CN106190954B - Hyperuricemia hepatocyte model and construction method thereof - Google Patents
Hyperuricemia hepatocyte model and construction method thereof Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
Abstract
The invention discloses a hyperuricemia hepatocyte model and a construction method thereof, wherein the construction method comprises the following steps of (1) digesting hepatocytes (LO 2) in logarithmic growth phase by trypsin to prepare a cell suspension, diluting the hepatocytes to 10 4 -10 6/mL after counting by a blood counting plate, placing the cells in a 37 ℃ and 5% (V/V) CO 2 cell incubator for culture after plating, 2) taking out a pore plate after 12-24 h, adding a solution of adenosine with the dosage of 0.5-2 mmol/L, and continuing to incubate for 12-24 h, and 3) adding 0.2-0.4 xanthine oxidase into each IU and continuing to incubate for 12-24 h to obtain the hyperuricemia hepatocyte model, and the experimental result shows that the optimal density of the cell plating plate is 10 6/mL, the optimal dosage of adenosine is 1.5 mmol/L.
Description
Technical Field
The invention belongs to the technical field of cell model construction, and particularly relates to a hyperuricemia hepatocyte (LO 2) model and a construction method thereof.
Background
uric acid is a metabolite of endogenous purines and dietary purines. In the purine metabolism process of human body, ATP and the like 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 disturbed, uric acid content in a human body is increased, and it is generally considered that hyperuricemia is induced when the blood urate level is more than or equal to 417 mu mol/L. In recent years, the incidence of hyperuricemia has increased year by year, the age of onset has become low, and hyperuricemia is closely related to hypertension, hyperlipidemia, diabetes and the like, so that research on the pathogenesis of hyperuricemia and anti-gout drugs has become a focus of the medical community.
The currently clinically used drugs for treating gout can be classified into drugs for inhibiting uric acid production and drugs for promoting uric acid excretion according to their mechanisms of action. Most drugs that inhibit the production of uric acid (e.g., allopurinol) inhibit the conversion of hypoxanthine and xanthine into uric acid by inhibiting the activity of xanthine oxidase, thereby reducing the production of uric acid. Common drugs for promoting uric acid excretion are probenecid and benzbromarone. Although the medicines can effectively reduce the content of uric acid in a human body, the medicines have large toxic and side effects on the human body and certain damage to liver and kidney, and the clinical application of the medicines is limited to a certain extent. Therefore, a novel uric acid reducing drug with high efficiency, low toxicity and wider applicability is urgently needed clinically, and the establishment of a hyperuricemia model is a precondition for researching hyperuricemia. At present, hyperuricemia animal models are adopted at home and abroad to screen the uric acid reducing drugs and research the action mechanism of the uric acid reducing drugs.
Patent application publication No. CN1698906 discloses a method for copying a rat acute or persistent hyperuricemia model, which has a publication date of 2005-11-23 and is filed by Nanjing university of traditional Chinese medicine. The method is characterized in that SD and Wistar healthy rats are subjected to intragastric administration, subcutaneous injection and intraperitoneal injection, 100-2000 mg/kg of purine (hypoxanthine and xanthine) and 5-1000 mg/kg of uricase inhibitor (potassium oxonate) are simultaneously administered, the rats are administered by the same method and the drug dosage every 5-24 hours, and the administration lasts for one to two weeks, so that a stable acute or persistent rat hyperuricemia model can be obtained.
patent application publication No. CN1698907 discloses a method for copying a mouse acute hyperuricemia model, publication date is 2005-11-23, and applicant is Nanjing university of traditional Chinese medicine. The patent is that hypoxanthine or xanthine 100-1500 mg/kg and uricase inhibitor potassium oxonate 10-1500 mg/kg are given to Kunming and ICR healthy mice by means of gavage, subcutaneous injection or intraperitoneal injection, and experiments prove that a stable mouse hyperuricemia model can be obtained after 1 hour by using uricase inhibitor to match with purines.
