Method for identifying differential marker of IC50 dose of vitamin C for RAW264.7 and K562 cells

A technology of RAW264.7 and vitamins, applied in the field of 1HNMR metabolomics analysis technology, can solve the problems of unclear biological significance of metabolite changes, unknown metabolite changes, and unknown biological significance, and achieve reliable data preprocessing Fast, easy-to-use effects

Active Publication Date: 2017-09-15
SHANDONG NORMAL UNIV
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Problems solved by technology

At present, there are literatures using capillary-time-of-flight mass spectrometry (CE-TOFMS) to explore the IC 50 Effects of dosed vitamin C on related metabolites in glycolysis, tricarboxylic acid cycle (TCA) and pentose phosphate pathway (PPP), but the biological significance of metabolite changes is unclear; and vitamin C-induced metabolism in cells The biological changes are unknown, and the biological significance is also unknown

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  • Method for identifying differential marker of IC50 dose of vitamin C for RAW264.7 and K562 cells
  • Method for identifying differential marker of IC50 dose of vitamin C for RAW264.7 and K562 cells
  • Method for identifying differential marker of IC50 dose of vitamin C for RAW264.7 and K562 cells

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preparation example Construction

[0084] (2) Preparation of cell extract:

[0085] Preparation of vitamin C group cell extract: RAW264.7 or K562 cells in the corresponding vitamin C IC 50 Cultivate at a certain dose, add cold methanol after culturing to quench cell metabolism, then use methanol chloroform water extraction method for ultrasonic crushing and extraction, and collect the water phase;

[0086] Preparation of the cell extract of the blank group: culture RAW264.7 or K562 cells, add cold methanol after culture to quench the cell metabolism, and then use methanol chloroform water extraction method for ultrasonic crushing and extraction, and collect the water phase;

[0087] In the process of preparing cell extracts, for the tissue structure and components of RAW264.7 or K562 cells, in order to ensure that as much metabolite information as possible can be obtained from vitamin C group cells and blank group cells, the extraction reagent is preferably selected It is a mixed solvent of methanol, chloroform ...

Embodiment 1

[0140] 1. Method principle

[0141] High-concentration doses of vitamin C will affect the metabolic level of cells after acting on cells, and change the types and concentrations of intracellular metabolites. 1 H NMR technology can identify the differential metabolites after vitamin C treatment and the control group, and then use metabolomics analysis technology to analyze the obtained data with PCA, PLS-DA, OPLS-DA and other techniques to find differential metabolism thing. Qualitative analysis was performed through the Human Metabolome Database (HMDB) and the Biological Nuclear Magnetic Resonance Database (BMRB) to determine the species names of the differential metabolites. The identified differential metabolites were imported into the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and MetaboAnalyst (MetPA) for pathway enrichment analysis.

[0142] 2. Instruments and materials

[0143] Instruments and equipment:

[0144] Stat Fax-2000 microplate reader American ...

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Abstract

The invention discloses a method for identifying a differential marker of IC50 dose of vitamin C for RAW264.7 and K562 cells by applying 1H NMR technology and metabonomic methodology. The method comprises the following steps: (1) determining the IC50 dosage of vitamin C for RAW264.7 or K562 cells; (2) preparing a cell extract; (3) performing nuclear magnetic resonance pretreatment; (4) performing 1H nuclear magnetic resonance detection; (5) screening differential metabolites; and (6) performing metabolism pathway analysis. The method studies influence of different cell differential metabolites and metabolic pathway by applying H-nuclear magnetic resonance technology in combination with metabonomic methodology, and the basic research provides theoretical basis for studying the method for analyzing sub-toxicity of vitamin C for RAW264.7 or K562 cells.

Description

technical field [0001] The present invention relates to the identification of IC by metabolomics technology using nuclear magnetic resonance 50 Method of dosing vitamin C on differential markers of RAW264.7 and K562 cells, belonging to 1 Metabolomics analysis technique of H NMR. Background technique [0002] Metabolomics: [0003] Metabolomics is an analytical technique for identifying and quantifying small-molecule metabolites. It uses modern ultra-high performance liquid chromatography / high-resolution mass spectrometry to analyze the composition profile of metabolites in body fluids. Technology to integrate information, compare and analyze biological differential metabolites at the system and overall level. [0004] At present, the commonly used detection instruments include high performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), nuclear magnetic resonance (NMR), infrared spectroscopy (IR), etc., among which NMR technology has hig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/82G01N33/569G01N33/574G01N33/50G01N24/08G01N1/28G01N1/40G06F19/24
CPCG01N1/28G01N1/4055G01N1/4077G01N24/088G01N33/5014G01N33/505G01N33/5055G01N33/56966G01N33/57426G01N33/82G01N2001/4061G01N2001/4094G01N2570/00G16B40/00
Inventor 耿越李慧陆洋
Owner SHANDONG NORMAL UNIV
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