CN114107172A - Method for constructing non-infectious gastric mucosa injury cell model and application - Google Patents
Method for constructing non-infectious gastric mucosa injury cell model and application Download PDFInfo
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Abstract
The invention provides a method for constructing a non-infectious gastric mucosa injury cell model and application, and belongs to the technical field of cell model construction. The invention provides a method for constructing a non-infectious gastric mucosa injury cell model, which comprises the following steps: taking the concentration of 2 × 104‑2×105Performing conventional culture on the logarithmic phase GES-1 cells per mL, and adding ethanol or H after the cells adhere to the wall2O2Culturing the culture solution for 6-24h, and removing the injury solution to obtain the induced gastric mucosa injury cell model. The invention uses ethanol and H2O2Compared with animal models, the cell model is easier to obtain, the operation is simple and convenient, the high-throughput screening of samples can be realized, the modeling time of the method is short, the stability of the constructed model is good, and the method can be applied to research and development of products for protecting stomach and protecting stomachOr noninfectious gastric mucosa injury mechanism.
Description
Technical Field
The invention belongs to the technical field of cell model construction, and particularly relates to a method for constructing a non-infectious gastric mucosa injury cell model and application thereof.
Background
Once the dynamic balance is broken, the generated gastric acid gradually damages the stomach wall to cause damage of the mucous membrane, and the diseases related to the damage of the gastric mucosa such as gastritis and gastric ulcer are caused. With the progress of society and times, the pace of life is accelerated, and the incidence rate of stomach diseases shows a rapid increasing trend under the influence of bad living habits, gastric ulcer also becomes common digestive diseases in internal medicine, is one of the most serious gastrointestinal diseases in the world at present, and can be caused by a plurality of factors, but compared with the infectious gastric ulcer caused by helicobacter pylori, the non-infectious gastric ulcer diseases caused by oxidative stress caused by alcoholism, smoking and active oxygen, long-term intake of non-steroidal anti-inflammatory drugs, mental stress and other factors are more common in daily life.
The underlying cause of noninfectious gastric mucosal damage is an imbalance in gastric mucosal defense and erosion factors. In general, physical or chemical damage to the defense barrier of the gastric mucosa is an important factor in the development of non-infectious gastric ulcers. In addition to the defense barrier, the antioxidant system is also an important factor in protecting the structure and function of the gastric mucosa. In healthy humans, the balance of Reactive Oxygen Species (ROS) is maintained through the nuclear factor Nrf2 pathway. When stimulated by external factors, the excessive production of ROS and the reduction of antioxidant activity lead to imbalance of antioxidant system, which in turn causes chemical damage such as cell membrane lipid peroxidation to gastric mucosa. And exogenous H2O2Can cause oxidative stress and increase intracellular reactive oxygen species. Oxygen radicals are thought to play an important role in the formation and development of gastritis or gastric cancer. Thus, construction H2O2InducedThe gastric mucosa injury model has important practical significance.
With the improvement of the living standard of people, the consumption proportion of ethanol (alcohol) by residents in China is continuously increased, the alcohol intake is close to the level of developed countries, and the proportion of strong alcohol is high. Ethanol enters blood through gastrointestinal absorption and directly contacts with gastric mucosa, so that the metabolism and function of the gastric mucosa are changed, the normal physiological microenvironment of the gastric mucosa is damaged, the barrier of the gastric mucosa is damaged, the permeability of cell membranes is increased, and the gastric mucosa is damaged. Research shows that the incidence of acute gastric mucosal injury caused by ethanol is increasingly high. Therefore, ethanol is also not negligible as a gastric mucosal lesion.
In recent years, the protective effect of polysaccharide substances such as hericium erinaceus and dendrobium on gastric mucosal injury or gastric ulcer in animal models is discovered, but because the animal models have long experimental period and are complicated to operate and relate to the ethical problem of animals, the experimental samples are large in demand and difficult to screen in large quantities. The existing research also lacks the evaluation of the activity of polysaccharide samples from different sources on protecting the gastric mucosa on the same model. Therefore, the construction of the non-infectious gastric mucosa injury cell model which is stable, short in modeling time and simple and convenient in method and can be used for developing gastric protection products has great practical significance.
