Tobacco product lung injury evaluation method based on murine lung organs

Abstract

The invention discloses a tobacco product lung injury evaluation method based on murine lung organs, which comprises the following steps: step A, preparing a trapping substance by using a tobacco product to obtain the tobacco product trapping substance; step B, performing induction culture on the mouse lung tissue by using an MAOs culture medium to obtain mouse lung organs; step C, pre-poisoning the lung organs of the mouse by using the tobacco product traps to determine IC50 and poisoning time of the tobacco product traps; and D, setting a concentration gradient according to the IC50 of the tobacco product traps, contaminating the lung organs of the mouse, and evaluating the lung injury caused by the tobacco product traps according to the physiological index change of the lung organs of the mouse. According to the method for evaluating the lung injury of the tobacco product by using the mouse lung organ, the influence of exposure of the tobacco product on the lung injury can be simulated, the effect difference between different tobacco products is reflected, and a more accurate evaluation method is provided for toxicological research of the tobacco product.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biology, and particularly relates to a method for evaluating lung injury of tobacco products based on murine lung organoids. Background Art

[0002] Today, with the accelerating globalization process, tobacco products, as one of the most stubborn threats in the field of public health, have triggered a health crisis that has transcended geographical boundaries. According to the World Health Organization, the number of deaths caused by tobacco-related diseases globally exceeds 8 million annually, with chronic obstructive pulmonary disease and lung cancer being the main causes of death. Harmful substances in tobacco smoke (such as tar, nicotine, polycyclic aromatic hydrocarbons, etc.) cause irreversible damage to the respiratory tract and alveolar structure through the synergistic action of multiple targets and multiple mechanisms. Among them, at the airway level, oxidative stress substances in the smoke generated by tobacco product aspiration can directly damage ciliated epithelial cells, resulting in a decrease in the ciliary beat frequency (the amplitude can reach 40 - 60% of the normal value), weakened motility, and stimulation of the mucus glands under the airway mucosa, leading to a significant decrease in mucus clearance ability. This change in the microenvironment promotes goblet cell hyperplasia and hypersecretion of mucus glands, forming mucus plugs that block small airways, directly causing gas exchange disorders and drug trafficking infections, leading to chronic obstructive pulmonary disease and gradually decreasing lung function. At the alveolar structure level, the imbalance of the protease / antiprotease system in tobacco leads to the degradation of elastic fibers in the alveolar septum, causing the alveoli to gradually expand and fuse to form emphysema. At the immune regulation level, the smoke generated by tobacco product aspiration can activate the NLRP3 inflammasome, triggering a cascade reaction of pro-inflammatory factors, thereby affecting pulmonary immune defense, triggering pulmonary inflammation, and causing progressive dyspnea such as pulmonary fibrosis, pneumothorax, or asthma in patients, seriously affecting the quality of life.

[0003] Currently, the evaluation of the lung damage and toxic effects of tobacco products is mainly based on cell and animal models. The cell model operation has the advantages of simplicity and a large detection throughput. However, due to the overly single type of cell model, during the in vitro culture process, cells will gradually lose their original state in vivo, and there are significant differences in the cell morphology, biological functions, and genetics of in vitro culture compared to in vivo cells. There are great limitations when applied to the study of lung injury caused by tobacco products. Although animal models can be used as one of the alternatives, there are ethical controversies and practical dilemmas in the study of tobacco products. They are restricted by various aspects such as complex operations, long experimental cycles, and high costs, making it difficult to detect in real-time and unable to conduct high-throughput screening experiments.

