A method for constructing and evaluating an in vitro cell co-culture model for evaluating the degree of tobacco smoke-induced inflammatory response

Abstract

The present invention discloses a method for constructing and evaluating an in vitro cell co-culture model for evaluating the degree of tobacco smoke-induced inflammatory response, and belongs to the technical field of tobacco and cigarette smoke safety evaluation. The present invention cultivates beas-2b cells in the upper chamber of a co-culture plate to achieve a gas-liquid interface, then treats them with a poison, stains human lung macrophages with CFDA SE viable cell dye, and places the poisoned beas-2b cells and the stained human lung macrophages in the upper and lower chambers of the co-culture plate for co-culture, respectively. The cells are observed and counted using a fluorescence microscope to evaluate the migration of macrophages, and then the expression of inflammatory factors in the culture medium of the upper and lower chambers is measured respectively. The inflammatory changes in the co-culture system are evaluated, thereby evaluating the degree of tobacco smoke-induced inflammatory response.

Description

Technical Field

[0001] The present invention relates to a method for constructing and evaluating an in vitro cell co-culture model for evaluating the degree of tobacco smoke-induced inflammatory response, and belongs to the technical field of tobacco and cigarette smoke safety evaluation. Background Art

[0002] Cigarette smoke causes inflammation in the airways and lungs, and cigarette smoke condensate (CS) can cause tissue damage in the respiratory tract. This damage is implicated in the development of CS-induced airway disease and related lung diseases, such as chronic obstructive pulmonary disease (COPD). Airway epithelial cells are the primary target of CS. Currently, most studies investigating rapid in vitro COPD risk assessment models mimic the response of lung cells to CS, with a significant portion utilizing alveolar, bronchial, and airway epithelial cell systems. For example, epithelial cells (mouse alveolar type II epithelial cells, bronchial epithelial cells) or transformed cell lines are used. The airways of COPD patients harbor inflammatory cells, including neutrophils, macrophages, T lymphocytes, and dendritic cells. Macrophages are considered one of the primary cell types in COPD and play a crucial role in the pathophysiology of COPD.

[0003] Numerous studies have demonstrated a close connection between pathological changes and a disrupted immune system, particularly the monocyte-macrophage system. Macrophages are a type of white blood cell that participates in the development and progression of inflammation and repairs and remodels tissues through functions such as phagocytosis, antigen presentation, and immune regulation. Studies have demonstrated that M2 phenotype polarization is enhanced in the lungs of smokers and even higher in COPD patients, suggesting that M2 macrophages play a crucial role in the pathogenesis of smoking-induced COPD.

[0004] Some studies have examined the extent of inflammatory responses by phagocytizing depleted uranium particles by macrophages and then applying the resulting supernatant to bronchial epithelial cells. However, cell culture supernatant treatment of lung and bronchial epithelial cells is insufficient for assessing the inflammatory process in COPD. Furthermore, there are few reports on the interaction and mechanisms between macrophages and damaged airway epithelial cells when stimulated by CS. Summary of the Invention

[0005] The present invention aims to provide a method for constructing an in vitro cell co-culture model for evaluating the extent of tobacco smoke-induced inflammatory responses. This method can better simulate the physiological environment in the body and be used to assess disease risk.

[0006] The present invention also provides a method for evaluating the degree of inflammatory response induced by tobacco smoke.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A method for constructing an in vitro cell co-culture model for evaluating the degree of tobacco smoke-induced inflammatory response comprises the following steps:

[0009] (1) Beas-2b cells were cultured in the upper chamber of a co-culture plate and then exposed to the toxin;

[0010] (2) The stained macrophages are placed in the lower chamber of the co-culture plate and co-cultured with the beas-2b cells treated with the toxin in step (1) to obtain an in vitro cell co-culture model.

[0011] In step (1), beas-2b cells are placed in the upper chamber of the co-culture plate and the culture medium used includes BEBM basal medium and cytokines.

