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A kind of hyperuricemia hepatocyte model and its construction method

A hyperuricemia and construction method technology, which is applied in the field of hyperuricemia hepatic cell (LO2) model and its construction, can solve the problem of establishing a hyperuricemia model with hepatic cells that has not yet been seen, and achieves a short time-consuming, The effect of good repeatability and simple operation

Active Publication Date: 2019-12-10
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Before this application was filed, there was no report on the establishment of a hyperuricemia model using liver cells

Method used

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  • A kind of hyperuricemia hepatocyte model and its construction method
  • A kind of hyperuricemia hepatocyte model and its construction method
  • A kind of hyperuricemia hepatocyte model and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Construction of a liver cell model of hyperuricemia: This experiment was set as a blank control group and a model group.

[0042] Model group: (1) Hepatocytes in the logarithmic growth phase were taken, digested with trypsin, and blown with a pipette to make a cell suspension. After counting on a hemocytometer, the cell concentration was adjusted to 10 by dilution. 4 cells / mL, mix well, inoculate in 24-well plate, place at 37°C, 5% (V / V) CO 2 cultured in a cell culture incubator;

[0043](2) After 12 hours, take out the 24-well plate, discard the cell supernatant, wash twice with PBS, add the freshly prepared adenosine solution prepared with the basal medium without fetal bovine serum as the solvent, adenosine The dosage is 0.5 mmol / L, and placed in the cell culture incubator to continue incubation for 12 h;

[0044] (3) Take out the 24-well plate, add 0.2 IU xanthine oxidase to each well, place it in a cell culture incubator and continue to incubate for 12 hours to o...

Embodiment 2

[0049] Construction of a hyperuricemia cell model:

[0050] (1) Digest the hepatocytes in the logarithmic growth phase with trypsin, blow with a pipette to make a cell suspension, and dilute the cell density to 10 after counting on a hemocytometer 5 cells / mL, mix well, inoculate in 24-well plate, place at 37 °C, 5% (V / V) CO 2 cultured in a cell culture incubator;

[0051] (2) After 12 hours, take out the 24-well plate, discard the cell supernatant, wash with PBS for 3 times, add the adenosine solution prepared by using the basal medium without fetal bovine serum as the solvent, and adenosine The dosage of glycosides was 1 mmol / L, and the incubation was continued for 18 h in the cell culture incubator;

[0052] (3) Take out the 24-well plate, add 0.2 IU xanthine oxidase to each well, place it in a cell culture incubator and continue to incubate for 12 hours to obtain a hyperuricemia hepatocyte model.

[0053] The cell supernatant was taken out, and its uric acid content was ...

Embodiment 3

[0055] Construction of a hyperuricemia cell model:

[0056] (1) Digest the hepatocytes in the logarithmic growth phase with trypsin, blow with a pipette to make a cell suspension, and dilute the cell density to 10 after counting on a hemocytometer 5 cells / mL, mix well, inoculate in 24-well plate, place at 37 °C, 5% (V / V) CO 2 cultured in a cell culture incubator;

[0057] (2) After 12 hours, the cell supernatant was discarded, washed twice with PBS, and the freshly prepared adenosine solution prepared with the basal medium without fetal bovine serum as the solvent was added. The dosage of adenosine was 1 mmol / L, placed in the cell culture incubator for 24 h;

[0058] (3) Take out 24 wells of the plate, add 0.3 IU xanthine oxidase to each well, place them in a cell culture incubator and continue to incubate for 18 hours to obtain a hyperuricemia liver cell model.

[0059] The cell supernatant was taken out, and its uric acid content was analyzed by high performance liquid ph...

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Abstract

The invention discloses a hyperuricemia hepatocyte model and a construction method of the hyperuricemia hepatocyte model. The construction method comprises the following steps: (1) carrying out trypsin digestion on hepatocytes (LO2) in the logarithmic phase, thus preparing a cell suspension, after counting by adopting a blood counting chamber, diluting the hepatocytes till the concentration is 10<4>-10<6>pic / mL, and after paving on a board, carrying out culturing in a 5% (V / V) of CO2 cell culture box at the temperature of 37 DEG C; (2) after 12-24 h, taking out the pore board, adding the solution of adenosine with the use amount being 0.5-2 mmol / L, carrying out further incubation for 12-24 h; (3) adding 0.2-0.4 IU of xanthine oxidase into each hole, and carrying out further incubation for 12-24 h, thus obtaining the hyperuricemia hepatocyte model. The experimental result shows that the optimal density of cell pavement on the board is 10<6>pic / mL, and the optimal use mount of the inductor, namely, adenosine is 1.5 mmol / L. The obtained hyperuricemia hepatocyte model can be used for screening medicines for reducing uric acid, and has the advantages that the operation is simple, the needed time is short, and the efficiency is high compared with the hyperuricemia animal model.

Description

technical field [0001] The invention belongs to the technical field of cell model construction, in particular to a hyperuricemia hepatocyte (LO 2 ) model and its construction method. Background technique [0002] Uric acid is a metabolite of endogenous purines and dietary purines. In the process of purine metabolism in the human body, ATP and others undergo a series of reactions to generate hypoxanthine and xanthine, and uric acid is generated under the action of xanthine oxidase. When purine metabolism is disordered, the uric acid content in the human body will increase. It is generally believed that when the blood urate level is ≥ 417 μmol / L, hyperuricemia will be induced. In recent years, the incidence of hyperuricemia has been increasing year by year, and the age of onset is getting younger. At the same time, hyperuricemia is closely related to hypertension, hyperlipidemia, and diabetes. Therefore, the research on the pathogenesis of hyperuricemia and anti-gout drugs i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12Q1/02
CPCC12N5/067G01N33/5008
Inventor 任娇艳刘丹
Owner SOUTH CHINA UNIV OF TECH
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