Disclosure of Invention
In view of this, the present invention aims to provide a method for constructing a non-infectious gastric mucosal injury cell model, which has the advantages of short modeling time, simple method and stable constructed model.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a method for constructing a non-infectious gastric mucosa injury cell model, which comprises the following steps: taking the concentration of 2 × 104-2×105Culturing the individual/mL logarithmic phase GES-1 cells, and adding ethanol or H after the cells adhere to the wall2O2Culturing the culture solution for 6-24h, and removing the injury solution to obtain the gastric mucosa injury cell model.
Preferably, the concentration of ethanol in the culture solution is 1% -7%.
Preferably, H is contained in the culture solution2O2Is in a concentration of 0.125mM to 1 mM.
Preferably, the concentration of GES-1 cells is 2X 10 when ethanol is contained in the culture medium4-1×105one/mL.
Preferably, when the culture medium contains H2O2When the concentration of GES-1 cells is 5X 104-2×105one/mL.
The invention also provides a non-infectious gastric mucosa injury cell model constructed according to any one of the methods.
The invention also provides application of the method or the cell model in the development of gastric protection products.
Preferably, the stomach protecting product comprises edible fungus polysaccharide.
Preferably, the method comprises the following steps: mixing edible fungus polysaccharide and GES-1 cells, culturing for 24H, and adding ethanol or H according to model construction method2O2Co-culturing, discarding the injury solution, rinsing with PBS for 2 times, adding Alamar Blue coloring agent in the dark, incubating for 4-8h, measuring the light absorption values at 570nm and 600nm before and after Alamar Blue discoloration by an enzyme labeling instrument, calculating the cell proliferation rate, and evaluating the protective effect of different edible fungus polysaccharides on gastric mucosa.
The invention also provides an application of the method or the cell model in the research of a non-infectious gastric mucosal injury mechanism.
The invention has the beneficial effects that: the gastric mucosa injury model disclosed in the prior art is mainly an animal model, while the cell model aims at helicobacter pylori infection models, and a single injury factor is mainly researched in the same research, but ethanol and H are firstly constructed in the invention2O2The non-infectious gastric mucosa injury cell model induced by the two injury factors has the characteristics of short modeling time and good model stability. Compared with an animal model, the cell model constructed by the invention is easy to obtain, is simple and convenient to operate, and can realize high-throughput screening of samples. The cell model constructed by the invention can realize research and development tests and noninfectiveness of products for protecting stomach and protecting stomachIn the study of the mechanism of gastric mucosal injury.
Drawings
FIG. 1 shows the protective effect of different edible fungus polysaccharides on ethanol-induced GES-1 cell damage, wherein, # # P < 0.01 compared to the blank group, # P < 0.05, # P < 0.01 compared to the model group;
FIG. 2 shows polysaccharide pairs H of different edible fungi2O2Induced GES-1 cell damage protection, wherein, # P < 0.01 compared to the blank group, P < 0.05, # P < 0.01 compared to the model group.
Detailed Description
The invention provides a method for constructing a non-infectious gastric mucosa injury cell model, which comprises the following steps: taking the concentration of 2 × 104-2×105Culturing the individual/mL logarithmic phase GES-1 cells, and adding ethanol or H after the cells adhere to the wall2O2Culturing the culture solution for 6-24h, and removing the injury solution to obtain the gastric mucosa injury cell model.
The invention adopts two widely-existing damage factors to construct a non-infectious gastric mucosa damage cell model for the first time, wherein the non-infectious gastric mucosa damage factor is ethanol or H2O2When the non-infectious mucosal injury factor is ethanol, the initial concentration of GES-1 cells is preferably 2X 104-1×105Preferably, the concentration of ethanol in the culture solution is 1% -7%, more preferably 5% -6%, and the culture time is preferably 6 h. When the non-infectious mucosal injury factor is H2O2When the initial concentration of GES-1 cells is 5X 104-2×105Per mL, H in the culture medium2O2Is preferably 0.125mM-1mM, more preferably 0.75mM-1mM, and the time of the incubation is preferably 6 h.
