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Oxidized fraction of extracellular dna as a biomarker of stress and methods for using the same

A cellular, oxidative technology, applied in the direction of biochemical equipment and methods, extracellular fluid diseases, chemical instruments and methods, etc.

Inactive Publication Date: 2016-08-10
安娜·巴兰诺娃 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, damaged DNA released from irradiated cells may cause distant effects that are suspected to depend on the actions of the immune system, especially those mediated by TLRs

Method used

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  • Oxidized fraction of extracellular dna as a biomarker of stress and methods for using the same
  • Oxidized fraction of extracellular dna as a biomarker of stress and methods for using the same
  • Oxidized fraction of extracellular dna as a biomarker of stress and methods for using the same

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0133] cell culture

[0134] ER / PR-positive MCF-7 breast cancer cells were purchased at ATCC, Manassas, USA (catalogue number: HTB-22). Human embryonic lung fibroblasts were retrieved from the biological sample collection maintained by the Research Center for Medical Genetics (Russian Academy of Medical Sciences collection) and grown as described in [7]. Ethical approval for the use of primary human cells was obtained from the Committee for Medical and Health Research Ethics of Research Center for Medical Genetics, Russian Academy of Medical Sciences (2012, approval number 5).

[0135] MCF-7 cells were cultured in DMEM medium supplemented with 10% (v / v) fetal calf serum, 2 mM L-glutamine, 100 units / mL penicillin, and 100 μg / mL streptomycin. cells in a 5% CO 2 Grow at 37 °C under a humidified atmosphere of air. Cells were grown in slide flasks for 24h or 72h before treatment with DNA probes.

example 2

[0137] Flow Cytometry

[0138] Before flow cytometry, cells were washed in EDTA solution and then treated with 0.25% trypsin under controlled light microscopy. Cells were transferred to Eppendorf tubes, washed with medium, then centrifuged and resuspended in PBS. Cell staining using various antibodies was performed as follows. Briefly, to fix cells, paraformaldehyde (Sigma) was added at a final concentration of 2% for 10 minutes at 37°C. Cells were washed three times with 0.5% BSA-PBS and permeabilized with 0.1% Triton X-100 (Sigma) in PBS or with 70% ethanol for 15 minutes at 4°C. Cells (approximately 50×10 3 ) were washed three times with 0.5% BSA-PBS and washed with 1-2 μg / mL FITC-γH2AX (Ser139) antibody (Temecula California), FITC-Ki-67 antibody, PCNA, 8 -oxodG, EEA1, AIM2, TLR9, NRF2, NF-κB (p65), S529NF-κB (p65) and STAT3 antibody (Abcam) were stained for 3h, then washed again three times with 0.5% BSA-PBS, and Staining was performed with 1 μg / mL of a secondary FITC...

example 3

[0142] fluorescence microscope

[0143] Cell images were acquired using an AxioScope Al microscope (Carl Zeiss).

[0144] Immunocytochemistry.

[0145] MCF-7 cells were fixed in 3% formaldehyde (4°C) for 20 minutes, washed with PBS and then permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 0.5% BSA in PBS 1 h, and incubated overnight at 4°C with FITC-γH2AX (Ser139), 8-oxodG, NRF2, STAT3, NF-κB (p65), AIM2 antibodies. After washing with 0.01% Triton X-100 in PBS, MCF-7 cells were incubated with FITC / PE goat anti-mouse IgG for 2 h at room temperature, washed with PBS and then stained with DAPI.

[0146] Intracellular localization of labeled DNA fragments.

[0147] gDNA 红 , gDNA 红-氧化 and pBR322 绿 The labeled fraction (50 ng / ml) was added to the culture medium for 30 minutes. Cells were washed three times with PBS, fixed in 3% paraformaldehyde (4°C) for 20 minutes, washed with PBS and stained with 2 μg / mL DAPI. To analyze the...

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Abstract

The present invention relates to methods of treating and diagnosing oxidative damage in a subject comprising administering an agent that binds oxidized extracellular nucleic acid, and methods of treating diseases and conditions in a subject comprising administering an adjuvant therapy comprising an agent that binds oxidized extracellular nucleic acid. The oxidized fraction of extracellular DNA can also be detected through electrochemical methods or by mass- spectrometry.

Description

technical field [0001] The present invention relates generally to the field of redox biology. In particular, the present invention relates to the use of isolated oxidized extracellular DNA moieties in body fluids as biomarkers of stress in humans, and to the use of agents such as antibodies or fragments thereof that bind to the oxidized extracellular DNA moieties for diagnosis and Methods of treating diseases and conditions. Oxidized extracellular DNA fractions can also be detected electrochemically or by mass spectrometry. Background technique [0002] Many chronic diseases are accompanied by increased overall oxidation of genomic DNA. Under oxidative stress, DNA bases are susceptible to oxidation, the most common products of which are thymidine glycol and 8-hydroxy-2′-deoxyguanosine (8-oxodG). In fact, the 8-oxodG is the most widely used "marker" of oxidative DNA damage. This 8-oxodG is formed in DNA by direct oxidation, or can be incorporated into DNA by DNA polymeras...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/44
CPCC12Q1/6883C12Q1/6886C12Q2600/142A61P13/12A61P19/02A61P25/00A61P25/16A61P25/18A61P25/28A61P27/02A61P29/00A61P35/00A61P3/06A61P43/00A61P7/06A61P9/00A61P9/02A61P9/10A61P9/12A61P3/10C07K16/44C12Q1/6876C12Q2600/112
Inventor 安娜·巴兰诺娃斯韦特兰娜·科斯特约克纳塔利娅·韦科
Owner 安娜·巴兰诺娃
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