[0004] From 2D cell culture (a traditional cell culture method that suspends cells on a flat culture dish and allows cells to attach and grow on a planar surface) to 3D organoid models, the research paradigm for tobacco product-related lung injury is undergoing a profound transformation. 3D lung organoids have multiple cell types and can reproduce the characteristics of the airway. Existing evaluation methods have great limitations when applied to the evaluation of the lung injury caused by heated cigarettes, and the traditional exposure method is extremely likely to result in false-negative results. Therefore, it is necessary to establish an evaluation method for the lung injury of tobacco products using lung organoids, with the expectation of quickly and effectively discovering the toxicity and injury indicators of tobacco products and providing strong support for the mechanism of action of the multicellular interaction network in the lung injury response. Summary of the Invention

[0005] The purpose of this patent is to establish an evaluation method for the lung injury of tobacco products using lung organoids, with the expectation of quickly and effectively discovering the toxicity and injury indicators of tobacco products and providing strong support for the mechanism of action of the multicellular interaction network in the lung injury response.

[0006] To solve the above technical problems:

[0007] This patent provides a method for evaluating the lung injury of tobacco products based on murine lung organoids, including the following steps: Step A: Prepare a trap using a tobacco product to obtain a tobacco product trap; Step B: Induce and culture mouse lung tissue using MAOs medium to obtain mouse lung organoids; Step C: Pre-expose the mouse lung organoids to the tobacco product trap to determine the IC 50 of the tobacco product trap and the exposure time; Step D: Set a concentration gradient according to the IC 50 of the tobacco product trap, expose the mouse lung organoids, and evaluate the lung injury caused by the tobacco product trap according to the changes in the physiological indicators of the mouse lung organoids.

[0008] Further, Step A includes: Step A-1: Equilibrate the tobacco product for 72 h, and use a smoking machine to aspirate the equilibrated tobacco product through a deep aspiration mode to obtain the mainstream smoke or aerosol of the tobacco product; Step A-2: Collect the mainstream smoke using a Cambridge filter, and extract the mainstream smoke rich on the Cambridge filter through the DMSO extraction method to obtain a tobacco product trap, where the tobacco product trap is a traditional cigarette smoke trap; or, collect the aerosol through a solvent-free trapping method to prepare a tobacco product trap, where the tobacco product trap is a heated cigarette aerosol trap.

[0009] Further, the temperature for equilibrating the tobacco product is 21-23 °C, and the humidity for equilibration is 58-62%.

[0010] Furthermore, the traditional cigarette smoke capture includes the smoke capture of reference cigarette 1R6F; the heated tobacco aerosol capture includes the aerosol capture of heated tobacco product HTP.

[0011] Furthermore, step B includes: Step B-1: After obtaining normal mouse lung tissue by dissection, use a blade to chop the mouse lung tissue to obtain pretreated lung tissue; subject the pretreated lung tissue to two digestion reactions, and centrifuge to obtain a cell mass precipitate; Step B-2: Resuspend the cell mass precipitate with MAOs medium to obtain a cell suspension, mix the cell suspension with Matrigel in a ratio of 4:6, add it to a cell culture plate, and place it in a constant temperature incubator for induced culture to obtain mouse lung organoids.

[0012] Furthermore, the two digestion reactions are the first digestion reaction and the second digestion reaction; the digestive solution in the first digestion reaction is Collagenase I digestive solution, and the digestion time is 15 - 30 min; the digestive solution in the second digestion reaction is DNaseI, and the digestion time is 10 - 15 min;

[0013] Furthermore, MAOs medium is based on DMEM medium or F12 medium, and add penicillin-streptomycin mixture, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution, L-alanyl-glutamine solution, recombinant human R-Spondin-1 protein, recombinant human Noggin protein, human leukocyte antigen B27 additive, ROCK inhibitor, TGF-β type I receptor (ALK5 / 4 / 7) inhibitor, selective p38 MAPK inhibitor, fibroblast growth factor 7, fibroblast growth factor 10, puromycin, human neuregulin-β1, N-acetylcysteine and nicotinamide to the basic medium.

