条纹中占多数的黑色素团块主要由黑素细胞产生,可以较好地模拟人类色素斑。相较于哺乳动物其繁殖成本低；并且其器官系统和基因序列与人类相似：另外斑马鱼的体表即有黑色素沉着，可随环境变化而迅速变化，易于监测并可通过显微镜进行分析定量。另外，作为模式生物的斑马鱼与人类在基因组构成和很多分子机制上相似，多种突变及转基因斑马鱼品系也有利于研究病理性色素沉着的细胞和分子机制。总之，斑马鱼黑色素细胞的可视性及其与人类黑色素细胞之间的共同保守性特征使得斑马鱼是研究黑色素生成的理想模型。
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，在亚砷酸钠溶液处理后可短期快速建立斑马鱼黑色素生成增加模型。为砷致皮肤色素沉着的机制研究提供简单、高效，低廉的生物模型。
本发明的第一个目的是提供一种斑马鱼黑色素沉着模型的构建方法，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小时后开灯换水，取出隔板，待30-60分钟后收集鱼卵；
2)收集到的鱼卵置于含有亚甲基蓝的培养皿中，挑选受精鱼卵进行分盘，加入胚胎培养液，恒温孵育；
3)恒温孵育后挑出发育状况优良的受精卵随机分为对照组和亚砷酸钠溶液组，其中，亚砷酸钠溶液的浓度为0.4～1.0mmol/L，进行恒温培养；
4)取步骤3)培养后的斑马鱼置于载玻片上进行显微镜观察，拍照并保存，与对照组相比，若亚砷酸钠溶液组的黑色素生成增加，得到斑马鱼黑色素沉着模型。
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4～6天。
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4～6小时。
进一步地，在步骤2)中，按80～120枚/盘的密度进行分盘。
进一步地，所述的亚砷酸钠溶液通过如下方法进行配制：
S1、配制母液：取亚砷酸钠粉末溶于水中，配置成80～120mmol/L的母液；
S2、配制工作液：取母液，加水配制成浓度为8～12mmol/L的亚砷酸钠溶液，再加入养鱼水，配置成所需浓度的亚砷酸钠溶液。
进一步地，对照组中添加养鱼水。
本发明中的胚胎培养液为能够使受精卵正常生长发育的培养液，也可使用养鱼水。
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。
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，在亚砷酸钠溶液处理后可短期快速建立斑马鱼黑色素生成增加模型。且由于斑马鱼生长周期短，容易繁殖，产卵量大，个体小，饲养容易，使得实验要求大大降低，为砷致皮肤色素沉着的机制研究提供简单、高效，低廉的生物模型。
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为使用不同浓度的亚砷酸钠构建斑马鱼黑色素沉着模型的图片和定量结果。
具体实施方式
溶液配制方法如下：
配制母液：称取亚砷酸钠粉末0.650g，加入50ml纯净水充分溶解；
配制工作液：取所述母液5μl，加水至50ml，得10mmol/l亚砷酸钠溶液；
取所述10mmol/l亚砷酸钠溶液0.06ml，加养鱼水至3ml，得0.2mmol/l亚砷酸钠溶液；
取所述10mmol/l亚砷酸钠溶液0.12ml，加养鱼水至3ml，得0.4mmol/l亚砷酸钠溶液
取所述10mmol/l亚砷酸钠溶液0.24ml，加养鱼水至3ml，得0.8mmol/l亚砷酸钠溶液。
其中，养鱼水的理化参数如下：
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(CaCO 3):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
实施例1：
一种斑马鱼黑色素沉着模型的构建方法，包括以下步骤：
1)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；隔夜后开灯换水，取出隔板，待30-60分钟后收集鱼卵；
2)收集到的鱼卵置于含有亚甲基蓝的培养皿中，挑选受精鱼卵，按100枚/盘的密度进行分盘，加入胚胎培养液，在28.5℃恒温孵育5小时；
3)恒温孵育后挑出发育状况优良的受精卵分布于6孔板中，随机分为一个对照组和三个浓度的亚砷酸钠溶液组，向空白对照组的每孔中加入3ml养鱼水，向三个亚砷酸钠溶液组中分别加入3mL不同浓度的亚砷酸钠溶液，浓度分别为0.2mmol/L，0.4mmol/L，0.8mmol/L，每孔30个卵，每组3个孔，随后放入恒温培养室培养；恒温孵育的温度为28.5℃，时间为 5天，每天以14小时光照/10小时黑暗的条件进行恒温孵育；
4)取步骤3)培养后的样品进行显微镜观察，与空白对照组相比，若亚砷酸钠溶液组的黑色素生成增加，即得到斑马鱼黑色素生成模型。
显微镜观察步骤：
a：取步骤3)中受精卵培养第5天的斑马鱼，放在载玻片上置于显微镜下进行观察；
b：将斑马鱼保持在竖直状态，先拍摄全身照，再选取头胸部进行拍摄，拍摄期间固定曝光时间；
c：每个浓度组随机选取十条鱼；
d：使用ImageJ软件进行图像处理。
e：使用excel和SPSS软件进行数据处理，比较各组的值。
结果如图1所示，A图是四个浓度组的代表性图片，可见黑色素斑点的大小明显增加；B图是对拍摄的斑马鱼图片使用ImageJ进行定量后的结果，可见与对照组相比，0.2mM无统计学差异，而0.4和0.8浓度组则呈浓度依赖式的递增，并且具统计学差异，由此可得砷诱导斑马鱼黑色素生成增加的模型构建完成。
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. 一种斑马鱼黑色素沉着模型的构建方法，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小时后开灯换水，取出隔板，待30-60分钟后收集鱼卵；
    2)收集到的鱼卵置于含有亚甲基蓝的培养皿中，挑选受精鱼卵进行分盘，加入胚胎培养液，恒温孵育；
    3)恒温孵育后挑出发育状况优良的受精卵随机分为对照组和亚砷酸钠溶液组，其中，亚砷酸钠溶液的浓度为0.4～1.0mmol/L，进行恒温培养；
    4)取步骤3)培养后的斑马鱼置于载玻片上进行显微镜观察，拍照并保存，与对照组相比，若亚砷酸钠溶液组的黑色素生成增加，得到斑马鱼黑色素沉着模型。
  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4～6天。
  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4～6小时。
  7. 根据权利要求1所述的构建方法，其特征在于，在步骤2)中，按80～120枚/盘的密度进行分盘。
  8. 根据权利要求1所述的构建方法，其特征在于，所述的亚砷酸钠溶液通过如下方法进行配制：
    S1、配制母液：取亚砷酸钠粉末溶于水中，配置成80～120mmol/L的母液；
    S2、配制工作液：取母液，加水配制成浓度为8～12mmol/L的亚砷酸钠溶液，再加入养鱼水，配置成所需浓度的亚砷酸钠溶液。
  9. 根据权利要求1所述的构建方法，其特征在于，对照组中添加养鱼水。
  10. 根据权利要求8或9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。
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