[0012] The above-mentioned cytokines include at least one of BPE, Hydrocortisone, hEGF, Epinephrine, Insulin, Transferrin, Triiodothyronine, Retinoic Acid, and GA.

[0013] The poisoning treatment operation in step (1) includes the following steps:

[0014] A. Collect the smoke condensate of the smoke to be tested and fully dissolve it in a solvent to obtain the poisoning mother solution;

[0015] B. Add the poisoned mother solution to the culture medium of beas-2b cells to obtain the poisoned solution;

[0016] C. Add the toxin solution into the upper chamber of the co-culture plate to infect beas-2b cells.

[0017] The exposure time in step C is 1.5 to 2.5 hours.

[0018] The concentration of the poison solution in step C ranges from 0 to 400 μg / ml.

[0019] The macrophages described in step (2) are human lung macrophages.

[0020] The co-cultivation time in step (2) is 0.5 to 10 hours.

[0021] The staining of macrophages in step (2) comprises the following steps:

[0022] 1) suspending the macrophages cultured in step (1) with CFDA SE cell labeling solution and placing them in a centrifuge tube;

[0023] 2) adding CFDA SE cell storage solution to the centrifuge tube described in step 1) and mixing to obtain a mixed solution;

[0024] 3) Place the mixture in a CO2 incubator and incubate at 37°C for 10 minutes;

[0025] 4) Immediately after the incubation, add the DMEM high-glucose complex medium used for culturing macrophages, mix thoroughly, centrifuge, and remove the supernatant;

[0026] 5) Repeat washing 2-3 times with the DMEM high-glucose composite medium described in step 4), then add the DMEM high-glucose composite medium described in step 4), mix well, and incubate in the incubator described in step 3) for 5 minutes to promote the staining effect;

[0027] 6) After removal, centrifugation was repeated, the supernatant was removed, and the DMEM high-glucose composite medium described in step 4) was added and mixed;

[0028] 7) Observe the staining effect under a fluorescence microscope and perform normal cell culture;

[0029] The whole process was carried out under light-proof conditions.

[0030] The in vitro cell co-culture model used to evaluate the extent of tobacco smoke-induced inflammatory responses was developed based on the characteristics of Beas-2b cells that require air-liquid interface culture and the migration of macrophages under inflammatory stimulation. Air-liquid interface (ALI) culture is commonly used to produce apical-basolateral polarization in airway epithelial cells during in vitro culture. Air-liquid interface (ALI) culture of Beas-2b cells provides an excellent cell model for CS exposure research. The establishment of this model has significantly contributed to further understanding of the impact of air pollution on human health and has also been helpful in understanding the specific causes of COPD. It is also applicable to the study of other tobacco-related diseases that threaten human health.

[0031] A method for evaluating the degree of tobacco smoke-induced inflammatory response comprises the following steps:

[0032] (1) Beas-2b cells were cultured in the upper chamber of a co-culture plate and then exposed to the toxin;

[0033] (2) Placing the stained macrophages in the lower chamber of the co-culture plate and co-culturing with the beas-2b cells treated with the toxin in step 1);

[0034] (3) Select evaluation indicators and classify evaluation standards;

[0035] (4) Evaluate the degree of inflammatory response induced by tobacco smoke.

[0036] In the above method for evaluating the degree of tobacco smoke-induced inflammatory response, the operation of the poisoning treatment in step (1) is:

[0037] A. Collect the smoke condensate of the smoke to be tested and fully dissolve it in a solvent to obtain the poisoning mother solution;

[0038] B. Add the poisoned mother solution to the culture medium of beas-2b cells to obtain the poisoned solution;

[0039] C. Add the toxin solution into the upper chamber of the co-culture plate to infect beas-2b cells.

[0040] The evaluation indicators selected in step (3) are selected from the aspects of cell level and protein expression level.

[0041] The evaluation index selected at the cellular level is specifically the determination of cell number.

