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Construction method for zebra fish osteoporosis model and application thereof

An osteoporosis and construction method technology, applied in the field of biotechnology model construction, can solve problems such as high lethality, zebrafish skeletal developmental defects, zebrafish embryo teratogenicity, etc. Quantitative analysis, repeatable, stable and reliable results

Pending Publication Date: 2020-12-08
苏州木芮生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, prednisolone or dexamethasone are the main drugs used in the construction of zebrafish osteoporosis models, but the above two drugs are glucocorticoid drugs, and it is very easy to use them due to inaccurate dosage when making models. Excessive immunosuppression leads to teratogenicity and high lethality in zebrafish embryos
At present, some studies have found that high iron can also cause skeletal development defects in zebrafish, but whether the high iron-induced osteoporosis model in juvenile fish can be used to screen other drugs for the treatment of osteoporosis other than iron chelators is still unknown

Method used

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  • Construction method for zebra fish osteoporosis model and application thereof
  • Construction method for zebra fish osteoporosis model and application thereof
  • Construction method for zebra fish osteoporosis model and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Cartilage Staining of Juvenile Zebrafish

[0048] a: Take the zebrafish fertilized eggs cultured in step 3) on the 5th day, fix with 4% PFA for 24h, and use Tris buffer / MgCl 2 Rinse the sample with the solution for 10 minutes, suck up the solution, then add 1ml 0.04% Alcian blue / MgCl 2 The solution was soaked overnight at room temperature, shaken and dyed;

[0049] b: Use Tris buffer / MgCl containing 80%, 50%, and 25% ethanol in turn 2 Each solution was rinsed for 5 minutes for decolorization;

[0050] c: Rinse with an equal volume of 3% H 2 o 2 Bleach with 0.5% KOH to remove the pigment, and observe it every 10 minutes until the pigment is bleached;

[0051] d: After the pigment is rinsed, rinse with 25% glycerin / 0.1% KOH for 10 minutes, rinse 3-4 times, and then replace with a new 50% glycerin / 0.1% KOH solution to soak and store.

Embodiment 2

[0052] Example 2: Staining of teleosts in juvenile zebrafish

[0053] a: Take the zebrafish after 8 days of fertilized egg culture in step 3), fix with 4% PFA for 24h, and use Tris buffer / MgCl 2 Rinse with the solution for 10 minutes;

[0054] b: Equal volume of 3% H 2 o 2 Bleach with 0.5% KOH to remove the pigment, and observe it every 10 minutes until the pigment is bleached;

[0055] c: Rinse with 25% glycerol / 0.1% KOH for 10 minutes, rinse 2-3 times; then add 0.01% Alizarin Red S and soak for 30 minutes for hard bone staining;

[0056] d: Rinse with 50% glycerin / 0.1% KOH solution for 10 minutes after dyeing, rinse 2-3 times, replace with new 50% glycerin / 0.1% KOH solution and soak for storage.

[0057] Wherein, the pH value of the Tris buffer is preferably 7.5, and the concentration is preferably 100 mM.

[0058] Among them, the MgCl 2 The concentration of the solution is preferably 10 mM.

[0059] The zebrafish stained in Example 1 and Example 2 were observed under...

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Abstract

The invention relates to a construction method for a zebra fish osteoporosis model and application thereof. The construction method for the zebra fish osteoporosis model mainly comprises the steps ofzebra fish selection, CdCl2 solution treatment, cartilage dyeing and hard bone dyeing. Compared with a conventional molding method using prednisolone and dexamethasone, the method has the advantages that the teratogenesis rate and the fatality rate of zebra fishes can be remarkably reduced by using a CdCl2 solution for treating the zebra fishes; when the high-concentration CdCl2 solution is used for treating the zebra fishes, the mineralization area of zebra fish skeletons is remarkably reduced, the density of the zebra fish skeletons is remarkably reduced, skeletal development defects are caused, and expression of genes related to skeletal regulation and control is reduced; the method has the advantages of being simple, convenient, quick, high in success rate, good in repeatability, stable, reliable and the like, and can achieve qualitative analysis and quantitative analysis; and the constructed zebra fish model has more outstanding and obvious osteoporosis characteristics, and can bequickly and efficiently used for widely evaluating and screening different types of models for treating osteoporosis drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology model construction, and in particular relates to a construction method and application of a zebrafish osteoporosis model. Background technique [0002] Osteoporosis is a group of disorders characterized by decreased bone mass, abnormal bone microstructure and increased risk of fracture. This disease is a common and frequently-occurring disease of the elderly. With the acceleration of population aging process, the incidence rate of osteoporosis is getting higher and higher. Therefore, the method of constructing an effective osteoporosis model and its application are of great significance. [0003] Zebrafish is a tropical carp fish that has been gradually used as a bone disease model due to its small size, fast reproductive cycle, transparent embryos, rapid bone development, and high homology with human-related genes and signaling pathways. Compared with rats and mice, zebrafish has more advantages as...

Claims

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Application Information

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IPC IPC(8): A01K61/10A01K61/17
CPCA01K2267/035A01K61/10A01K61/17Y02A40/81
Inventor 王明勇
Owner 苏州木芮生物科技有限公司
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