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Application of amphimutation zebra fish in preparation of osteosclerosis animal model

A technology for osteopetrosis and animal models, applied in compound screening/testing, preparations for in vivo experiments, animal husbandry, etc., can solve brain malformation, unknown mechanism and interaction relationship of osteoclast development, lack of osteosclerosis disease animal models

Active Publication Date: 2021-01-22
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

More importantly, several recent clinical family studies have found that CSF-1R biallelic mutations can lead to severe brain malformations and osteopetrosis
Although PU.1 and FMS have been reported to play a role in osteoclastopenic osteopetrosis in mice, the mechanisms and interactions that specifically affect osteoclast development remain unknown.
And due to the cost of mouse models, breeding scale and operational limitations, there is still a lack of animal models of osteosclerosis that can be used for osteosclerosis drug evaluation and large-scale candidate compound screening

Method used

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  • Application of amphimutation zebra fish in preparation of osteosclerosis animal model
  • Application of amphimutation zebra fish in preparation of osteosclerosis animal model
  • Application of amphimutation zebra fish in preparation of osteosclerosis animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Pu.1 and FMS have a synergistic effect in the development of refunctional osteoclasts in zebrafish It is known that in mammals, osteoclasts are derived from macrophages. Both PU.1 and FMS are expressed in macrophages, and the abnormality of either gene will lead to abnormal osteoclasts, suggesting that they play an important role in osteoclast formation and maturation. However, whether PU.1 and FMS have a synergistic effect in osteoclast development is unknown. To answer this question, we conducted the following experiments:

[0082] 1) Frozen section experiment: zebrafish specimens were first fixed with 4% PFA (paraformaldehyde) (the fixation time depends on the size of the tissue, such as small fish tissues at room temperature for 2 hours, adult fish tissues at room temperature for 2 days), and then 30% (w / v) sucrose for dehydration (overnight dehydration at 4°C). After the tissue is completely dehydrated and sinks to the bottom of the tube, it can be emb...

Embodiment 2

[0086] Example 2: Compared with FMS, PU.1 mainly acts on zebrafish osteoclastogenesis

[0087] There are two possible reasons for the reduction of functional osteoclasts: one is a disorder of osteoclastogenesis, and the other is a disorder of multinucleated osteoclast maturation. To determine the cause of the reduced functional osteoclasts caused by PU.1 and FMS deficiency, we performed the following experiments:

[0088] The co-staining method of LCP and DAPI immunofluorescence is as follows:

[0089] ①The zebrafish were killed in ice water first, and then the body segment between the pectoral fin and pelvic fin was taken out with a scalpel under a dissecting microscope;

[0090] ② Fix the tissue in 4% PFA overnight;

[0091] ③The removed body was washed with PBST for 3×5mins,

[0092] ④Reuse 20mg / mL cathepsin (name: proteinase K; brand: Fermentas, ThermoFisher Scientific) to digest at room temperature for 2h;

[0093] ⑤ After that, wash with PBST for 3×5mins;

[0094] ⑥...

Embodiment 3

[0102] Example 3: Osteoclast reduction is partially caused by apoptosis

[0103] To identify the cellular mechanism by which pu.1 and fms deficiencies lead to osteoclast reduction, we performed co-staining of LCP with terminal deoxynucleotidyl transferase dUTP-labeled end-label (TUNEL) as follows:

[0104] (1) TUNEL staining:

[0105] ①The zebrafish were killed in ice water first, and then the body segment between the pectoral fin and pelvic fin was taken out with a scalpel under a dissecting microscope;

[0106] ②According to the instructions of the TUNEL kit (name: In-Situ Cell Death Detection Kit TMR red; brand: Roche; address: Basel, Switzerland; article number: 12156792910), wash the removed body with PBST for 3×5mins;

[0107] ③Use 20 mg / mL cathepsin (name: proteinase K; brand: Fermentas, ThermoFisher Scientific) to digest at room temperature for 2 hours;

[0108] ④ Wash with PBST for 3×5mins;

[0109] ⑤Use acetone:ethanol (1:2, v / v) solution to soak for 7 minutes at ...

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Abstract

The invention discloses an application of amphimutation zebra fish in preparation of an osteosclerosis animal model. The amphimutation zebra fish is pu.1 G242D; and the fmsj4e1 mutant zebra fish is amutant zebra fish in which 242th amino acid glycine in a DAB structural domain of a zebra fish pu.1 gene is substituted by aspartic acid and 614th amino acid valine in a fms gene kinase structural domain is substituted by methionine. The amphimutation zebra fish has osteosclerosis symptoms similar to those of a human body, so that the amphimutation zebra fish can be used as the animal model for osteosclerosis drug evaluation and large-scale candidate drug screening, in addition, scales of the amphimutation zebra fish can be used for osteosclerosis drug screening in a rapid reading and processing manner, and the application has the characteristics of high flux, short period, low cost and convenience in operation, and provides an effective way for screening osteosclerosis drugs.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to the application of a double mutant zebrafish in preparing an animal model of osteopetrosis. Background technique [0002] Bone development and bone remodeling programs are controlled by a precise balance between the bone formation function of osteoblasts (Ob) (forming new bone) and the bone resorption function of osteoclasts (Oclasts (Oc) (absorbing and digesting old bone). Completed. Bone resorption and bone formation mainly depend on the activities of Oc and Ob. The formation and maturation of Ob and Oc depend largely on transcription factors and cytokines. Therefore, the abnormality of these factors will break the balance between bone formation and bone resorption, resulting in osteoporosis (bone resorption > bone formation) and the occurrence of osteosclerotic diseases (bone formation > bone resorption). [0003] Osteosclerosis (also known as osteopet...

Claims

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Application Information

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IPC IPC(8): A01K67/027A61K49/00
CPCA01K67/0275A01K2217/03A01K2227/40A01K2267/03A61K49/0008
Inventor 刘伟张译月张文清黄志斌
Owner SOUTH CHINA UNIV OF TECH
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