[0042] The above-mentioned evaluation index selected from the protein expression level is specifically the determination of the expression level of the IL-6 inflammatory factor.

[0043] The expression of the IL-6 inflammatory factor was detected using an ELISA method. Standards were prepared according to the product instructions, and different concentration gradients of the standards were set. The kit procedures were followed, and the values ​​were analyzed and calculated using a microplate reader at a wavelength of 450 nm.

[0044] Beneficial effects of the present invention:

[0045] The present invention treats beas-2b cells with a poison solution and simultaneously stains human pulmonary macrophages. By constructing a co-culture model of the treated beas-2b cells and human pulmonary macrophages, the risk of CS exposure in chronic obstructive pulmonary disease and the specific etiology of COPD are assessed. Compared with the culture of airway epithelial cells alone, the co-culture system of beas-2b cells and human pulmonary macrophages can better simulate the physiological environment in the body and provide strong support for the evaluation of the degree of inflammatory response caused by smoke.

[0046] The evaluation method of the present invention is also applicable to the evaluation of other cigarette-related diseases that threaten human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is a flow chart of the technical solution;

[0048] Figure 2 is the survival rate of beas-2b cells at different toxin concentrations;

[0049] Figure 3 for macrophage culture and dye staining;

[0050] Figure 4 The fluorescence of macrophages in the lower chamber of co-culture migrated to the upper chamber after beas-2b infection and the number of macrophages entering the upper chamber;

[0051] Figure 5 The expression difference of inflammatory factor IL6 level after CS poisoning. DETAILED DESCRIPTION

[0052] The embodiments of the present invention will be further described below with reference to the accompanying drawings.

[0053] 1. Experimental Materials: Human bronchial epithelial cell line (beas-2b) and human pulmonary macrophage cell line. Beas-2b cells were purchased from American Type Culture Collection (Manassas, VA, USA); human pulmonary macrophages were purchased from CELLBIO (CBR-130027); the kit used for staining human pulmonary macrophages (CFDA SE Cell Proliferation and Tracing Detection Kit) was purchased from Nanjing Enjing Biotechnology Co., Ltd. and used strictly according to the manufacturer's instructions.

[0054] 2. Main experimental equipment: turntable smoke machine, biological safety cabinet, CO2 incubator, 96-well plate, Corning 6-well transwell (8μm), pipette, real-time fluorescence quantitative PCR instrument, and microplate reader.

[0055] 3. The co-culture system of the embodiment is specifically a transwell co-culture system, and the membrane pore size of the co-culture system is 5 to 8 μm.

[0056] 4. Fluorescent cell counting was performed using ImageJ software; PCR and ELISA data were analyzed and plotted using Prism 5.0.

[0057] Example 1

[0058] The method for constructing an in vitro cell co-culture model for evaluating the degree of tobacco smoke-induced inflammatory response in this example includes the following steps:

[0059] Technical solution flow chart as follows Figure 1 shown.

[0060] 1. Preparation of culture medium

[0061] (1) The beas-2b cell culture medium was BEGM BulletKit medium (CC-3171), purchased from Lonza, and used according to the instructions.

[0062] (2) The macrophage culture medium is DMEM high-glucose composite culture medium, which is prepared as follows: 90% DMEM high-glucose culture medium (DMEM high-glucose culture medium purchased from Hyclone Company, product number SH30022.01B) and 10% fetal bovine serum are mixed in a volume ratio of 9:1, and then 1% penicillin and streptomycin are added to the total volume ratio to shake well, package, and store at -20°C.

[0063] 2. Cell culture and passaging

[0064] Culture and passaging of beas-2b cells:

[0065] (1) Culture of beas-2b cells: In a cell culture room, aseptically add 5 ml of cell culture medium to a 15 ml centrifuge tube and a 100 × 20 mm culture dish in advance, and preheat in a 37°C CO2 incubator. Then, add the cell solution to the centrifuge tube, mix well, and centrifuge. Aspirate the supernatant, mix the cell pellet with 1 ml of culture medium, aspirate and add to the culture dish. Observe the cell morphology under a light microscope, and culture in an incubator. Remove the culture dish from the incubator regularly and observe under a light microscope. Culture for about 48 hours, aspirate the old culture medium, rinse the culture dish with DPBS, and add 5 ml of new culture medium.

