CN113265372A - Model for inducing mucus high secretion in vitro and construction method thereof - Google Patents
Model for inducing mucus high secretion in vitro and construction method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0688—Cells from the lungs or the respiratory tract
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2301—Interleukin-1 (IL-1)
Abstract
The invention relates to an in vitro induced mucus hypersecretion model and a construction method thereof. The invention adopts IL-1 beta to induce H292 cells in vitro, and detects the influence of IL-1 beta with different concentrations on the growth of the H292 cells in different time of culture through MTT; ELISA detects the content of H292 cell adhesive protein MUC5AC and inflammatory factor interleukin-8 (IL-8); PCR detection of MUC5AC mRNA expression level proves that IL-1 beta has no inhibiting effect on H292 cell growth, and can induce H292 cell to secrete inflammatory factor IL-8 and up-regulate MUC5AC gene expression and protein secretion. Therefore, the invention selects 5-10ng/mL IL-1 beta to induce H292 cells for 24H as an in vitro mucus high secretion model as an establishing condition, and can simply, conveniently and quickly obtain an effective mucus high secretion model. The in vitro mucus hypersecretion model provided by the invention can be used for researching pathological mechanisms of airway mucus secretion and researching and developing related medicaments, and has short period and simple and convenient operation compared with an animal model.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to an in-vitro induced mucus high secretion model and a construction method thereof.
Background
Airway epithelial cells have great relationship with the generation and development of inflammatory reaction in the airway and the secretion and removal of mucus, it plays an important regulatory role in the formation of airway mucus hypersecretion (Zhang Ling, Van Xiaoyun, Wusha, etc.. analysis of poly-L-arginine induced apoptosis of NCI-H292 cells and its possible mechanism [ J ]. J.clinics and experimental pathologists, 2016,32(10): 1135-1139; Lee L Y, Gu Q, LinAH, et al. air hypersensitivity induced by eosinophiel-derived functional proteins [ J ]. Pulm Pharmacol Ther,2019:101804), airway mucus hypersecretion is an important pathological feature of various chronic respiratory diseases, the main clinical manifestations of the cough are excessive phlegm and cough, and are accompanied by airflow limitation and lung function reduction, which are important factors for increasing morbidity and mortality of diseases such as asthma, emphysema and the like. The targeted therapy of pathological airway mucus is an important way for improving cough and dyspnea symptoms, reducing disease attack frequency and delaying disease progression, so that establishment of an effective cell mucus high secretion model is beneficial to better research of pathological airway mucus secretion mechanisms in vitro, and provides an experimental tool for research and development of related medicaments.
At present, most of research and development on medicines are mainly aimed at a mucus high secretion model of an animal rat, wherein the research and development include acrolein atomization inhalation mode, Lipopolysaccharide (LPS) injection and smoking method and the like; in the research on the establishment of the cell airway mucus high secretion model, a scholars established a model induced by Human Neutrophil Elastase (HNE), but the induced cell was a lung adenocarcinoma cell strain a549 (arrow, grand boat, liu qi, etc.. preliminary research on the establishment of the cell model for inducing airway mucus high secretion by human neutrophil elastase and the mechanism thereof [ J ]. journal of biological engineering of china, 2008, (03): 1-7.). At present, an airway mucus high secretion model established aiming at airway epithelial cells is still lacked.
Disclosure of Invention
The invention aims to provide a method for constructing a model for inducing mucus hypersecretion in vitro.
Another object of the present invention is to provide a model for inducing mucus hypersecretion in vitro.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for constructing an in vitro induced mucus hypersecretion model, which comprises the following steps:
1) h292 cell strain in logarithmic growth phase was inoculated in 96-well culture plate at 1X 10 cells per well4Placing the individual cells in a cell culture box for culture;
2) after 24h of culture, IL-1 beta with the final concentration of 5-10ng/mL is added, and the culture is continued for 24h in an incubator to obtain an externally induced mucus high secretion model.
Preferably, IL-1 beta with the final concentration of 5ng/mL is added after the culture for 24 hours in the step 2), and the culture is continued for 24 hours in an incubator to obtain an in vitro induced mucus hypersecretion model.