In the invention, too low or too high cell concentration, too low or high concentration of damage factors and long action time can cause the difficulty in stably establishing a cell model, and the problems of excessive cell damage or insufficient action concentration of the damage factors, unobvious screening effect of the constructed model sample and the like can occur. The noninfectious gastric mucosa injured cell model constructed by the method has good stability, and can realize high-throughput screening of samples.
The specific source of the GES-1 cells in the present invention is not particularly limited, and any commercially available product that is conventional in the art may be used. In the invention, GES-1 cells in logarithmic growth phase are preferably taken to construct cell models, the specific methods for cell recovery and cell subculture of the GES-1 cells are not particularly limited, and the conventional methods for cell recovery and cell subculture of the GES-1 cells in the field can be adopted.
The invention also provides application of the method or the cell model in the development of gastric protection products.
The specific type of the product for protecting the stomach is not particularly limited, and in the specific embodiment of the invention, the non-infectious gastric mucosa injury cell model constructed by the invention is preferably adopted to research the protection effect of edible fungus polysaccharide from different sources on the gastric mucosa. The type of the edible fungi is not particularly limited, and the conventional edible fungi in the field can be adopted.
In the present invention, when the correlation between the edible fungus polysaccharide species and the gastric mucosa is studied, the following steps are preferably included: mixing edible fungus polysaccharide and GES-1 cell, culturing, and adding ethanol or H according to model construction method2O2Co-culturing, removing damage liquid, rinsing with PBS for 2 times, adding Alamar Blue coloring agent in dark, incubating for 4-8h, measuring the light absorption values at 570nm and 600nm before and after Alamar Blue discoloration by an enzyme labeling instrument, calculating the cell proliferation rate, and evaluating the protective effect of different edible fungus polysaccharides on gastric mucosa according to the cell proliferation rate. As an implementable mode, the edible fungus polysaccharide can be prepared by adopting a water extraction and alcohol precipitation mode. Specifically, mixing edible fungi with water, boiling, filtering, mixing the filter residue with water, boiling, filtering, mixing the two filtrates, concentrating, adding ethanol solution, precipitating with ethanol, volatilizing ethanol, dissolving in water, and lyophilizing to obtain edible fungi polysaccharide. In the invention, the feed-liquid ratio of the edible fungi to the water is preferably 1:10-15, more preferably 1:12-13, the boiling time is preferably 1-3h, more preferably 2h, and the concentration ratio of the filtrate to the water is preferably 3:1-1:1, more preferably 2: 1. ColdWhen absolute ethyl alcohol is added for alcohol precipitation after the temperature is reduced to the room temperature, the concentration of the ethanol precipitation is preferably 75% (V/V), and the specific operation is preferably to add the absolute ethyl alcohol until the concentration of the solution ethanol is 75% (V/V).
The method or the cell model can be applied to research on non-infectious gastric mucosal injury mechanisms.
The present invention provides the following detailed description of the technical solutions provided by the present invention with reference to the examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Taking out the cell freezing tube from the liquid nitrogen tank, quickly placing the tube in a water bath kettle at 37 ℃ to be dissolved to a state without solid crystals, wiping the outer wall with 75% alcohol, transferring the tube to a 15ml sterile centrifuge tube containing 5ml of complete culture medium (DMEM medium containing 10% FBS), blowing and beating the tube uniformly by using a pipette, and centrifuging the tube for 5min at 1000 rmp. Discarding supernatant, collecting precipitated cells, adding 1ml complete medium to disperse cells, standing at 37 deg.C and 5% CO2Culturing in a cell culture box.
When the cell density in the culture bottle reaches about 80%, the old culture medium is sucked off, the culture bottle is lightly rinsed for 2 times by PBS, 2ml of 0.25% pancreatin digestive juice is added, the mixture is digested for 2min in an incubator at 37 ℃, the digestion condition of the cells is observed under a microscope after the cells are taken out, and if most of the cells become round and disperse, 5ml of complete culture medium is added to stop the digestion. Centrifuging at 1000rmp for 5min, discarding supernatant, and uniformly suspending cells according to the weight ratio of 1: 3 into new sterile culture bottles.