[0066] (2) Passaging of beas-2b cells: Prepare 1 ml of 0.05% trypsin in advance and preheat it to room temperature. Aspirate the old culture medium in the original culture dish, wash twice with DPBS, and then add the prepared trypsin. When the beas-2b cells change from an adherent state to a floating state under a light microscope, quickly neutralize the trypsin with 3 ml of serum-containing BEBM culture medium. Transfer the liquid to a centrifuge tube and centrifuge, then remove the supernatant. Resuspend the cells in 2 ml of culture medium and divide them evenly into the prepared culture dishes containing 5 ml of culture medium. Observe and culture.

[0067] Culture and passaging of human lung macrophages:

[0068] (1) Cultivation of macrophages: In the cell culture room, add 5 ml of cell culture medium to a 15 ml centrifuge tube and a 50 ml cell culture flask under sterile conditions in advance, and place them in a 37°C CO2 incubator for preheating. Then add the cell solution to the centrifuge tube, mix well, and centrifuge, and remove the supernatant. Mix the cell pellet with 1 ml of culture medium, pipette it into the cell culture flask, observe the cell morphology under a light microscope, place it in an incubator for culture, and regularly remove the culture flask from the incubator and observe it under a light microscope. When the culture medium in the flask turns orange-yellow and the cells are proliferating vigorously, add 5 ml of culture medium to the flask.

[0069] (2) Passaging of macrophages: Observe macrophage proliferation to a certain extent under a light microscope. Add 5 ml of cell culture medium to each of three 50 ml culture flasks in advance and preheat in a 37°C CO2 incubator. Aspirate the cell fluid in the original culture flask into a 15 ml centrifuge tube, centrifuge, and remove the supernatant. Mix with 3 ml of cell culture medium and divide evenly into three culture flasks for observation and culture.

[0070] 3. Preparation of toxic substances

[0071] Preparation and collection of CS: CS is 3R4F mainstream standard cigarettes. Smoke condensate is extracted using a smoking machine. The collected smoke condensate is fully dissolved in 10 mg / ml DMSO to obtain a 10 mg / ml smoke condensate solution, i.e., the poisoning solution mother solution, which is frozen at -80°C for use.

[0072] 4. Cell Processing

[0073] (1) Infection of beas-2b cells: 4×10 cells were seeded in each well of a six-well transwell co-culture plate. 5 Mature beas-2b cells were prepared with 3R4F toxin solution. To model the co-culture toxin solution, the 3R4F toxin solution was diluted with BEBM medium to different concentrations. By testing the survival rate of cells at different concentrations, the toxin concentration with a survival rate of more than 80% was selected as the optimal toxin concentration for beas-2b in the co-culture system. The dilution concentration of the 3R4F toxin solution was 0-400μg / ml, and the specific dilution concentrations were 25μg / ml, 50μg / ml, 100μg / ml, 150μg / ml, and 200μg / ml. Figure 2 shown.

[0074] in, Figure 2 The horizontal axes 1-6 of A represent the concentrations of the poison solution as 0, 25μg / ml, 50μg / ml, 100μg / ml, 150μg / ml, and 200μg / ml, respectively. Figure 2 The horizontal axes 1-3 in B represent the concentrations of the poison solution, 0, 100 μg / ml, and 300 μg / ml, respectively. Based on the results of cell survival rates at different dilution concentrations, 300 μg / ml was finally selected as the optimal poison concentration.

[0075] The infection solution was diluted to 300 μg / ml with BEBM medium. The old medium of the cells to be infected was aspirated, and 2 ml of new culture medium was added to the lower chamber of the co-culture plate and 1 ml of infection solution was added to the upper chamber of the co-culture plate. The cells were cultured in an incubator for 2 h (1.5 to 2.5 h).