Preferably, the culture Medium used in step 1) is RPMI Medium 1640 culture Medium, and the culture conditions are 37 deg.C and 5% CO2Culturing in a constant-temperature cell culture box.
Preferably, the culture Medium used in step 2) is RPMI Medium 1640 culture Medium, and the culture conditions are 37 deg.C and 5% CO2Culturing in a constant-temperature cell culture box.
The application also provides an in vitro induced mucus hypersecretion model obtained by the construction method.
The invention has the following beneficial effects:
the invention provides a method for constructing a model for inducing mucus secretion in vitro, which adopts IL-1 beta to induce H292 cells in vitro, and detects the influence of IL-1 beta with different concentrations on the growth of the H292 cells in different times of culture through MTT; ELISA detects the content of H292 cell adhesive protein MUC5AC and inflammatory factor interleukin-8 (IL-8); PCR detection of MUC5AC mRNA expression level proves that IL-1 beta has no inhibiting effect on H292 cell growth, and can induce H292 cell to secrete inflammatory factor IL-8 and up-regulate MUC5AC gene expression and protein secretion. Therefore, the invention selects 5-10ng/mL IL-1 beta to induce H292 cells for 24H as an in vitro mucus high secretion model as an establishing condition, and can simply, conveniently and quickly obtain an effective mucus high secretion model. The in vitro mucus hypersecretion model provided by the invention can be used for researching pathological mechanisms of airway mucus secretion and researching and developing related medicaments, and has short period and simple and convenient operation compared with an animal model.
Detailed Description
The invention will be further described with reference to specific embodiments, but the scope of the invention is not limited thereto; the test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.
The human airway epithelial cell line H292 used in the examples was purchased from cell bank of Shanghai Life sciences college of China academy of sciences, Cat: TCHU122, and was stored by molecular biology laboratory center of medicine university in south and river. RPMI Medium 1640 Medium (Solaibio, lot # 20171249), fetal bovine serum (Gemini, lot # A97E00G), Tetramethylazo salt (MTT) (Solaibio, lot # 303H0529), Recombinant Human IL-1 beta (Peprotech, lot # 0606B95), MUC5AC ELISA Kit (Wuhan Youer trade Co., Ltd., lot # L160525793), IL-8ELISA Kit (doctor DebBiol, lot # 13410811028), ultrapure RNA extraction Kit (Soxhlet, lot # 40267142), Quantscript RT Kit (Tiangen, lot # P5024), Trizol Total RNA extraction Kit (Invitrogen, lot # D14218-02).
CO2Cell incubator (Eppendorf Co., CO 170R-230-.
Example 1
MTT method for detecting influence of IL-1 beta on growth of human airway epithelial cells H292
1 cell culture and passaging
1.1 cell culture: after the human airway epithelial cell strain H292 is recovered, the cell strain is placed in a constant-temperature cell culture box with 37 ℃ and 5% CO2, the liquid is changed for 1 time in 1-2 days, the cell density is 80-90% after passage, and the cell state is gradually stabilized after the cell state is transferred to 3 rd-4 th generation, so that the cell strain can be used for experiments. 1.2 cell passage: and (3) passage when the cell density reaches 80-90%, washing with 3mL of PBS buffer solution, adding 2mL of trypsin digestive juice to uniformly spread the trypsin digestive juice on the bottom of the flask, standing at room temperature and keeping flat for 12min, adding fetal calf serum after complete digestion, gently blowing to make the cells fall off from the bottom of the culture flask, centrifuging at 1000rpm/min for 5min, blowing again and uniformly mixing, and placing in an incubator for culture.
2MTT colorimetric method for detecting influence of IL-1 beta on growth of H292 cells
Taking H292 cell strain in logarithmic growth phase to inoculate 96-hole culture plate, inoculating 1 × 10 cells in each hole4And setting a normal group (0ng/mL) and an IL-1 beta 4 concentration group (1.25ng/mL, 2.5ng/mL, 5g/mL and 10ng/mL), adding IL-1 beta with different concentrations in groups after the cells are cultured for 24 hours, continuously culturing for different times (6 hours, 12 hours, 24 hours and 48 hours) in an incubator, then adding a prepared 10% MTT solution, carefully removing liquid in each hole after the cells are cultured for 4 hours, adding 150 mu L DMSO in each hole, and immediately detecting the absorbance value of 570nm by using a microplate reader after shaking and dissolving.
