Construction method and application of osteoporosis zebra fish model

A technology of osteoporosis and construction methods, applied in the field of medicine, can solve the problems of low transgenic efficiency and low parental heritability

Active Publication Date: 2015-01-28
HOSPITAL AFFILIATED TO GUANDONG MEDICAL COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method has the disadvantages of low transgenic efficiency and low parental heritability

Method used

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  • Construction method and application of osteoporosis zebra fish model
  • Construction method and application of osteoporosis zebra fish model
  • Construction method and application of osteoporosis zebra fish model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The propagation of embodiment 1 zebrafish

[0048] Zebrafish (provided by the Zebrafish Model Animal Platform of the Affiliated Hospital of Guangdong Medical College) were fed in a special fish tank, and the sunlight cycle was controlled (14 hours of light and 10 hours of darkness). Put the male fish and the female fish (1:2) in the breeding tank in the evening of the previous day, collect the fertilized eggs the next day, place them in a petri dish, and add embryo water (5mM NaCl, 0.17mM KCl, 0.33mM CaCl 2 ,0.33mM MgSO 4 ), cultivated in a constant temperature incubator at 28.5°C. Zebrafish embryos generally do not need to be fed for 7 days.

Embodiment 2

[0049] The construction of embodiment 2 transgenic zebrafish

[0050] 1. Experimental method

[0051]Plasmid 5'Entry clone: ​​p5E-Osterix, Middle Entry clone: ​​pME-nls EGFP and 3'Entry clone: ​​p3E-Poly A were constructed using tol2-gateway transgenic technology. The above three plasmids were subjected to three-segment LR reaction to obtain the expression plasmid pDestTol2CG2-osterix-nls-EGFP-pA.

[0052] In the morning, after the fertilized egg embryos were collected, they were fixed on a plate with agar gel, and the expression plasmid pDestTol2CG2-osterix-nls-EGFP-pA was mixed with Tol2 mRNA (the final concentration was 50ng / μl), and microinjected into a cell stage in zebrafish embryo cells. The next day, screen the fish that report green fluorescence under a fluorescent microscope, and culture until the adult fish is the F0 generation. Afterwards, the F1 generation was obtained by crossing the F0 generation with the wild type, and so on, and the F3 generation was used f...

Embodiment 3

[0055] Example 3 Effects of different doses of DEX on wild-type and transgenic zebrafish bones (Glucocorticoid-induced osteoporosis, GIOP model construction)

[0056] 1. Experimental method

[0057] 1. Design of osteoporosis model

[0058] Take the wild-type and transgenic zebrafish embryos 3 days after fertilization and put them into a 12-well culture plate filled with embryo water (PTU should be added to the wild-type, and N-Phenylthiourea is used to inhibit pigmentation so as not to affect the staining results). 6-8 juvenile fish were divided into DMSO negative control group and dexamethasone DEX (Dexamethasone, MP company) model group. In the dexamethasone model group, three concentrations of 5, 10, and 20 μM were set in order to select the best model-making concentration. The experiment was repeated at least 3 times to ensure scientificity and reliability. Add 2 mL of embryonic water (containing vehicle or drug) to each well, and culture in a constant temperature incub...

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Abstract

The invention belongs to the technical field of medicines and particularly relates to a construction method and an application of an osteoporosis zebra fish model. The construction method comprises the following steps: constructing transgenetic zebra fish with green fluorescence of a skeleton report after obtaining a promoter of a specific transcription factor of osteoblast by virtue of applying a tol2-gateway technique; constructing the glucocorticoid induced osteoporosis zebra fish model by using the transgenetic zebra fish; and meanwhile, estimating the model by applying a bone-strengthening and kidney-tonifying traditional Chinese medicine herba epimedii general flavone. The osteoporosis model constructed by the method provided by the invention is convenient, feasible and effective, and is suitable for high throughput screening of anti-osteoporosis drugs. Compared with the conventional chemical staining methods, the construction method is more stable, time-saving and efficient.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to a construction method and application of a zebrafish model of osteoporosis. Background technique [0002] Osteoporosis (Osteoporosis, OP) is a group of diseases characterized by decreased bone mass, degeneration of bone microstructure and fracture risk. The harm caused by it has gradually attracted people's attention, and the active development of drugs for the prevention and treatment of osteoporosis has gradually become one of the active fields in the medical field. For drug development, experimental models are particularly important. The cell model has a single function link, which is difficult to reflect the overall view of the drug, and cannot evaluate the drug in vivo; traditional animals such as mice are used as drug screening models due to high costs and long modeling time, and it is impossible to observe and locate them in vivo Such defects limit the application of hi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63A01K67/027A61K31/573A61K49/00
Inventor 张晶晶黄洪新萧崇德曾荣
Owner HOSPITAL AFFILIATED TO GUANDONG MEDICAL COLLEGE
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