[0014] Furthermore, in MAOs medium, the concentration of penicillin-streptomycin mixture is 1×, the concentration of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution is 1×, the concentration of L-alanyl-glutamine solution is 1×, the final concentration of recombinant human R-Spondin-1 protein is 200 ng / mL, the final concentration of recombinant human Noggin protein is 300 ng / mL, the concentration of human leukocyte antigen B27 additive is 1×, the final concentration of ROCK inhibitor is 50 μM, the final concentration of TGF-β type I receptor (ALK5 / 4 / 7) inhibitor is 500 nM, the final concentration of selective p38 MAPK inhibitor is 1 μM, the final concentration of fibroblast growth factor 7 is 5 ng / mL, the final concentration of fibroblast growth factor 10 is 20 ng / mL, the final concentration of puromycin is 100 μg / mL, the final concentration of human neuregulin-β1 is 5 nM, the final concentration of N-acetylcysteine is 1 mM, and the final concentration of nicotinamide is 10 mM.

[0015] Further, step C specifically includes: performing CCK-8 and LDH cytotoxicity tests on mouse lung organoids using tobacco product captives to determine the IC 50 .

[0016] Further, the physiological indicators of mouse lung organoids include changes in reactive oxygen species levels and changes in tissue structure.

[0017] Further, a high-content cell imaging system is used to detect the reactive oxygen species in the mouse lung organoids after exposure to toxins, and the change in the reactive oxygen species level is used to evaluate the oxidative stress level of the tobacco product captives on the mouse lung organoids; a microscope is used to detect the tissue structure of the mouse lung organoids after exposure to toxins, and HE staining is used to evaluate the effect of the tobacco product captives on the tissue structure in the mouse lung organoids.

[0018] Further, the physiological indicators also include antibody detection. Different types of antibodies are used to stain different types of cells in the organoids to explore the effects on different cells after exposure to toxins.

[0019] This patent provides a method for evaluating lung injury of tobacco products using mouse lung organoids, which can simulate the effects of tobacco product exposure on lung injury. The exposure injury effects include changes in the reactive oxygen species level and tissue structure of the lung organoids. The effect differences between different tobacco products are reflected through the exposure injury effects, providing a more accurate evaluation method for the toxicological research of tobacco products. Description of the Drawings

[0020] The above content of this patent and the following specific implementation manners will be better understood when read in conjunction with the drawings. It should be noted that the drawings are only examples of the claimed technical solutions.

[0021] Figure 1 It is a characteristic morphology diagram of mouse airway organoids on the 5th day of induced culture;

[0022] Figure 2 It is an identification diagram of characteristic markers of mouse airway organoids (Acetylated α-tubulin is acetylated microtubule protein α, the green-labeled part; MUC5AC is a mucus-cilia marker, the red-labeled part; Hoechst is the cell nucleus, the blue-labeled part);

[0023] Figure 3 It is a cilia structure diagram in mouse airway organoids (where the red circle indicates the cilia structure of mouse airway organoids);

[0024] Figure 4Cytotoxicity results of mouse lung organoids detected by CCK-8 kit (where the abscissa is TPM, i.e., total particulate matter of mainstream cigarette smoke, with the unit of μg / mL; the ordinate is the control ratio, with the unit of %; blue is the smoke trap of reference cigarette 1R6F; red is the aerosol trap of representative heated cigarette HTP);

[0025] Figure 5 Cytotoxicity results of mouse lung organoids detected by LDH kit (where the abscissa is TPM, i.e., total particulate matter of mainstream cigarette smoke, with the unit of μg / mL; the ordinate is the control ratio, with the unit of %; blue is the smoke trap of reference cigarette 1R6F; red is the aerosol trap of representative heated cigarette HTP);

[0026] Figure 6 Fluorescence analysis of mouse airway organoids after 48 h of exposure to tobacco product traps (where 1R6F is a reference cigarette; HTP is a representative heated tobacco product; Ctrl is a blank control; ROS is a reactive oxygen species fluorescent probe; 33342 is the fluorescent dye Hoechst 33342, used for staining the nuclei of living cells; Merge is the merged image of the same field of view after staining with ROS reactive oxygen species fluorescent probe and fluorescent dye Hoechst 33342 respectively, and the scale bar is 100 μm);