The results are as follows:
compared with a normal control group, IL-1 beta in different concentrations has no inhibition effect on H292 cells, no statistical significance on the difference, and no statistical significance in comparison among groups (P > 0.05). See table 1.
TABLE 1 Effect of different concentrations of IL-1. beta. on H292 cell growth by incubation for different periods of time: (
n＝5)
Example 2
ELISA method for detecting influence of IL-1 beta on mucin MUC5AC secreted by H292 cells 1, inoculating H292 cell strain in logarithmic growth phase into 96-well culture plate, inoculating 1 × 10 cells into each well4Setting normal group (0ng/mL) and IL-1 beta 4 concentration groups (1.25ng/mL, 2.5ng/mL, 5ng/mL and 10ng/mL), grouping and adding IL-1 beta with different concentrations after culturing cells for 24h, collecting supernatant in holes after continuously culturing for different time (6h, 12h, 24h and 48h) in an incubator, centrifuging at 1000rpm for 5min, subpackaging and storing at 4 ℃ for later use; washing the cells in the wells with 4 deg.C precooled PBS for 3 times, adding 100 μ L cell lysate into each well, transferring the cell lysate after lysis into a 1.5mL centrifuge tube, centrifuging at 14000rpm for 20min at 4 deg.C, subpackaging, and storing at-20 deg.C for later use.
2. Preparing a standard product: the reagents and specimens were moved to room temperature prior to use and the standards were formulated exactly as described by dilution of MUC5AC standard at 7 concentrations within 15min prior to use. Adding 1mL of sample diluent into a standard sample tube, and dissolving to obtain a solution with the concentration of 20000 pg/mL; after the dilution to 5000pg/mL, 7 EP tubes were taken, and 0.5mL of sample diluent was added to each tube, and the samples were diluted in duplicate to 5000pg/mL, 2500pg/mL, 1250pg/mL, 625pg/mL, 312pg/mL, 156pg/mL, 78pg/mL, and 0pg/mL (standard diluent).
3. Sample loading and incubation: and setting a standard hole, a blank hole and a sample adding hole according to the experimental requirements, wherein the standard hole is provided with 7 holes, and sequentially adding 100 mu L of diluted standard substances with different concentrations. Adding 100 mu L of standard dilution into a blank hole, adding 100 mu L of sample to be detected into a sample adding hole, arranging 3 multiple holes, covering a film, and incubating for 1h at 37 ℃.
4. Adding working solution, washing a plate, developing and terminating reaction: pouring out the liquid in the holes, and drying the liquid on the absorbent paper as much as possible without washing; and the prepared antibody working solution (diluted with special antibody diluent 1:99 before use) is added rapidly. Adding 100 μ L of the extract into each well, and covering with a film and incubating at 37 deg.C for 60 min; discarding liquid in the hole, rapidly adding 350 μ L/hole of cleaning solution (concentrated cleaning solution is diluted to 1 × with ultrapure water for use), soaking for 1-2min, discarding liquid, spin-drying, washing the plate for 3 times, washing unbound protein, but not drying the plate at any time; then adding 100 mu L of working solution of the enzyme conjugate (diluted by diluent 1:99 before use), and incubating at 37 ℃ for 35 min; and (3) discarding liquid in the holes, carrying out spin-drying, washing the plate for 5 times, adding the balanced TMB substrate solution, carrying out light-proof reaction at 37 ℃ for a proper time (when the front 3-4 holes of the standard holes have obvious gradient blue and the rear 3-4 holes are not obvious, the reaction can be stopped), adding 50 mu L of stop solution into each hole to stop the reaction, and immediately detecting the OD value by using an enzyme-labeling instrument at the wavelength of 450 nm.
5. And drawing a standard curve according to the standard hole, and calculating the content of MUC5AC in the sample according to the standard curve.
The results are as follows:
compared with the normal group, IL-1 beta acts for 6h, and the level of IL-1 beta histone MUC5AC is increased (P is less than 0.01) at 1.25ng/mL, 5ng/mL and 10 ng/mL; the effect is 12h and 24h, the level of the four concentrations of IL-1 beta histone MUC5AC is increased (P <0.05), and the level is most obvious at 5ng/mL IL-1 beta group at 24h (P < 0.01); at 48h, the water levels of IL-1. beta. histone MUC5AC at various concentrations were reduced compared to the other time-length values. See table 2.