Patent application publication No. CN103977007A discloses a method for constructing a tree shrew model for acute hyperuricemia, the publication date is 2014-08-13, and the applicant is the institute of medical biology of Chinese academy of medicine sciences. The patent is that an adult healthy tree shrew of 1-3 years old is injected with oteracil potassium suspension in the abdominal cavity, the dosage is 40-100 mg/kg according to the weight of the tree shrew, and the tree shrew model with acute hyperuricemia can be obtained.
Patent application publication No. CN104388467A discloses "a method for constructing a model of spontaneous hyperuricemia and its use", publication date 2015-03-04, applicant is expensive in Li. According to the patent, the uricase gene of a target mouse is knocked out by using transcription activator-like effector nuclease to knock out the gene, so that a spontaneous hyperuricemia mouse model is obtained, the survival time of the mouse is long, and long-term experiments on the mouse are facilitated.
Patent application publication No. 103860562A discloses "a pharmaceutical composition for establishing hyperuricemia animal model and application thereof", publication date 2014-06-18, applicant Zeng. The patent adopts ethambutol and uric acid for molding, the molding animals are rhesus monkeys, the molding time is short, the model is stable, the success rate is high, and a stable and reliable acute hyperuricemia model can be obtained.
As described above, many studies on hyperuricemia have been conducted to construct a hyperuricemia model by injecting purine compounds, uricase inhibitors, and the like, based on animal models. However, existing animal models have many imperfect places, such as complicated operation and long experimental period, which bring great inconvenience to gout research, and although the commonly used model animals such as mice and rats belong to the same genus of mammals as human beings, the model animals have far different classification status, belong to lower rodent animals, have far different anatomical and physiological statuses from human beings, and have certain limitation on the data referential property obtained by the animal models.
the liver is a main place for purine metabolism to generate uric acid, liver cells (LO 2) are selected as modeling cells to construct a hyperuricemia cell model, and the model can be used for rapid screening of uric acid-reducing drugs, and is simple in operation, short in time consumption and high in efficiency compared with an animal model.
Before the present application, no report on the establishment of a hyperuricemia model using hepatocytes has been found.
Disclosure of Invention
In view of the requirement of developing a hyperuricemia model for rapidly screening uric acid lowering drugs, the invention provides a hyperuricemia hepatocyte model and a construction method thereof by taking hepatocytes as modeling cells.
A method for constructing a hyperuricemia hepatocyte model comprises the following steps:
(1) Selecting hepatic cells (LO 2) in logarithmic growth phase, digesting, making into cell suspension, counting by a blood counting plate, diluting the cell suspension, mixing uniformly, adding into a pore plate, and culturing in a cell culture box;
(2) After plating for 12-24 h, taking out the pore plate, removing cell supernatant, cleaning, adding an inducer adenosine, and placing in a cell culture box for continuous culture for 12-24 h;
(3) taking out the pore plate from the cell culture box, adding xanthine oxidase, placing in the cell culture box, and continuously incubating for 12-24 h to obtain a hyperuricemia liver cell model; and (3) determining the content of uric acid by adopting a reverse high-performance liquid phase.
further, the digestion in step (1) is carried out by using trypsin; and blowing and beating the cells on the bottle wall by using a suction pipe to prepare cell suspension.
Further, in step (1), after the cell suspension is diluted, the concentration of the cells is 10 4 -10 6 cells/mL, preferably 10 6 cells/mL.
Further, the washing in the step (2) refers to washing with polybutylene succinate (PBS) for 2-3 times.
Further, the dosage of the adenosine in the step (2) is 0.5-2 mmol/L, and preferably 1.5 mmol/L; adenosine is added in the form of adenosine solution, and the adenosine solution is prepared by taking a basic culture medium without fetal calf serum as a solvent, and is used as it is and mixed when in use.
Further, in the step (3), the xanthine oxidase is added in an amount of 0.2-0.4 IU per well, preferably 0.3 IU per well.
A hyperuricemia hepatocyte model prepared by any one of the preparation methods.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) The invention establishes the hyperuricemia cell model by utilizing the liver cells (LO 2) for the first time, and has the advantages of simple operation, effectiveness, simplicity, convenience, sensitivity, good repeatability and shorter time consumption.
