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Zebra fish vascular injury model for screening vascular injury resisting medicament as well as building method and application thereof

A technology of vascular injury and establishment method, applied in the field of medicine, can solve the problems of unestablished, uncertain treatment of angiogenesis diseases, etc.

Inactive Publication Date: 2012-06-20
UNIVERSITY OF MACAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantage of this method is that potential angiogenesis drugs can be directly and quickly discovered according to the special morphology of angiogenesis blood vessels, but the fly in the ointment is that the drugs screened by this method are uncertain for the treatment of angiogenesis diseases, in other words In other words, drugs that are effective in normal models may not be effective in pathological models. Therefore, drug screening in pathological models is more direct and meaningful in terms of drug therapeutic effects.
However, unfortunately, in the zebrafish, an excellent model for studying angiogenesis, a significant, stable and recognized pathological model of blood vessels has not yet been established.

Method used

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  • Zebra fish vascular injury model for screening vascular injury resisting medicament as well as building method and application thereof
  • Zebra fish vascular injury model for screening vascular injury resisting medicament as well as building method and application thereof
  • Zebra fish vascular injury model for screening vascular injury resisting medicament as well as building method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Modeling of Zebrafish Embryo Vascular Injury and Rapid Screening of Drugs Promoting Angiogenesis

[0037] Experimental animals: Zebrafish (Danio rerio) embryos (1dpf) one day after fertilization were used as materials. Wild-type and transgenic zebrafish were provided by the Zebrafish International Resource Center (Oregon, PO), where the wild-type was the AB strain, and the transgenic zebrafish was Tg (fli-1: EGFP), and the transgenic zebrafish was the fli-1 The green fluorescent protein gene is inserted into the gene, so the expression of fli-1 gene in transgenic vascular endothelial cells will also express green fluorescent protein, so Tg(fli-1:EGFP) will express green fluorescent protein in the whole endothelial cells , and the feeding regimen was carried out as described in the Zebrafish Book. The fertilized eggs are obtained from the mating of male and female fish in the early morning, and then the fertilized eggs are placed in the embryo culture medium a...

Embodiment 2

[0043] Example 2 The VRI-induced vascular injury model of zebrafish embryos is used in the rapid screening experiment of pro-angiogenic drugs Calycoisoflavone can restore the vascular injury (disappearance) caused by vascular growth factor receptor tyrosine kinase inhibitor (VRI)

[0044] Transgenic zebrafish Tg(fli-1:EGFP) embryos 28 hours after fertilization were treated with 300nM / L vascular growth factor receptor tyrosine kinase inhibitor (VRI) for 3 hours, and then the VRI solution was washed away. And wash 1-2 times with pure deionized water, and culture the fish in the embryo culture solution containing 0.1% DMSO and the drug that needs to be screened (taking calycosin as an example), add 12.5, 25, 50 μ M / L calycosin respectively, and respectively Observation by fluorescent confocal microscope at 2, 3, and 4 days old showed that after VRI pretreatment and cultured in 0.1% DMSO embryo culture medium, the intersegmental blood vessels and dorsal longitudinal blood vessels g...

Embodiment 3

[0045] Example 3 Changes in the level of zebrafish genes induced by VRI

[0046] Experimental method: Flk1-A is also called KDR1, VEGFR2 like, Flk1-B is also called KDR, VEGFR2, and Flt1 is also called VEGFR1. As a receptor of vascular growth factor (VEGF), it is activated by VEGF to induce intracellular signals for angiogenesis Response, and such receptors are also specifically expressed in vascular endothelial cells, so the expression of such receptors can be used as a key indicator of the state of endothelial cells. In this experiment, 30 wild-type zebrafish embryos 28 hours after fertilization were taken from each group. After pretreatment with 300nM / L VRI for 3 hours, the VRI solution was removed, washed 1-2 times with pure deionized water, and then distributed in 6 wells. In the plate, 30 zebrafish embryos are guaranteed for each well, each well is a group, and 12.5, 25, and 50 μM concentrations of calycein are added respectively, and the total RNA is extracted after 24 ...

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Abstract

The invention discloses a zebra fish vascular injury model for screening a vascular injury resisting medicament as well as a building method and application thereof. According to the invention, a fertilized zebra fish embryo is treated with a vascular growth factor receptor inhibitor to obtain zebra fish suffering from vascular injury so as to build the zebra fish vascular injury model, then the zebra fish is treated with a detected medicament for 1-3 days, and the gene expression level of the vascular endothelial growth factor receptors (Flk-1A, Flk1-B and Flt1) in the zebra fish tissues is detected by morphological observation and real-time quantitative PCR (polymerase chain reaction) so as to judge the treating effect of the detected medicament on the zebra fish vascular injury. Experiment results prove that the zebra fish vascular injury model built according to the method provided by the invention has extremely high model forming rate, and can simulate in-vivo insufficient angiogenesis environment to quickly screen medicaments for promoting angiogenesis; the effect is easy to observe and is obvious; the medicament dosage in the experiment process is low; and the experiment results are accurate and strong in repeatability. The model can be used for screening potential medicaments in batch with high working efficiency.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to a method for establishing a zebrafish vascular injury model and a method for applying the model to screening anti-vascular injury drugs. Background technique [0002] Angiogenesis refers to the process by which adults form new blood vessels by sprouting on the basis of primitive vascular plexuses or existing blood vessels. It has the function of maintaining normal growth and balance of the human body. It plays an important role in the occurrence, development and outcome of many diseases , plays an important role in prognosis. In recent years, the research on regulating angiogenesis has become a hot spot in clinical treatment. Angiogenesis mainly involves the degradation of basement membrane, migration, proliferation and tube formation of endothelial cells, among which the proliferation of endothelial cells is one of the most important indicators of angiogenesis. At the same ti...

Claims

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Application Information

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IPC IPC(8): A01K61/00A61K49/00G01N21/64C12Q1/68
CPCY02A40/81G01N2800/32G01N2800/7014A61K49/0008
Inventor 李铭源黎晌许贝文
Owner UNIVERSITY OF MACAU
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