[0076] (2) Staining of human lung macrophages:

[0077] 1) Suspend 5×10 cells in 1 ml of CFDA SE cell labeling solution 6 The mature macrophages were placed in a 15 ml centrifuge tube.

[0078] 2) Dilute the CFDA SE cell stock solution (1000×) to 2× with CFDA SE cell labeling solution.

[0079] 3) Add 1 ml of CFDA SE cell storage solution (2×) to the 15 ml centrifuge tube in step 1) and mix gently.

[0080] 4) Place the centrifuge tube in a 37°C CO2 incubator and incubate for 10 minutes. Immediately after taking out, add 10 ml of DEDM high-glucose complex medium, mix well, and centrifuge. Remove the supernatant and wash repeatedly with 5 ml (5-10 ml) of DEDM high-glucose complex medium.

[0081] 5) Add 5 ml (5-10 ml) of DEDM high-glucose complex medium, mix well, and incubate in a 37°C CO2 incubator for 5 minutes to promote the staining effect. Remove the tube and centrifuge again, remove the supernatant, add 5 ml of high-glucose complex medium, and mix well.

[0082] 6) Observe under a fluorescence microscope (e.g. Figure 3 As shown), normal cell culture was performed.

[0083] The whole process was carried out under light-proof conditions.

[0084] 5. Co-culture of beas-2b cells and human lung macrophages

[0085] Beas-2b cells were co-cultured with human lung macrophages in six transwell co-culture plates, numbered 1, 2, 3, 4, 5, and control. Experimental design: Group 1 was co-cultured for 40 minutes, Group 2 for 80 minutes, Group 3 for 120 minutes, Group 4 for 160 minutes, Group 5 for 200 minutes, and the control group for 240 minutes. The control group was not treated with the toxin, while the remaining five groups were treated with the toxin at a concentration of 300 μg / ml. Based on the fluorescence effects observed at different incubation times, a 2-hour incubation time was selected as the optimal time.

[0086] After 2 h of co-culture, the co-culture system was taken out and the infection solution in the upper chamber and the culture medium in the lower chamber of the corresponding experimental well were aspirated. 2 ml of new BEBM culture medium containing 1% FBS was added to the upper and lower chambers of the co-culture system, and 1 / 6×5×10 6 The stained mature macrophages were placed in an incubator for co-culture, and fluorescent images of the upper chamber were taken under a fluorescence microscope.

[0087] Example 2

[0088] The method for evaluating the degree of tobacco smoke-induced inflammatory response comprises the following steps:

[0089] Steps 1-4 are the same as in Example 1.

[0090] 5. Co-culture of macrophages and beas-2b cells

[0091] Macrophages co-cultured with Beas-2b cells: Six-well co-culture plates were numbered 1, 2, 3, 4, 5, and control. The experimental design was as follows: Group 1 was co-cultured for 40 minutes, Group 2 for 80 minutes, Group 3 for 120 minutes, Group 4 for 160 minutes, Group 5 for 200 minutes, and the control group for 240 minutes. The control group was not treated with the toxin, while the remaining five groups were treated with the toxin at a concentration of 300 μg / ml. Based on the fluorescence effects observed at different incubation times, the optimal incubation time was 2 hours.

[0092] After the corresponding culture time, the six-well co-culture plate was taken out and the fluorescence image of the upper chamber was taken under a fluorescence microscope (the results are shown in the figure). Figure 4 A) from Figure 4 As can be seen from A, when the toxin concentration was 300 μg / ml, the number of macrophages migrating from the lower chamber to the upper chamber was significantly higher than that in the control group, indicating that the beas-2b cells in the upper chamber can promote the upward migration of macrophages under the action of the toxin solution, thereby protecting airway epithelial cells.