(2) the method can be used for screening and developing the medicament for reducing the uric acid by establishing the hyperuricemia cell model, and has very important significance for evaluating the drug effect and action mechanism of the relevant anti-hyperuricemia medicament on the hyperuricemia disease.
Drawings
FIG. 1 is a schematic diagram of the uric acid metabolic pathway;
FIG. 2 shows uric acid standard yeast;
FIG. 3 is a high performance liquid phase diagram of the cell fluid of the white control group and the model group in example 1;
FIG. 4 is a diagram showing the uric acid concentration in the supernatant of model group cells according to various embodiments;
FIG. 5 is a graph showing the concentrations of uric acid in cell supernatants of the blank control group, the model group, and the positive control group in example 6.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments and drawings.
The experimental methods used in the following examples are all conventional methods unless otherwise specified.
The material reagents and the like used in the following modes are commercially available unless otherwise specified.
1. experimental cell, hepatocyte LO 2, from university of Guang Chinese medicine
2. molding medicine: adenosine, available from Guangzhou Shuicheng Biotech Ltd
3. An experimental instrument: high performance liquid chromatography, available from Shimadzu corporation of Japan
The hepatocyte LO 2 was cultured in DMEM medium, 10 wt% fetal bovine serum and 1 wt% double antibody were added, and the mixture was placed in a 37 ℃ and 5% (V/V) CO 2 incubator for static culture.
FIG. 1 shows the uric acid metabolic pathway, and it is understood from FIG. 1 that adenosine produces inosine and hypoxanthine by the action of adenosine deaminase and purine nucleoside phosphorylase, and then produces xanthine and uric acid by the action of xanthine oxidase.
FIG. 2 shows uric acid standard koji, and the amount of uric acid produced can be quantified based on the uric acid standard koji shown in FIG. 2.
Example 1
The construction of a hyperuricemia hepatocyte model comprises the following steps: the experiment was set as a blank control group and a model group.
Taking hepatic cells in logarithmic phase, digesting with trypsin, blowing and beating with a suction tube to prepare cell suspension, counting by a blood counting plate, adjusting the cell concentration to 10 4/mL by dilution, mixing uniformly, inoculating into a 24-pore plate, and culturing in a 37 ℃ and 5% (V/V) CO 2 cell culture box;
(2) after 12 h, taking out the 24-hole plate, discarding cell supernatant, washing with PBS for 2 times, adding a prepared adenosine solution which is prepared by taking a basal culture medium without fetal calf serum as a solvent, wherein the dosage of the adenosine is 0.5 mmol/L, and placing the adenosine solution in a cell culture box for continuous incubation for 12 h;
(3) And taking out the 24-hole plate, adding 0.2 IU of xanthine oxidase into each hole, and placing the plate in a cell culture box for continuous incubation for 12 hours to obtain the hyperuricemia hepatocyte model.
Blank control group: the medium was added without adenosine solution, and the rest was the same as the model group.
Taking out cell supernatant, and analyzing uric acid content with high performance liquid chromatography, wherein the high performance liquid phase diagram and the uric acid content diagram are shown in FIG. 3 and FIG. 4, respectively.
From fig. 3, it can be seen that the blank control group does not produce uric acid, and the model group produces uric acid, further verifying that adenosine in fig. 1 can finally produce uric acid through the action of a series of enzymes.
example 2
Construction of a hyperuricemia cell model:
(1) Subjecting hepatocytes in logarithmic growth phase to pancreatin digestion, blowing and beating by a suction pipe to prepare cell suspension, counting by a blood counting plate, diluting the cell density to 10 5 cells/mL, uniformly mixing, inoculating into a 24-well plate, and culturing in a 37 ℃ and 5% (V/V) CO 2 cell culture box;
(2) after 12 h, taking out the 24-hole plate, discarding cell supernatant, washing with PBS for 3 times, adding prepared adenosine solution prepared by taking a basal culture medium without fetal calf serum as a solvent, wherein the dosage of the adenosine is 1 mmol/L, and placing the adenosine solution in a cell culture box for continuous incubation for 18 h;
(3) And taking out the 24-hole plate, adding 0.2 IU of xanthine oxidase into each hole, and placing the plate in a cell culture box for continuous incubation for 12 hours to obtain the hyperuricemia hepatocyte model.