[0027] Figure 7 Quantitative statistical chart of the average fluorescence intensity of ROS probe in mouse airway organoids after 48 h of exposure to tobacco product traps (where the abscissa is TPM, i.e., total particulate matter of mainstream cigarette smoke, with the unit of μg / mL; the ordinate is the control ratio, with the unit of %; blue is the smoke trap of reference cigarette 1R6F; red is the aerosol trap of representative heated cigarette HTP);

[0028] Figure 8 HE staining change diagram of the tissue structure of mouse airway organoids under different exposure doses of different tobacco product traps (where A is the effect of 1R6F on the tissue structure of airway organoids detected by HE staining under different exposure doses; B is the effect of HTP on the tissue structure of airway organoids detected by HE staining under different exposure doses; Ctrl is a blank control, and the scale bar is 100 μm). Detailed implementation mode

[0029] The detailed features and advantages of the present invention are described in detail in the following detailed implementation mode. The content is sufficient for any person skilled in the art to understand the technical content of the present invention and implement it accordingly. And according to the specification, claims and drawings disclosed in this specification, those skilled in the art can easily understand the related purposes and advantages of the present invention.

[0030] It should be noted that in this specification, similar reference numerals and letters denote similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0031] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will further describe in detail the embodiments of the present invention with reference to the drawings. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels unless otherwise specified.

[0032] (1) Source of sample materials

[0033] In this embodiment, the mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0034] The reference cigarette 1R6F and the representative heated cigarette (code HTP) were both purchased on the market.

[0035] (2) Source of reagents and consumables

[0036] Table 1 Sources and catalog numbers of reagents and consumables required for the experiment

[0037]

[0038]

[0039]

[0040] (3) Source of instruments and equipment

[0041] Table 2 Sources and models of instruments and equipment required for the experiment

[0042] Name Manufacturer Model Heated Cigarette Smoking Machine Qingdao Yizhong Technology Co., Ltd. NSM 100 High-Speed Centrifuge Beckman Coulter, Inc. (USA) AllegraTM 64R Type -80°C Ultra-Low Temperature Refrigerator Thermo Fisher Scientific (Germany) 906GP Incubator Thermo Fisher Scientific (Germany) 160i High-Content Cell Imaging System PerkinElmer, Inc. (USA) Operetta CLS Microtome Shanghai Leica Instruments Co., Ltd. RM2016 Microscope Nikon (Japan) Nikon Eclipse E100 Microplate Reader Tecan Group Ltd. (Switzerland) SPARK

[0043] Example

[0044] A method for evaluating lung injury of tobacco products based on murine lung organoids, comprising the following steps:

[0045] S1. Preparation of the tobacco product trap, specifically referring to the following steps:

[0046] 1. Place the reference cigarette 1R6F and the representative heated cigarette HTP of tobacco products purchased on the market in a temperature- and humidity-controlled room for 72 h before suction. The temperature for tobacco product equilibration is 21 - 23 °C, and the humidity for equilibration is 58 - 62%.

[0047] 2. After 72 hours of equilibration, it is used for subsequent suction by a smoking machine. The suction conditions conform to the Canadian deep suction mode recommended by the World Health Organization's Tobacco Product Regulation Research Group, that is, the suction volume is 55 mL, the suction frequency is 30 s, and the suction parameters with 100% blocked ventilation holes generate 1R6F mainstream cigarette smoke and HTP aerosol on the smoking machine.

[0048] 3. Use a Cambridge filter to collect TPM (Total Particulate Matter in mainstream cigarette smoke) of 1R6F mainstream cigarette smoke. The Cambridge filter is a glass fiber sheet fixed with an organic binder (polyacrylate) and can effectively retain total particulate matter at room temperature.

[0049] 4. Use the DMSO (Dimethyl sulfoxide) extraction method to extract the 1R6F mainstream cigarette smoke rich on the Cambridge filter to obtain the 1R6F cigarette smoke trap. The specific steps are as follows: Place the device with the Cambridge filter rich in 1R6F mainstream cigarette smoke into a filter in-situ high-speed centrifuge, perform high-speed centrifugation extraction with DMSO, collect the centrifuged liquid, and filter and sterilize it with a 0.22 μm sterile filter membrane, which is the 1R6F cigarette smoke trap.