TABLE 2 Effect of different concentrations of IL-1. beta. on the secretion of mucin MUC5AC by H292 cells at different times: (
n＝5)
Note: in comparison with the normal group,*group P<0.05,；**Group P<0.01
Example 3
ELISA method for detecting influence of IL-1 beta on secretion of inflammatory factor IL-8 by H292 cells
1. Taking H292 cell strain in logarithmic growth phase to inoculate 96-hole culture plate, inoculating 1 × 10 cells in each hole4Setting normal group (0ng/mL) and IL-1 beta 4 concentration groups (1.25ng/mL, 2.5ng/mL, 5ng/mL and 10ng/mL), grouping and adding IL-1 beta with different concentrations after culturing cells for 24h, and continuing culturing in the incubator for different times (6h, 1h and 12h, 24h and 48h), collecting supernatant in the holes, centrifuging for 5min at 1000rpm, subpackaging and storing at 4 ℃ for later use; washing the cells in the wells with 4 deg.C precooled PBS for 3 times, adding 100 μ L cell lysate into each well, transferring the cell lysate after lysis into a 1.5mL centrifuge tube, centrifuging at 14000rpm for 20min at 4 deg.C, subpackaging, and storing at-20 deg.C for later use.
2. Preparing a standard product: the reagents and specimens were moved to room temperature prior to use and the standards were formulated exactly as described by dilution of MUC5AC standard at 7 concentrations within 15min prior to use. Adding 1mL of sample diluent into a standard sample tube, and dissolving to obtain a solution with the concentration of 20000 pg/mL; after the dilution to 5000pg/mL, 7 EP tubes were taken, and 0.5mL of sample diluent was added to each tube, and the samples were diluted in duplicate to 5000pg/mL, 2500pg/mL, 1250pg/mL, 625pg/mL, 312pg/mL, 156pg/mL, 78pg/mL, and 0pg/mL (standard diluent).
3. Sample loading and incubation: and setting a standard hole, a blank hole and a sample adding hole according to the experimental requirements, wherein the standard hole is provided with 7 holes, and sequentially adding 100 mu L of diluted standard substances with different concentrations. Adding 100 mu L of standard dilution into a blank hole, adding 100 mu L of sample to be detected into a sample adding hole, arranging 3 multiple holes, covering a film, and incubating for 1h at 37 ℃.
4. Adding working solution, washing a plate, developing and terminating reaction: pouring out the liquid in the holes, and drying the liquid on the absorbent paper as much as possible without washing; and the prepared antibody working solution (diluted with special antibody diluent 1:99 before use) is added rapidly. Adding 100 μ L of the extract into each well, and covering with a film and incubating at 37 deg.C for 60 min; discarding liquid in the hole, rapidly adding 350 μ L/hole of cleaning solution (concentrated cleaning solution is diluted to 1 × with ultrapure water for use), soaking for 1-2min, discarding liquid, spin-drying, washing the plate for 3 times, washing unbound protein, but not drying the plate at any time; then adding 100 mu L of working solution of the enzyme conjugate (diluted by diluent 1:99 before use), and incubating at 37 ℃ for 35 min; and (3) discarding liquid in the holes, carrying out spin-drying, washing the plate for 5 times, adding the balanced TMB substrate solution, carrying out light-proof reaction at 37 ℃ for a proper time (when the front 3-4 holes of the standard holes have obvious gradient blue and the rear 3-4 holes are not obvious, the reaction can be stopped), adding 50 mu L of stop solution into each hole to stop the reaction, and immediately detecting the OD value by using an enzyme-labeling instrument at the wavelength of 450 nm.
5. And drawing a standard curve according to the standard hole, and calculating the content of MUC5AC in the sample according to the standard curve.
The results are as follows:
compared with the normal group, the IL-1 beta acts for 24 hours, and the IL-8 level of the groups with 1.25ng/mL, 2.5ng/mL, 5ng/mL and 10ng/mLIL-1 beta is obviously increased (P is less than 0.01); compared with the normal group, IL-1 beta acted for 24h, and IL-1 beta group acted for 48h, the increase of 1.25ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL IL-1 beta group was obvious (P < 0.01). See table 3.