The cell supernatant was removed and analyzed for uric acid content in HPLC, the results are shown in FIG. 4.
Example 3
Construction of a hyperuricemia cell model:
(1) Subjecting hepatocytes in logarithmic growth phase to pancreatin digestion, blowing and beating by a suction pipe to prepare cell suspension, counting by a blood counting plate, diluting the cell density to 10 5 cells/mL, uniformly mixing, inoculating into a 24-well plate, and culturing in a 37 ℃ and 5% (V/V) CO 2 cell culture box;
(2) After 12 h, removing cell supernatant, washing with PBS for 2 times, adding prepared adenosine solution prepared by taking a basal culture medium without fetal calf serum as a solvent, wherein the dosage of adenosine is 1 mmol/L, and placing the solution in a cell culture box for continuous incubation for 24 h;
(3) And taking out 24 plate holes, adding 0.3 IU xanthine oxidase into each hole, placing the holes in a cell culture box, and continuously incubating for 18 hours to obtain the hyperuricemia hepatocyte model.
the cell supernatant was removed and analyzed for uric acid content in HPLC, the results are shown in FIG. 4.
Example 4
Construction of a hyperuricemia cell model:
(1) subjecting hepatocytes in logarithmic growth phase to pancreatin digestion, blowing and beating by a suction pipe to prepare cell suspension, counting by a blood counting plate, diluting the cell density to 10 6 cells/mL, uniformly mixing, inoculating into a 24-well plate, and culturing in a 37 ℃ and 5% (V/V) CO 2 cell culture box;
(2) After 18 h, removing cell supernatant, washing with PBS for 3 times, adding prepared adenosine solution prepared by taking a basal culture medium without fetal calf serum as a solvent, wherein the dosage of adenosine is 1.5mmol/L, and placing the solution in a cell culture box for continuous incubation for 24 h;
(3) And taking out 24 plate holes, adding 0.3 IU xanthine oxidase into each hole, placing the holes in a cell culture box, and continuously incubating for 24 hours to obtain the hyperuricemia hepatocyte model.
The cell supernatant was removed and analyzed for uric acid content in HPLC, the results are shown in FIG. 4.
Example 5
Construction of a hyperuricemia cell model:
(1) subjecting hepatocytes in logarithmic growth phase to pancreatin digestion, blowing and beating by a suction pipe to prepare cell suspension, counting by a blood counting plate, diluting the cell density to 10 6 cells/mL, uniformly mixing, inoculating into a 24-well plate, and culturing in a 37 ℃ and 5% (V/V) CO 2 cell culture box;
(2) After 24 h, taking out the 24-well hole, removing cell supernatant, washing with PBS for 3 times, adding an adenosine solution prepared by taking a basal culture medium without fetal calf serum as a solvent, wherein the dosage of the adenosine is 2 mmol/L, and placing the adenosine solution in a cell culture box for continuous incubation for 24 h;
(3) And taking out 24 plate holes, adding 0.4 IU xanthine oxidase into each hole, placing the holes in a cell culture box, and continuously incubating for 24 hours to obtain the hyperuricemia hepatocyte model.
The cell supernatant was removed and analyzed for uric acid content in HPLC, the results are shown in FIG. 4.
As can be seen from fig. 4, all of the model groups produced uric acid. As can be seen from examples 1 and 2, uric acid production increased significantly with cell plating density and prolonged incubation time with inducer adenosine; it is understood from examples 2 and 3 that the production of uric acid increases with an increase in the incubation time of the inducer adenosine and the incubation time after the addition of xanthine oxidase; it can be seen from examples 3, 4 and 5 that although the uric acid content produced by the model group increased with the increase of the concentration of the inducer adenosine, when the adenosine concentration increased to 1.5mmol/L, the concentration of the inducer adenosine was further increased, and the uric acid content did not increase significantly because the amount of adenosine converted was constant for a certain period of time and cell density, and thus the final uric acid content did not increase significantly.