[0050] 5. Collect and extract the HTP aerosol by solvent-free trapping method to prepare a heated tobacco aerosol trap, that is, the HTP aerosol trap. The specific steps are as follows: Through an aerosol trapping device, directly trap the aerosol from the HTP tobacco product, and in a highly centrifuged manner, directly centrifuge the aerosol trap physically, which is the HTP aerosol trap.

[0051] 6. Aliquot the 1R6F cigarette smoke trap and the HTP aerosol trap into 2 mL cryotubes and store them in a -80 °C ultra-low temperature refrigerator for subsequent experiments.

[0052] S2. Construction of mouse lung organoids, the specific reference steps are as follows:

[0053] 1. After obtaining normal mouse lung tissue by dissection, use a blade to cut the mouse lung tissue into small pieces smaller than 2 mm 3 to obtain pretreated lung tissue.

[0054] 2. Add 2 mL of Collagenase I digestion solution with a concentration of 2 mg / mL to a petri dish, disperse the pretreated lung tissue, and then transfer all of it to a 15 mL centrifuge tube containing the remaining 4 mL of Collagenase I digestion solution for subsequent two digestion reactions.

[0055] 3. Place the 15 mL centrifuge tube containing the pretreated lung tissue and Collagenase I digestion solution in a 37°C constant temperature incubator for the first digestion reaction to dissociate the pretreated lung tissue cells for the first time. The time for the first digestion reaction is 15 - 30 min.

[0056] 4. After the first digestion reaction, directly add 50 μL of DNase I to the 15 mL centrifuge tube in step 3 above, and place it in a 37°C constant temperature incubator for the second digestion reaction to dissociate the pretreated lung tissue cells for the second time. The time for the second digestion reaction is 10 - 15 min.

[0057] 5. Filter the pretreated lung tissue after the second digestion reaction through 100 μm and 40 μm cell strainers into a 50 mL centrifuge tube, and transfer all of it to a new 15 mL centrifuge tube.

[0058] 6. Centrifuge at 500 rpm for 10 min, discard the supernatant to obtain the precipitated cell mass after dissociation.

[0059] 7. Resuspend the precipitated cell mass obtained in step 6 above with a small amount of MAOs medium (MAOs medium is based on DMEM or F12 medium and specific growth factors and small molecule induction reagents are added to it, mainly including: 1× penicillin - streptomycin mixture, 1× 4 - (2 - hydroxyethyl)-1 - piperazineethanesulfonic acid buffer (HEPES), 1× L - alanyl - L - glutamine solution (GlutMAX), 200 ng / mL recombinant human R - Spondin - 1 protein (R - spondin - 1), recombinant human Noggin protein (Noggin) with a final concentration of 300 ng / mL, 1× human leukocyte antigen B27 additive (B27), 50 μM ROCK inhibitor (Y27632), 500 nM TGF - β type I receptor (ALK5 / 4 / 7) inhibitor (A8301), 1 μM selective p38 MAPK inhibitor (SB202190), fibroblast growth factor 7 (FGF - 7) with a final concentration of 5 ng / mL, fibroblast growth factor 10 (FGF - 10) with a final concentration of 20 ng / mL, Primocin with a final concentration of 100 μg / mL, human neuregulin - β1 (Human Heregulin - β1) with a final concentration of 5 nM, N - acetylcysteine with a final concentration of 1 mM, and nicotinamide with a final concentration of 10 mM) to obtain a cell suspension, and count the cell mass in the cell suspension.

[0060] 8. Mix the cells at a ratio of 40 μL cell suspension + 60 μL Matrigel, and seed the cells in the center of a 48-well plate at a rate of 30 μL per well, with approximately 300 cell clusters seeded per well.

[0061] 9. Place the 48-well cell culture plate in a 37°C constant temperature incubator to gel for about 10 minutes, then add 200 μL of MAOs culture medium to each well, and place it in a 37°C constant temperature incubator again for induction culture to obtain mouse lung organoids.

