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A method for evaluating oxidative stress in vivo based on detection of oxidative damage to mitochondrial DNA in peripheral blood

A technology for oxidative damage and mitochondria, applied in the field of molecular diagnosis, can solve the problems of low sensitivity, difficult promotion and application, and high cost

Active Publication Date: 2017-09-29
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently commonly used detection methods mainly include: 1) use spectrophotometry to detect MDA, SOD, CAT, reduced / oxidized GSH, GPx, etc., the method is simple and relatively cheap, but poor in stability; 2) use ELISA to detect MDA, HNE, 8-OHdG, etc., this method is more reliable, but the cost is relatively high
In addition, high-performance liquid chromatography-electrochemical (HPLC-ECD) and gas chromatography-mass spectrometry (GC-MS) can be used for the detection of 8-OHdG levels, but due to the need for related instruments, cumbersome operations, and low sensitivity, etc., Difficult to popularize and apply

Method used

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  • A method for evaluating oxidative stress in vivo based on detection of oxidative damage to mitochondrial DNA in peripheral blood
  • A method for evaluating oxidative stress in vivo based on detection of oxidative damage to mitochondrial DNA in peripheral blood
  • A method for evaluating oxidative stress in vivo based on detection of oxidative damage to mitochondrial DNA in peripheral blood

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Primer Design

[0039]According to the standard sequence of human mtDNA (NC_012920) in the NCBI gene bank, primers were first designed for the mtDNA D-loop region (displacement loop). The D-loop region contains the replication initiation region for mtDNA heavy chain synthesis, is the control region for mtDNA replication, and is also the binding site for mtDNA and mitochondrial membranes. It is susceptible to lipid peroxides and is a high-incidence region for mtDNA mutations. The base mutation rate is 6-8 times higher than other regions. The D-loop region includes three hypervariable regions, hypervariable region I (np16024-16383), hypervariable region II (np57-372) and hypervariable region III (np438-574). Therefore, in order to ensure the specificity of amplification, these three hypervariable regions should be avoided when designing primers.

[0040] The fragment amplified by the DLP1 primer shown in Table 1 does not cover the hypervariable region, while t...

Embodiment 2

[0044] Example 2 Construction of cell model

[0045] HUEVC cell culture: RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin-streptomycin solution, 37°C, 5% CO 2 Cultivation; after the cells grow to the full of the culture dish, they are subcultured. When subcultured, wash the cells twice with PBS, digest with trypsin at 37°C for 1-2 minutes, add 1640 medium and blow the cells to make them fall off, collect the cells, wash the cells twice with PBS, and continue to cultivate;

[0046] Oxidative stress cell model: add a certain amount of H 2 o 2 , so that the final concentration is 50, 100, 150, 200μM; antioxidant group: 200μM H 2 o 2 and antioxidant MtioQ, the final concentrations were 100, 200, 300nM; except H 2 o 2 , HUEVC cells under the same culture conditions as the control group. Cells were collected after 2 h of action, and after the protein concentration was measured by BCA method, MDA (malondialdehyde), SOD (superoxide dismutase), CAT (catalase), G...

Embodiment 3

[0050] Example 3 Detection of Oxidative Damage Levels of Intracellular and Extracellular mtDNA

[0051] Extract various H 2 o 2 Cell DNA treated with MitoQ concentration, the cell supernatant free DNA was extracted by magnetic bead method. A certain amount of DNA and hOGG were bathed in water at 137°C for 2h. The same amount of DNA was used as a control without adding hOGG1, and other conditions were the same. Then use the above 5 pairs of primers to carry out qPCR reaction respectively, and calculate the difference of Ct value between the DNA samples from the same source with and without hOGG1, so as to reflect the content of 8-OH-dG, and indirectly reflect the level of DNA oxidative damage .

[0052] Preparation of hOGG1 enzyme digestion reaction system

[0053]

[0054] Mix well, 37°C water bath for 2h, 65°C enzyme inactivation 15mim

[0055] Fluorescent quantitative PCR

[0056] 1) Prepare PCR reaction system

[0057]

[0058] 2) PCR reaction conditions:

[...

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Abstract

The invention provides an application of a reagent capable of detecting peripheral blood mitochondria DNA oxidative damage in preparation of a reagent for evaluating in-vivo oxidation stress, and a method for evaluating in-vivo oxidation stress based on detection of peripheral blood mitochondria DNA oxidative damage.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis. Specifically, the present invention provides the application of the reagent for detecting oxidative damage of mitochondrial DNA in peripheral blood to the preparation of reagents for evaluating oxidative stress in vivo, and a method for evaluating oxidative stress in vivo based on detecting oxidative damage of mitochondrial DNA in peripheral blood. Background technique [0002] Oxidative stress refers to excessive production of reactive oxygen species (ROS) or reactive nitrogen species (RNS) in the body and / or weakening of the body's antioxidant capacity and insufficient removal of ROS or RNS, resulting in increased ROS or RNS in the body and damage It disrupts the normal imbalance of oxidation / reduction in the body, leading to the pathological process of oxidative damage to tissues and cells. Therefore, evaluating oxidative stress in vivo is of great significance in disease diagnosi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q1/6888
Inventor 叶薇刘楚汤晓君吕建新
Owner WENZHOU MEDICAL UNIV
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