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Method for screening DOX (doxorubicin)-cardiotoxicity-resistant active substances through two-color fluorescence labeling

An anti-doxorubicin, two-color fluorescence technology, applied in the direction of fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of single information, not conducive to comprehensive evaluation of the effect of compound cells, leakage of screening, etc., to reduce dosage, screening and evaluation The results are comprehensive and rigorous, and the effect of eliminating experimental errors

Inactive Publication Date: 2014-08-06
ZHEJIANG UNIV
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Problems solved by technology

[0007] The disadvantage of this method is that when single-color fluorescent labeling is used in drug screening research, the information obtained is single, which is not conducive to a comprehensive evaluation of the effect of compounds on cells, and there is a phenomenon of missing screening.
[0008] At present, there are no relevant reports introducing a high-content screening method for simultaneously labeling cells with multiple fluorescent dyes, and determining the degree of cell damage and the protective effect of drugs through cell fluorescence imaging

Method used

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  • Method for screening DOX (doxorubicin)-cardiotoxicity-resistant active substances through two-color fluorescence labeling
  • Method for screening DOX (doxorubicin)-cardiotoxicity-resistant active substances through two-color fluorescence labeling
  • Method for screening DOX (doxorubicin)-cardiotoxicity-resistant active substances through two-color fluorescence labeling

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Embodiment Construction

[0063] (1) FDA and Hoechst marker cell density linear inspection

[0064] H9c2 cells in the logarithmic growth phase were digested with a digestive solution containing 0.25% trypsin and 0.02% EDTA, and were treated with different cell densities (0, 10, 100, 500, 1000, 2000, 4000, 6000, 8000, 10000 per well) were planted in the middle 60 wells of a 96-well plate, and the periphery was replaced with PBS to avoid edge effects, and placed in a cell culture incubator for 24h, 48h, and 72h respectively; the culture medium in each well was discarded, protected from light, and respectively Single-color fluorescent labeling of cells with different culture time was performed with FDA at a concentration of 10 μg / mL and Hoechst at a concentration of 10 μg / mL, incubated in a cell culture incubator for 10 min, discarded the culture medium, washed once with PBS, blotted dry, and placed under a fluorescent inverted microscope Fluorescent images are read on the platform.

[0065] The fluoresc...

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Abstract

The invention discloses a method for screening DOX (doxorubicin)-cardiotoxicity-resistant active substances through two-color fluorescence labeling. The method comprises the following steps: myocardial cells are taken and added into to-be-screened substances for pre-protection; DOX is added for damage after the pre-protection is finished; a cell membrane integrity fluorescent probe and a cell nucleus fluorescent probe which have different excitation and emission wavelengths are added simultaneously into the damaged myocardial cells for fluorescence labeling; fluorescence images of the cells are collected and analyzed after dying is finished, and the protection rates of the to-be-screened substances for cell membranes and cell nucleuses of the myocardial cells are calculated; and the active substances whose protection rates for the cell membranes and cell nucleuses of the myocardial cells are all higher than 15% are screened out from the to-be-screened substances. Two fluorescent probes are adopted to label the myocardial cells simultaneously, the protection effects of the to-be-screened substances for the myocardial cells are surveyed from different action mechanisms, the screening and evaluation method has the characteristics of fastness, economy, high throughput and high content, and the screening and evaluation results are more comprehensive and precise.

Description

technical field [0001] The invention belongs to the field of drug screening and evaluation methods, and in particular relates to a method for screening anti-doxorubicin myocardial toxicity active substances with two-color fluorescent labels. Background technique [0002] Doxorubicin (DOX) is one of the broad-spectrum anti-tumor drugs commonly used in clinical practice, and is widely used in the treatment of solid tumors, leukemia, lymphoma, breast cancer and other tumors. However, doxorubicin will produce cumulative, dose-dependent myocardial toxicity, and further develop into irreversible myocardial damage, eventually leading to congestive heart failure, thus seriously inhibiting its clinical application. [0003] The mechanism of doxorubicin-induced cardiotoxicity has not been fully elucidated so far. It is mainly believed that doxorubicin-induced cardiotoxicity is the result of multiple factors, including free radical damage, apoptosis, mitochondrial damage, calcium ion o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 程翼宇赵筱萍王毅
Owner ZHEJIANG UNIV
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