TABLE 3 Effect of different concentrations of IL-1. beta. on IL-8 secretion from H292 cells over different periods of time: (
n＝5)
Note: in comparison with the normal group,*group P<0.05,；**Group P<0.01
Example 4
PCR method for detecting influence of IL-1 beta on expression of MUC5AC mRNA of H292 cell
Taking H292 cell seed 6-well plate, 2mL culture medium per well approximately containing 5X 105Placing the cells in CO2Culturing in an incubator, setting a normal group, an IL-1 beta 2.5ng/mL group, an IL-1 beta 5ng/mL group and an IL-1 beta 10ng/mL group, adding IL-1 beta with different concentrations in groups after cells are cultured in the incubator for 24 hours to be completely attached to the wall, and continuously culturing in the incubator for 24 hours to collect samples.
1. Extraction of Total RNA from cells
The cell culture medium in the 6-well plate was discarded, and the cells were washed with PBS buffer, and PBS was discarded. Adding 1mL Trizol extraction reagent into each hole, uniformly mixing, standing for 5min, fully cracking all cells in the holes, and then moving into an enzyme-free tube. Adding 150 mu 1 of chloroform into the tube, slightly turning the chloroform upside down to mix the chloroform and the tube evenly, and standing the tube for 3 to 5min at room temperature. Placing into a high-speed low-temperature centrifuge, and centrifuging at 13000rpm for 20 min. After centrifugation, the upper layer contained total RNA, and 400. mu.L of the total RNA was carefully removed and placed in an enzyme-free tube. Adding isopropanol, mixing, and standing at room temperature for 12 min. Centrifuge at 13000rpm for 10min at 4 ℃. Carefully discard the supernatant and add 75% ethanol. Centrifuging at 4 deg.C and 13000rpm for 5min, discarding supernatant, and naturally air drying at room temperature on absorbent paper. Add 50. mu.l DEPC water and mix well, use the ultraviolet spectrophotometer to determine RNA purity and concentration rapidly. And subpackaging the samples to avoid repeated freeze thawing, and storing at-80 ℃ for later use.
2. Reverse transcription of RNA into cDNA
Unfreezing the template RNA on ice, unfreezing the reagent and the primer at room temperature, immediately placing on ice, vortexing, shaking uniformly, and centrifuging briefly for use. The preparation reaction system is shown in table 1, the mixed solution is prepared on ice according to the reverse transcription system, the mixed solution is fully mixed by vortex oscillation and then is subpackaged in a single reaction tube, an RNA template is added, RNase-Free water is supplemented, and the total volume of the reverse transcription system is 20 mu l. Incubating at 37 deg.C for 60min, recovering to room temperature, packaging reverse transcription product, and storing at-20 deg.C.
TABLE 1 reverse transcription reaction System
3. Real time PCR reaction
PCR amplification primers and internal reference sequences are shown in Table 2. The template RNA was thawed on ice, the reagents required were thawed at room temperature and rapidly placed on ice. The reaction system should be prepared into Mix and mixed fully, then the Mix is loaded into a single reaction tube, the mixture is thoroughly mixed after the preparation is finished, and the mixture is centrifuged briefly. Placing the reaction system in a fluorescent quantitative PCR instrument for PCR reaction; pre-denaturation at 95 ℃ for 15 min; denaturation at 95 ℃ for 10sec and 60 ℃ for 1min for 40 cycles. The image analysis software takes the ratio of the target gene to the reference gene as the relative expression value of the target gene.
TABLE 4 PCR amplification primers and internal reference sequences
Statistical analysis: the experimental data for each experimental group were analyzed using SPSS22.0 statistical software. Experimental data adoption
The form of (a) is described, and statistical comparisons are made using one-way variance in conformity with a normal distribution. If the variance of the detected samples is checked for all with LSD, the variance is checked for all with Dunnett T3. Statistical analysis by P<0.05 shows that the difference has statistical significance, and a statistical chart is drawn.