As can be seen from fig. 3 and 4, the hyperuricemia hepatocyte model was successfully modeled.
Example 6
experiments were set as blank control, model group and positive control.
Digesting hepatic cells in logarithmic phase by trypsin, blowing and beating the hepatic cells into cell suspension by a suction tube, counting by a blood counting plate, diluting the cell density to 10 6 cells/mL, uniformly mixing, inoculating the cell suspension into a 24-pore plate, wherein each pore is 1 mL, and culturing in a 37 ℃ and 5% (V/V) CO 2 incubator;
(2) after 18 h, taking out the 24-hole plate, removing cell supernatant in the 24-hole plate, adding PBS (phosphate buffer solution) for cleaning for 3 times, adding prepared adenosine solution which is prepared by taking a basal culture medium without fetal calf serum as a solvent into all the holes, wherein the dosage of the adenosine is 1.5mmol/L, and placing the holes in a cell culture box for continuous culture for 24 h;
(3) Taking out the plate holes, adding 0.3 IU xanthine oxidase into each hole, placing the plate holes in a cell culture box, and continuously incubating for 24 hours to obtain the hyperuricemia hepatocyte model.
Blank control group: medium was added without adenosine, and the conditions were the same as for the model group.
positive control group: probenecid and febuxostat were added in an amount of 10. mu. mol/L, respectively, and the other conditions were the same as in the model group.
Taking out cell supernatant, and measuring uric acid content in the cell supernatant by high performance liquid chromatography, wherein the uric acid content is shown in figure 5.
As can be seen from FIG. 5, the content of uric acid generated after adding the positive drugs probenecid and febuxostat is remarkably reduced compared with the model group. The hyperuricemia hepatocyte model has the efficacy of uric acid reduction evaluation, and can be further used for screening uric acid reduction medicines.
Claims (7)
1. A method for constructing a hyperuricemia hepatocyte model is characterized by comprising the following steps:
(1) Selecting hepatic cells in logarithmic growth phase, digesting to prepare cell suspension, counting by a blood counting chamber, diluting the cell suspension, uniformly mixing, adding into a pore plate, and culturing in a cell culture box, wherein the concentration of the cells after the cell suspension is diluted is 10 4 -10 6 cells/mL;
(2) After plating for 12-24 h, taking out the pore plate, discarding cell supernatant, cleaning, adding an inducer adenosine, and placing in a cell culture box for continuous incubation for 12-24 h; the dosage of the adenosine is 0.5-2 mmol/L, the adenosine is added in the form of adenosine solution, the adenosine solution is prepared by taking a basal culture medium without fetal calf serum as a solvent, the adenosine solution is used as it is and is mixed evenly when in use;
(3) taking out the pore plate, adding xanthine oxidase 0.2-0.4 IU per pore, placing in a cell culture box, and incubating for 12-24 hr to obtain hyperuricemia hepatocyte model; and (3) determining the content of uric acid by adopting a reverse high-performance liquid phase.
2. The method for constructing a hyperuricemia hepatocyte model according to claim 1, wherein in the step (1), the digestion is performed by using trypsin; and blowing and beating the cells on the bottle wall by using a suction pipe to prepare cell suspension.
3. the method according to claim 1, wherein in step (1), the concentration of the cells after the cell suspension is diluted is 10 6 cells/mL.
4. The method for constructing a hyperuricemia hepatocyte model according to claim 1, wherein in the step (2), the washing is performed 2-3 times by using polybutylene succinate.
5. the method for constructing a hyperuricemia hepatocyte model according to claim 1, wherein in the step (2), the adenosine is used in an amount of 1.5 mmol/L.
6. the method according to claim 1, wherein in step (3), xanthine oxidase is added in an amount of 0.3 IU per well.
7. a hyperuricemia hepatocyte model produced by the method according to any one of claims 1 ~ 6.
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