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A method for preparing a zebrafish osteoporosis model and its alizarin red staining method

A technology of osteoporosis and zebrafish, applied in the field of animal models of osteoporosis, can solve the problems of not being able to simulate osteoporosis well, zebrafish embryo teratogenicity, and high lethality, and achieve obvious characteristics of osteoporosis, Effects of increased total bone mass damage and decreased expression

Active Publication Date: 2019-05-10
MINNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, prednisolone and 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 doses during model building. Excessive immunosuppression leads to teratogenicity and high lethality in zebrafish embryos
At present, some studies have found that deferoxammonium or hepcidin can repair the skeletal development defects of zebrafish caused by high iron, but whether the high iron-induced osteoporosis model in juvenile fish can be used to screen for other treatments for osteoporosis other than iron chelators? drug unknown
In addition, the zebrafish used for modeling at this stage is generally selected as a juvenile zebrafish, which is quite different from the physiological stage of the patient when osteoporosis occurs (osteoporosis mainly occurs in adulthood, especially in the middle-aged and elderly stages). , does not model osteoporosis well

Method used

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  • A method for preparing a zebrafish osteoporosis model and its alizarin red staining method
  • A method for preparing a zebrafish osteoporosis model and its alizarin red staining method
  • A method for preparing a zebrafish osteoporosis model and its alizarin red staining method

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Embodiment 1

[0059] 1. Establish a staining method for zebrafish juvenile Alizarin Red:

[0060] (1) Prepare the reagents needed for dyeing: a) Alizarin red solution: weigh 0.5g of Alizarin Red powder dissolved in 100ml of 95% ethanol, the final concentration is 0.5%; b) 0.7% KOH solution: weigh 0.7g The KOH solid was dissolved in 100 ml of distilled water and prepared into a 0.7% KOH solution.

[0061] (2) The specific steps of Alizarin Red staining:

[0062] a) Sample collection and fixation: Collect the juveniles on the 6th day after fertilization, put the juveniles in a 1.5ml EP tube, and fix them in 4% PF at 4°C overnight, not more than one week;

[0063] b) Depigmentation: remove 4% PF, wash 3 times with PBST for 5 minutes each time; add depigmentation solution (0.5%KOH / 3%H 2 O 2 ) Until the melanin and yellow pigment are not visible with naked eyes, replace the solution 1 to 2 times as needed. The whole process takes 30 min to 1 h. In order to prevent the tube from expanding and bursting du...

Embodiment 2

[0075] 1. Ferric ammonium citrate induces the establishment of a zebrafish juvenile osteoporosis model

[0076] Refer to the following method to establish an osteoporosis model of zebrafish juveniles induced by ferric ammonium citrate: Ferric ammonium citrate (FAC) is used as a model drug, and the juveniles 48h after fertilization are exposed to different concentrations of FAC solution. FAC concentrations are 50, 100, 200, 500 μg·ml -1 , While setting up ordinary culture medium (Holfreter H 2 0) is the control group, cultured in a 6-well plate at 28.5°C for 4 days until the zebrafish skull is formed, during which the dressing is changed every 12h.

[0077] 2. Evaluation of the osteoporosis model of juvenile zebrafish

[0078] (1) Statistics of teratogenicity and lethality: After modeling, the teratogenicity and lethality of zebrafish juveniles are counted, and the results are shown in Table 1. It can be seen that when using ferric ammonium citrate for modeling, the teratogenicity ra...

Embodiment 3

[0091] Alendronate can well repair FAC-induced osteoporosis in juvenile fish

[0092] Juvenile repair: Collect the zebrafish juveniles 48h after fertilization and randomly divide them into 4 groups, each with at least 30 pieces: the control group is cultured in normal fish water until the 10th day; the model is 500μg·ml -1 FAC treatment to the 10th day; the negative drug control group is FAC treatment for 4 days, then change to H 2 O was cultured for 4 days; the positive drug repair group was FAC treatment for 4 days, and then replaced with 0.1% positive drug alendronate for 4 days. The 4 groups of juvenile fish on the 10th day after treatment were collected and stained with Alizarin Red, Calcein and Alcian Blue to detect the repair of hard bone and cartilage development. The results are as follows Figure 5 Shown. Experimental results show that whether it is from bone density, degree of mineralization or cartilage development, alendronate can extremely repair the reduction of bon...

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Abstract

The invention provides a method of preparing an osteoporosis model of a zebra fish and an alizarin red dyeing method thereof. According to the modeling method provided by the invention, a juvenile zebra fish or an adult zebra fish is treated by using a high concentration Fe<3+> solution to obtain the osteoporosis model. The model has obvious and prominent characteristics of osteoporosis, and can be used for screening various drugs for treating osteoporosis in a broad spectrum. The dyeing method provided by the invention optimizes the alizarin red dyeing method of the juvenile zebra fish or the adult zebra fish, and the dyeing effect of the zebra fish, especially the adult zebra fish, is improved, so that the adult zebra fish can be used for preparing the osteoporosis model.

Description

Technical field [0001] The present invention relates to the field of osteoporosis animal models, in particular to a method for preparing a zebrafish osteoporosis model and an alizarin red staining method. Background technique [0002] Osteoporosis is a type of disease characterized by bone loss, abnormal bone tissue microstructure, and increased risk of fractures. This disease is a common and frequently-occurring disease in the elderly. With the acceleration of the aging process of the population, the incidence of osteoporosis is getting higher and higher. 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. [0003] At present, the animals used to prepare osteoporosis models mainly include rats, mice, and zebrafish; due to their small size, fast growth cycle, and low cost of feeding and maintenance, zebrafish are used as bones compared to rat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K33/26A61K31/295A61K49/00A01K61/10
CPCA01K61/00A61K33/26A61K49/0008Y02A40/81
Inventor 薛钰潘裕添张文娟许璟瑾李秀敏
Owner MINNAN NORMAL UNIV
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