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Method for screening mitochondria targeted compounds by using zebra fish

A compound and mitochondrial technology, applied in the field of compound (chemical) detection, can solve the problems of difficult quantitative analysis, complicated experimental operation, cell damage, etc., and achieve the effect of avoiding interaction, simple administration method, and short sexual maturity cycle

Active Publication Date: 2011-02-09
南京新环检测科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The experimental operation of this method is complicated, it is easy to cause cell damage, and it cannot reflect the absorption, distribution, metabolism and excretion of the drug in the whole organism, and cannot reflect the real situation of the drug in the body.
[0011] (3) Cell transfection: transfect cells with cDNA encoding membrane protein labeled with green fluorescent protein (GFP), although this method can Applied to living cells and dynamic observation, but the process is more complicated and the processing capacity is low
[0016] (1) The detection at the cell level adopts the method of cell culture. Although this method is effective and requires low cost, the in vitro cells lack the metabolic cycle transformation of the whole organism and circulating distribution in the body, which cannot truly reflect the overall biological activity;
[0017] (2) The detection at the molecular level needs to separate the mitochondria, which also lacks the absorption, distribution, metabolism and excretion process of the drug in the organism

Method used

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  • Method for screening mitochondria targeted compounds by using zebra fish
  • Method for screening mitochondria targeted compounds by using zebra fish
  • Method for screening mitochondria targeted compounds by using zebra fish

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0181] (1) Selection of zebrafish

[0182] The 4dpf zebrafish were observed under a dissecting microscope, and the normally developed zebrafish were picked and transferred into 48-well plates, 10 in each well.

[0183] (2) Compound treatment

[0184] Set up 8 experimental groups: 5 drug treatment groups to be tested, 1 mitochondrial damage positive control group, 1 solvent control group, and 1 blank control group. Remove the culture water in the microwell plate, and add 1mL doxorubicin with concentrations of 0.1μM, 1μM, 10μM, 100μM, and 1000μM to the five drug treatment groups to be tested respectively. [26] solution; in the positive control group, 1 mL of etoposide solution with a concentration of 50 μM was added; in the solvent control group, 1 mL of DMSO with a concentration of 0.1% was added; in the blank control group, 1 mL of aquaculture water was added. Incubate for 24 hours in a constant temperature incubator at 28°C.

[0185] (3) Dyeing treatment

[0186] The solv...

Embodiment 2

[0203] (1) Selection of zebrafish

[0204] The 4dpf zebrafish were observed under a dissecting microscope, and the normally developed zebrafish were picked and transferred to a 48-well plate, with 10 fish per well.

[0205] (2) Compound treatment

[0206] Set up 8 experimental groups: 5 groups treated with environmental toxic compounds to be tested, 1 mitochondrial damage positive control group, 1 solvent control group, and 1 blank control group. Remove the breeding water in the microwell plate, and add 1 mL of the environmentally toxic compound Nonglin at a concentration of 0.1 μM, 1 μM, 10 μM, 100 μM, and 1000 μM to the five treatment groups of environmentally toxic compounds to be tested. [27] solution; in the positive control group, 1 mL of staurosporine solution with a concentration of 50 μM was added; in the solvent control group, 1 mL of DMSO with a concentration of 0.1% was added; in the blank control group, 1 mL of aquaculture water was added. Incubate for 24 hours ...

Embodiment 3

[0225] The present embodiment carries out according to the following steps:

[0226] (1) Selection of zebrafish

[0227] The 4 dpf zebrafish were observed under a dissecting microscope, and the normally developed zebrafish were picked and transferred to a 96-well plate, with one fish per well.

[0228] (2) Compound treatment

[0229] Eight experimental groups were set up: 5 candidate antineoplastic drug treatment groups, 1 mitochondrial damage positive control group, 1 solvent control group, and 1 blank control group. Remove the culture water in the microwell plate, and add 150 μL of the candidate antineoplastic drug teniposide at concentrations of 0.1 μM, 1 μM, 10 μM, 100 μM, and 1000 μM to the five candidate antitumor drug treatment groups. [28] solution; 150 μL of 50 μM etoposide solution was added to the positive control group; 150 μL of 0.1% DMSO was added to the solvent control group; 150 μL of aquaculture water was added to the blank control group. Incubate for 24 ho...

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Abstract

The invention relates to the field of compound detection, in particular to a method for screening mitochondria targeted compounds by using zebra fish. The method mainly comprises the following steps of: zebra fish selection, compound treatment, dyeing treatment and quantitative/qualitative analysis, wherein the dyeing treatment is that the dyeing treatment is performed on the zebra fish, on whichthe compound treatment is performed, for 0.5 to 1 hour at the temperature of 28 DEG C by taking JC-1 or DASPEI as a staining agent. The method covers the shortage that mitochondrial damage cannot be detected in vivo in the prior art by using an in vivo experimental analysis method for screening the mitochondria targeted compounds by using the zebra fish, is convenient, swift, economic and practical, and has the characteristics of high flux and high specificity.

Description

technical field [0001] The invention relates to the field of compound (chemical) detection, in particular to a simple, economical and high-throughput method for screening mitochondrial targeting compounds by using zebrafish. Background technique [0002] Mitochondria are important organelles in eukaryotic cells and participate in many important physiological and biochemical processes such as intracellular tricarboxylic acid cycle, fatty acid metabolism, and oxidative phosphorylation. Mitochondria play an important pivotal role in cell apoptosis. A series of metabolic processes in mitochondria are closely related to cell apoptosis, such as excessive production of oxygen free radicals, release of cytochrome C, and abnormal opening of mitochondrial membrane permeability transition pores. wait. The mitochondrial membrane has a variety of ion channels that can mediate ion transport, and the regulation of ion channels may affect the function of mitochondria and even cells. Mitoc...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N21/78G01N1/30G01N21/64
Inventor 彭恩泽
Owner 南京新环检测科技有限公司
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