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Method of preparing osteoporosis model of zebra fish and alizarin red dyeing method thereof

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: 2017-05-31
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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  • Method of preparing osteoporosis model of zebra fish and alizarin red dyeing method thereof
  • Method of preparing osteoporosis model of zebra fish and alizarin red dyeing method thereof
  • Method of preparing osteoporosis model of zebra fish and alizarin red dyeing method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0060] (1) Reagents required for preparation of dyeing: a) Alizarin red solution: weigh 0.5g of Alizarin red powder and dissolve 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 100ml of distilled water to prepare a 0.7% KOH solution.

[0061] (2) Specific steps of alizarin red staining:

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

[0063] b) Depigmentation: absorb 4% PF, wash 3 times with PBST, 5min each time; add depigmentation solution (0.5%KOH / 3%H 2 o 2 ) until the melanin and yellow pigment are invisible to the naked eye, and the solution is replaced 1-2 times in the middle as needed. The whole process takes 30min-1h. In order to prevent the tube from bursting d...

Embodiment 2

[0075] 1. Ferric ammonium citrate induced osteoporosis model of juvenile zebrafish

[0076]The zebrafish juvenile osteoporosis model induced by ferric ammonium citrate was established according to the following method: Ferric ammonium citrate (FAC) was used as the drug for modeling, and juvenile fish 48 hours after fertilization were selected to be exposed to different concentrations of FAC solutions. FAC concentrations were 50, 100, 200, 500 μg·ml -1 , and set common culture medium (Holfreter H 2 0) was the control group, cultured in a 6-well plate at 28.5°C for 4 days until the zebrafish skull was formed, during which the dressing was changed every 12 hours.

[0077] 2. Evaluation of zebrafish juvenile osteoporosis model

[0078] (1) Statistics on teratogenic rate and lethal dose: After modeling, the teratogenic rate and lethal rate of juvenile zebrafish were counted, and the results are shown in Table 1. It can be seen that when ferric ammonium citrate is used for modeli...

Embodiment 3

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

[0092] Juvenile fish restoration: Collect zebrafish juveniles 48 hours after fertilization and divide them into 4 groups randomly, with at least 30 fish in each group, respectively: the control group is cultured in normal fish culture water until the 10th day; -1 FAC was treated until the 10th day; the negative drug control group was treated with FAC for 4 days, and then replaced with H 2 O cultured for 4 days; the positive drug restoration group was treated with FAC for 4 days, and then replaced with 0.1% positive drug alendronate sodium for 4 days. The juveniles of the 4 groups on the 10th day after the treatment were collected and stained with alizarin red, calcein and Alcian blue to detect the repair of cartilage and cartilage development. The results are as follows: Figure 5 shown. The experimental results show that alendronate sodium can excellently repair the decreased bone calcification a...

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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 invention relates to the field of animal models of osteoporosis, in particular to a method for preparing a zebrafish osteoporosis model and an alizarin red staining method thereof. 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, constructing an effective osteoporosis drug screening 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 feeding and maintenance costs, compared with rats and mice, zebrafish is th...

Claims

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Application Information

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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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