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Dyeing method of yolk sacs and/or adipose cells and applications thereof

A technique for adipocytes and fish eggs, which is applied in the field of staining and application of yolk sac and/or adipocytes, and can solve problems such as no related reports

Inactive Publication Date: 2013-01-02
INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the above-mentioned characteristics of zebrafish, if it is used for drug screening, there will be many advantages, but there is no relevant report so far

Method used

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  • Dyeing method of yolk sacs and/or adipose cells and applications thereof
  • Dyeing method of yolk sacs and/or adipose cells and applications thereof
  • Dyeing method of yolk sacs and/or adipose cells and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: Observation of Zebrafish Yolk Sac Adipocytes

[0070] Nutrition for zebrafish embryos and larvae is provided by the yolk sac. In the experiment, it was found that there is a large amount of fat in the yolk sac, which is clearly visible under the microscope.

[0071] Obtain zebrafish fertilized eggs: Select sexually mature zebrafish females (zebrafish are from the School of Life Sciences, Peking University), pick out females with swollen abdomen, sink to the bottom of the fish tank and do not want to move, and 1:1 with males The ratio is placed in the zebrafish ovipositor, and the plate is pulled out the next morning, allowing it to fertilize freely. 4-6 hours after fertilization, the fertilized eggs are taken out, washed, and placed in the Holt Buffer of a 24-well plate at 28.5°C Cultivation, usually after 48-72 hours of incubation, it can be hatched, and it can be used for experiments after hatching.

[0072] 3) Observation method: After the zebrafish larv...

Embodiment 2

[0073] Example 2: Using Oil Red O to stain zebrafish juvenile adipocytes

[0074] 1) Staining: Wash the newly hatched zebrafish larvae obtained according to the method described in Example 1 with culture fluid (Holt Buffer), add Oil Red O / acetone solution, which is to dye the mother solution in a concentration gradient of 1 -50ng / ml (the specific concentration is 1, 5, 10, 20, 30, 40, 50ng / ml) is diluted with Holt Buffer solution, mixed well and added to a 24-well plate. For the convenience of observation, 30mg / L of PTU (sigma product) was added to prevent pigmentation. The medium was changed once a day for 3 consecutive days.

[0075] 2) Staining observation: the zebrafish was taken out, anesthetized with 3×Tricaine (sigma product) solution, and the fluorescence changes were observed under a fluorescence microscope.

[0076] 3) Results: The high concentration (50ng / ml) of the staining solution is slightly darker than the low concentration (20ng / ml), and the color is yellowi...

Embodiment 3

[0077] Example 3: Juvenile Zebrafish Adipocytes Stained with Oil Red O for Efficacy Evaluation of Weight Loss Drugs

[0078] 1) Dyeing: the dyeing method is the same as step 1) in Example 2, and the concentration of the staining solution is 20 ng / ml.

[0079] 2) Administration of weight-loss drugs: add pre-diluted weight-loss drug mother solution to the staining solution at a certain concentration, mix well and add to 24-well plate. At the same time, the wells without weight-loss drugs were set up as controls. For the convenience of observation, PTU with a final concentration of 30mg / L was added to prevent pigmentation. The medium was changed once a day for 3 consecutive days. The experimental and control groups each included 10-20 zebrafish.

[0080] 3) Staining and drug effect observation: the zebrafish was taken out, anesthetized with 3×Tricaine, and the fluorescence changes were observed under a fluorescence microscope.

[0081] Results: The fluorescence of the yolk sa...

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Abstract

The invention relates to a dyeing method of yolk sacs and / or adipose cells, and particularly relates to a dyeing method of the yolk sacs and / or adipose cells of zebra fish, and a weight-reducing medicine screening model constructed by utilizing the method. Specially, resin dye is added in a culture solution for culturing juvenile fish of the zebra fish, the juvenile fish is incubated, the dyeing condition of the adipose cells of the zebra fish can be observed by a microscope, and the effect of the weight-reducing medicine can be judged according to the fluorescence intensity. The invention also relates to applications of the zebra fish used for screening the weight-reducing medicine. According to the method provided by the invention, the rapidness, intuition, flexibility and high efficiency can be realized, the high throughput screening can be conducted, and a new approach is provided for the construction of an animal model for evaluating the weight-reducing medicine.

Description

technical field [0001] The invention relates to a staining method of yolk sac and / or fat cells, in particular to a staining method of zebrafish yolk sac and / or fat cells, and a zebrafish weight loss drug screening model constructed by using the method. Background technique [0002] Obesity has become one of the risk factors for human health. With the improvement of material living conditions, the incidence of obesity has been increasing year by year. It not only affects posture and activity, but also easily causes hyperlipidemia, atherosclerosis, hypertension, coronary heart disease, diabetes and other diseases. According to incomplete statistics, more than 10% of the world's population suffers from obesity, especially in developing countries. The obesity population in my country's urban population has exceeded 30%, and Beijing has exceeded 40%, and there is a tendency to expand. Therefore, it is imperative to prevent and control obesity, and it is imperative to develop we...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K61/00G01N21/64
CPCY02A40/81
Inventor 赵宝全孙曼霁
Owner INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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