[0093] The number of macrophages entering the upper chamber was detected by cell counting. The results were as follows: Figure 4 As shown in Figure B, the horizontal axes 1-4 represent control group 1, control group 2, 300μg / ml-1, and 300μg / ml-2, respectively. Control group 1 and control group 2 are two parallel control groups, and 300μg / ml-1 and 300μg / ml-2 are two parallel experimental groups with a toxin concentration of 300μg / ml.

[0094] from Figure 4 B shows that the number of macrophages entering the upper chamber in the two experimental groups with a toxin concentration of 300 μg / ml was significantly higher than that in control group 1 and control group 2.

[0095] 6. ELISA: Add the cell co-culture supernatant sample to the sample well at 100ul / well, set up a blank well, and prepare a standard curve at the same time. Seal the plate with a sealing film and incubate at 37°C for 90min. After washing the plate three times with 300ul / well, add the diluted biotinylated antibody at 100ul / well, cover with a sealing film, and incubate at 37°C for 90min. After washing the plate three times, add the diluted Streptavidin-HRP at 100ul / well, cover with a sealing film, place in a humidified box, and incubate at 37°C for 30min. After washing the plate three times, add TMB at 100ul / well, develop for 15min, and quickly add the stop solution at 100μl / well. Read the value at a wavelength of 450nm using an enzyme reader. The results are as follows: Figure 5 shown.

[0096] from Figure 5 It can be seen that when the concentration of the poison solution was 300 μg / ml, the protein expression level of IL-6 inflammatory factor was significantly higher than that of the control group.

[0097] This application completed the co-culture of beas-2b and human lung macrophage cell line, and exposed beas-2b to CS culture, with a CS exposure concentration of 300 μg / ml. After the beas-2b cells were infected at the air-liquid interface culture, they could attract the macrophages co-cultured in the lower chamber to migrate to the upper chamber, and the ELISA test showed that the concentration of the inflammatory factor IL-6 in the experimental group was significantly different from that in the control group.

[0098] This method can be applied to in vitro exposure models of cigarette mainstream smoke, sidestream smoke, ambient smoke, and other smoke aerosols.

Claims

1. A method for evaluating the degree of tobacco smoke-induced inflammatory response for non-diagnostic purposes, characterized in that: The following steps are involved: 1) Beas-2b cells were cultured in the upper chamber of a co-culture plate and then exposed to the toxin. The exposure procedure was as follows: A. Collect the smoke condensate of the smoke to be tested and fully dissolve it in a solvent to obtain the poisoning mother solution; B. Add the poisoned mother solution to the culture medium of beas-2b cells to obtain the poisoned solution; C. Add the toxin solution diluted to 300 μg / mL into the upper chamber of the co-culture plate to infect beas-2b cells; 2) Place human lung macrophages stained with CFDA-SE live cell dye in the lower chamber of the co-culture plate and co-culture with the beas-2b cells treated with the toxin in step 1) for 0.5-10 hours; 3) Selecting evaluation indicators at the cellular level and protein expression level to divide the evaluation grade standards. The evaluation indicator selected at the cellular level specifically includes the measurement of cell number, which is the measurement of the fluorescence of macrophages migrating from the co-culture lower chamber to the upper chamber after beas-2b infection and the number of macrophages entering the upper chamber. The evaluation indicator selected at the protein expression level specifically includes the measurement of the expression level of the inflammatory factor IL-6; 4) evaluate the extent of tobacco smoke-induced inflammatory response; In step 1), beas-2b cells are placed in the upper chamber of the co-culture plate and the culture medium used includes BEBM basal medium and cytokines; the cytokines include at least one of BPE, Hydrocortisone, hEGF, Epinephrine, Insulin, Transferrin, Triiodothyronine, Retinoic Acid, and GA; In step 2), the culture medium for human lung macrophages is DMEM high-glucose complex medium; The co-culture system is a Transwell co-culture system, and the membrane pore size of the co-culture system is 5 to 8 μm; beas-2b cells are cultured at an air-liquid interface.

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