The results are as follows:
compared with the normal group, the MUC5AC mRNA expression of the IL-1 beta group is obviously up-regulated after the IL-1 beta intervention is carried out for 24 hours at 2.5ng/mL, 5ng/mL and 10ng/mL, and the expression is most obvious in the IL-1 beta group at 10ng/mL (P <0.01), see Table 5.
TABLE 5 Effect of different concentrations of IL-1 beta on the expression of MUC5AC mRNA in H292 cells cultured for 24H
Note: in comparison with the normal group,*group P<0.05,；**Group P<0.01
Example 5
Examples 1-4 compare the effect of different concentrations of IL-1. beta. on H292 cells at different times and find that none of them had inhibitory effect on H292 cells. IL-1 β acted for 12h, 24h, 1.25ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL IL-1 β group MUC5AC levels were elevated (P <0.05), and acted 24h most significantly with 5ng/mL IL-1 β group (P < 0.01); after IL-1 beta acts for 24h, IL-8 levels of 1.25ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL IL-1 beta groups are obviously increased (P <0.01), and are most significant with 5ng/mL IL-1 beta groups. IL-1 beta at different concentrations intervenes in H292 cells for 24H, the mRNA expression of MUC5AC in IL-1 beta groups of 2.5ng/mL, 5ng/mL and 10ng/mL is obviously up-regulated, and the expression is most significant in IL-1 beta group of 10ng/mL (P < 0.01). Combining the results, the invention adopts IL-1 beta with the concentration of 5-10ng/mL to induce H292 cells for 24H to establish an in vitro mucus hypersecretion model. The embodiment is a specific embodiment of an in vitro mucus hypersecretion model, and the construction method comprises the following steps:
after the human airway epithelial cell line H292 is recovered, the cell line is placed at 37 ℃ and 5 percent CO2Changing the solution for 1 time in a constant-temperature cell culture box after 1-2 days, carrying out passage when the cell density reaches 80-90%, washing with 3mL of PBS buffer solution, adding 2mL of trypsin digestive juice to uniformly spread the trypsin digestive juice on the bottom of the culture bottle, standing at room temperature for 12min, adding fetal calf serum after complete digestion, gently blowing to make the cells fall off from the bottom of the culture bottle, centrifuging at 1000rpm/min for 5min, blowing again to uniformly mix, and placing in the culture box for culture. According to the experimental grouping design, H292 cells in the logarithmic growth phase are taken to inoculate 96-well culture plates, and each well is inoculated with 1 multiplied by 104And (3) after the cells are cultured for 24 hours, adding IL-1 beta with the final concentration of 5ng/mL (in other embodiments, the final concentration of the added IL-1 beta is 5-10ng/mL), and continuously culturing for 24 hours in an incubator to obtain the H292 cell mucus high secretion model. The user can further culture according to self research needs, make relevant research observation, and collect WB, PCR and other experimental indexes related to cell detection.
Claims (5)
1. A method for constructing a model for inducing mucus hypersecretion in vitro is characterized by comprising the following steps:
1) h292 cell strain in logarithmic growth phase was inoculated in 96-well culture plate at 1X 10 cells per well4Placing the individual cells in a cell culture box for culture;
2) and adding IL-1 beta with the final concentration of 5-10ng/mL after culturing for 24h, and continuously culturing for 24h in an incubator to obtain an in-vitro induced mucus high secretion model.
2. The method for constructing the model for inducing mucus hypersecretion in vitro according to claim 1, wherein IL-1 β with a final concentration of 5ng/mL is added after culturing for 24 hours in step 2), and the culture is continued for 24 hours in the incubator, thereby obtaining the model for inducing mucus hypersecretion in vitro.
3. The method for constructing model of inducing mucus hypersecretion in vitro according to claim 1, wherein said method comprisesThe culture Medium adopted in the step 1) is RPMI Medium 1640 culture Medium, and the culture condition is 37 ℃ and 5% CO2Culturing in a constant-temperature cell culture box.
4. The method for constructing the model for inducing mucus hypersecretion in vitro according to claim 1, wherein the culture Medium used in the step 2) is RPMI Medium 1640 culture Medium, and the culture conditions are 37 ℃ and 5% CO2Culturing in a constant-temperature cell culture box.
5. An in vitro induced mucus hypersecretion model obtained by the construction method according to any one of claims 1 to 4.
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