GES-1 cells in the logarithmic growth phase were inoculated into 96-well cell culture plates to a cell concentration of 2X 104one/mL, 180. mu.L per well, 5% CO at 37 ℃2Culturing for 12h, after the cells adhere to the wall, adding DMEM culture solution containing 5% ethanol concentration (w/v), 20 mu L of each well, after the ethanol acts on the cells for 6h, discarding the injury solution (supernatant), rinsing for 2 times by PBS, adding Alamar Blue coloring agent with 0.075mM in a dark place, after incubating for 4h, measuring absorbance values at 570nm and 600nm before and after Alamar Blue discoloration by a microplate reader, and calculating the cell proliferation rate according to the following formula for 6 repeats of each group: growth rate (%)(117216×A570sample-80586×A600sample)/(117216×A570control-80586×A600control). When the proliferation rate of the GES-1 cells is about 50%, the constructed non-infectious gastric mucosal lesion GES-1 cell model is good in stability.
Example 2
The difference from example 1 is that the ethanol concentration is 6%, and the rest is the same as example 1.
Example 3
The difference from example 1 is that the ethanol concentration is 7%, and the rest is the same as example 1.
Example 4
The difference from example 1 is that the time of ethanol acting on the cells is 12h, and the rest is the same as example 1.
Example 5
The difference from example 1 is that the time of ethanol acting on the cells is 12h, the ethanol concentration is 6%, and the rest is the same as example 1.
Example 6
The difference from example 1 is that the time of ethanol acting on the cells is 12h, the ethanol concentration is 7%, and the rest is the same as example 1.
Example 7
The difference from example 1 is that the cell concentration of GES-1 cells is 5X 104one/mL, the rest being the same as in example 1.
Example 8
The difference from example 1 is that the cell concentration of GES-1 cells is 5X 104The concentration of ethanol in the solution per mL is 6%, and the rest is the same as that in example 1.
Example 9
The difference from example 1 is that the cell concentration of GES-1 cells is 5X 104The concentration of ethanol in the solution per mL is 7%, and the rest is the same as that in example 1.
Example 10
The difference from example 1 is that the cell concentration of GES-1 cells is 1X 105The cells were treated with ethanol for 12 hours per mL, as in example 1.
Example 11
The difference from example 1 is that the cell concentration of GES-1 cells is 1X 105The concentration of ethanol is 4 percent, the time for the ethanol to act on the cells is 12 hours, and the rest is the same as the example 1.
Comparative example 1
The difference from example 1 is that the ethanol concentration is 0%, and the rest is the same as example 1.
The results of cell proliferation rates of examples 1 to 11 and comparative example 1 are shown in Table 1.
TABLE 1 modeling results for different GES-1 cell concentrations, ethanol concentrations, and action times
Example 12
GES-1 cells of example 1 in the logarithmic growth phase were seeded in a 96-well cell culture plate to give a cell concentration of 5X 104one/mL, 180. mu.L per well, 5% CO at 37 ℃2Culturing for 12H, adding 0.5mM H2O220 μ L per well of DMEM medium for H2O2After 6 hours of cell activation, the injury solution (supernatant) was discarded, washed with PBS for 2 times, and after incubating with 0.075mM Alamar Blue stain added in the dark for 4 hours, absorbance values at 570nm and 600nm before and after Alamar Blue discoloration were measured with a microplate reader, and the cell proliferation rate was calculated according to the formula described in example 1. When the proliferation rate of the GES-1 cells is about 50%, the non-infectious gastric mucosal injury cell model is successfully constructed, and the constructed non-infectious gastric mucosal injury GES-1 cell model has good stability.
Example 13
The difference from example 12 is H in DMEM medium2O2Was added to the solution in (1) at a concentration of 0.75mM, as in example 12.
Example 14
And embodiments thereof12 differs in DMEM medium H2O2Was 1mM, as in example 12.
Example 15
The difference from example 12 is H in DMEM medium2O2In a concentration of 1mM, H2O2The time for cell activation was 12 hours, as in example 12.
Example 16
The difference from example 12 is that the concentration of GES-1 cells is 1X 105one/mL, the rest being the same as in example 12.
Example 17
The difference from example 12 is that the concentration of GES-1 cells is 1X 105Per mL, in DMEM Medium H2O2Was added to the solution in (1) at a concentration of 0.75mM, as in example 12.
Example 18
The difference from example 12 is that the concentration of GES-1 cells is 1X 105Per mL, in DMEM Medium H2O2In a concentration of 1mM, H2O2The time for cell activation was 24 hours, as in example 12.
Example 19
The difference from example 12 is that the concentration of GES-1 cells is 1X 105Per mL, in DMEM Medium H2O2In a concentration of 0.75mM, H2O2The time for cell activation was 24 hours, as in example 12.
Example 20
The difference from example 12 is that the concentration of GES-1 cells is 2X 105Per mL, in DMEM Medium H2O2Was 1mM, as in example 12.
Comparative example 2
The difference from example 12 is H in DMEM medium2O2Was 0mM, as in example 12.
The cell proliferation rate results of examples 12 to 20 and comparative example 2 are shown in Table 2.
TABLE 2 different GES-1 cell concentrations, H2O2Modeling results of concentration and action time
Example 21
Taking Hericium erinaceus, Dictyophora phalloidea eggs, shiitake mushrooms, straw mushrooms, Stropharia rugoso-annulata, Agaricus bisporus, oyster mushrooms, Hypsizygus marmoreus and needle mushrooms, respectively, removing unqualified fruiting bodies and impurities such as mildewing, metamorphism and the like, drying at 55 ℃, crushing, and sieving with a 100-mesh sieve to prepare fruiting body dry powder for later use.
Respectively taking 200g of fruit body powder, extracting for 2h by boiling water according to the material-liquid ratio of 1:15, filtering, collecting filtrate, continuously extracting filter residues for 2h by boiling water according to the material-liquid ratio of 1:15, filtering, combining the filtrates, concentrating according to the concentration ratio of 2:1, cooling, centrifuging, slowly adding absolute ethyl alcohol into supernatant to ensure that the final concentration of the ethyl alcohol in the whole system is 75%, precipitating with ethanol for 8h, centrifuging, washing the precipitate with ethanol, volatilizing the ethanol, adding water for dissolving, and freeze-drying for later use to obtain the edible fungus polysaccharide.
Evaluation of gastric mucosa protection activity of different edible fungus polysaccharides
Weighing 10mg of each edible fungus polysaccharide, shaking and dissolving with PBS for 10min, centrifuging at 12000r for 30min, and diluting into polysaccharide solutions of 50 μ g/mL, 200 μ g/mL and 500 μ g/mL (final concentration). The method for establishing the model in the embodiment 1 is the same as the method for establishing the model in the embodiment 1 by taking the GES-1 cells growing in the logarithmic phase and carrying out culture steps of digestion, centrifugation, heavy suspension and the like. After the cells adhere to the wall, adding polysaccharide solutions with different concentrations into each hole, dividing the cells into a blank group (without adding polysaccharide solution and ethanol), an ethanol model group (without adding polysaccharide solution) and a polysaccharide solution administration group with different concentrations (with adding polysaccharide solutions with different concentrations), after 24 hours of action, adding 5% ethanol into the rest groups except the normal group, after 6 hours of action, discarding the injury solution, rinsing for 2 times by PBS, adding 0.075mM Alamar Blue coloring agent in a dark place, incubating for 6 hours, and measuring the light absorption values at 570nm and 600nm before and after Alamar Blue color change by using an enzyme labeling instrument. The cell growth rate was calculated according to the formula described in example 1. The results are shown in FIG. 1.
Example 22
The difference from example 21 is that after the cells are attached, different concentrations of polysaccharide solution are added to each well, and the cells are divided into blanks (without polysaccharide solution and H)2O2)、H2O2Model group (without adding polysaccharide solution) and polysaccharide solution administration group (with adding polysaccharide solution of different concentration), after 24H of action, adding 0.75mM H into the other groups except normal group2O2Otherwise, the same procedure as in example 21 was repeated. The results are shown in FIG. 2.
As can be seen from figures 1 and 2, the hericium erinaceus polysaccharide, the dictyophora indusiata (egg) and the stropharia rugoso-annulata have obvious protective effects on gastric mucosa injury, and particularly, the hericium erinaceus polysaccharide has the best activity of protecting the gastric mucosa in the tested edible fungus polysaccharide. Meanwhile, the dictyophora indusiata and the dictyophora indusiata eggs are found to have the function of protecting gastric mucosa for the first time and may be related to the viscous polysaccharide contained in the dictyophora indusiata and the dictyophora indusiata eggs. While oyster mushroom, hypsizygus marmoreus and flammulina velutipes have no obvious or no effect on protecting gastric mucosa under the action concentration. As can be seen from comparison of the effects of different edible fungus polysaccharides on protecting gastric mucosa in two models, different edible fungus polysaccharides have different ways of playing the role of protecting gastric mucosa, such as straw mushroom polysaccharide and agaricus bisporus polysaccharide, which have relatively weak protection effect in an ethanol-induced damage model and have relatively weak protection effect in an H model2O2In the induced injury model, the gastric mucosa protection activity is better, and the possibility that the gastric mucosa protection effect is exerted by regulating and controlling oxidative stress reaction is revealed. The different edible fungus polysaccharides have different action ways for protecting the gastric mucosa, for example, the hericium erinaceus polysaccharide shows higher gastric mucosa protection effect in two models, and the hericium erinaceus polysaccharide is suggested to possibly play a role by regulating and controlling inflammatory reaction and oxidative stress; while it is possible for volvariella volvacea polysaccharide and agaricus bisporus polysaccharide to play a role by regulating oxidative stress. The construction of the non-infectious gastric mucosa injury model can provide technical support for accurately screening and protecting gastric mucosa raw materials.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for constructing a non-infectious gastric mucosa injury cell model is characterized by comprising the following steps: taking the concentration of 2 × 104-2×105Culturing the individual/mL logarithmic phase GES-1 cells, and adding ethanol or H after the cells adhere to the wall2O2Culturing the culture solution for 6-24h, and removing the injury solution to obtain the induced gastric mucosa injury cell model.
2. The method according to claim 1, wherein the concentration of ethanol in the culture solution is 1% to 7%.
3. The method of claim 1, wherein the culture medium is H2O2Is in a concentration of 0.125mM to 1 mM.
4. The method according to claim 1, wherein the concentration of GES-1 cells is 2X 10 when ethanol is contained in the culture medium4-1×105one/mL.
5. The method according to claim 1, wherein the culture medium contains H2O2When the concentration of GES-1 cells is 5X 104-2×105one/mL.
6. A non-infectious gastric mucosal lesion cell model constructed according to the method of any one of claims 1 to 5.
7. Use of the method of any one of claims 1 to 5 or the cell model of claim 6 in the development of a gastroprotective gastric product.
8. The use according to claim 7, wherein the stomach protecting product comprises edible fungus polysaccharides.
9. Use according to claim 8, characterized in that it comprises the following steps: mixing edible fungus polysaccharide and GES-1 cells, culturing for 24H, adding ethanol or H into the other groups except normal group according to model construction method2O2Co-culturing, discarding the injury solution, rinsing with PBS for 2 times, adding Alamar Blue coloring agent in the dark, incubating for 4-8h, measuring the light absorption values at 570nm and 600nm before and after Alamar Blue discoloration by an enzyme labeling instrument, calculating the cell proliferation rate, and evaluating the protective effect of different edible fungus polysaccharides on gastric mucosa.
10. Use of the method of any one of claims 1 to 5 or the cell model of claim 6 for the study of the mechanisms of noninfectious gastric mucosal lesion.
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| CN117503800B (en) * | 2024-01-04 | 2024-04-05 | 北京益华生物科技有限公司 | Gastric mucosa epithelial cell extract and preparation method and application thereof |
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