[0062] The results are as follows Figures 1 to 3 As shown, around the 5th day after MAOs induction, the mouse lung organoids reached the optimal state ( Figure 1 ); Immunofluorescence identification of characteristic markers showed that MAOs could successfully express MUC5AC, a characteristic marker of secretory cells, and Acetylated α-tubulin, a characteristic marker of ciliated cells, indicating that the constructed MAOs at least contained characteristic cell types of lung tissue ( Figure 2 ); and the obtained MAOs can achieve ciliation ( Figure 3 ). The results showed that mouse lung organoids were successfully constructed.

[0063] S3. Pre-experimental study of mouse lung organoids. The specific steps are as follows:

[0064] 1. Tobacco product captures (i.e., 1R6F smoke captures and HTP aerosol captures) were used to pre-contaminate the mouse lung organoids constructed in step S2 using the CCK-8 (Cell Counting Kit-8) or LDH (lactate dehydrogenase) cytotoxicity assay to determine the IC value of tobacco products. 50 (Halfmaximal inhibitory concentration) and exposure time.

[0065] 2. CCK-8 kit to detect cytotoxicity

[0066] (1) The concentration gradient of 1R6F smoke capture was set as: 1.0 mg / mL, 0.9 mg / mL, 0.8 mg / mL, 0.7 mg / mL, 0.5 mg / mL, 0.3 mg / mL, 0.25 mg / mL, 0.2 mg / mL, 0.15 mg / mL, 0.1 mg / mL, 0.05 mg / mL and 0. A 96-well plate was used, and 100 μL of 1R6F smoke capture of the corresponding concentration was added to each well. Three parallel tests were set for each concentration gradient, and the experiment was repeated three times independently.

[0067] (2) Set the concentration gradient of the HTP aerosol trap as: 20 mg / mL, 17 mg / mL, 14 mg / mL, 11 mg / mL, 8 mg / mL, 7 mg / mL, 5 mg / mL, 3 mg / mL, 1 mg / mL, and 0. Using a 96-well plate, add 100 μL of the HTP aerosol trap with the corresponding concentration to each well. Set three parallel detections for each concentration gradient and conduct 3 independent replicate experiments.

[0068] After 48 h of exposing the mouse lung organoids to the toxin, aspirate the toxin solution, and add 100 μL of 10% CCK-8 medium to each well. Incubate in a 37 °C constant temperature incubator for 2 - 3 h. After incubation, place the 96-well plate into an ELISA reader and detect the absorbance of each well with 450 nm as the reference wavelength.

[0069] The calculation formula for the cell viability of the mouse lung organoids after being exposed to the toxin is as follows:

[0070] Cell viability = average absorbance of the test substance group / average absorbance of the blank group × 100%.

[0071] 3. Detect cell toxicity using an LDH kit

[0072] (1) Set the concentration gradient of the 1R6F cigarette smoke trap as: 1.0 mg / mL, 0.9 mg / mL, 0.8 mg / mL, 0.7 mg / mL, 0.6 mg / mL, 0.5 mg / mL, 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, and 0. Using a 96-well plate, add 100 μL of the 1R6F cigarette smoke trap with the corresponding concentration to each well. Set three parallel detections for each concentration gradient and conduct 3 independent replicate experiments;

[0073] (2) Set the concentration gradient of the HTP aerosol trap as: 20 mg / mL, 18 mg / mL, 15 mg / mL, 12 mg / mL, 9 mg / mL, 6 mg / mL, 4 mg / mL, 2 mg / mL, 1 mg / mL, and 0. Using a 96-well plate, add 100 μL of the HTP aerosol trap to each well. Set three parallel detections for each concentration gradient and conduct 3 independent replicate experiments.

[0074] After 48 h of exposing the mouse lung organoids to the toxin, transfer 80 μL of the toxin solution containing the 1R6F cigarette smoke trap and the HTP aerosol trap from each well to a new 96-well plate. Then, add 80 μL of the LDH detection working solution to each well in the new 96-well plate, mix well with the toxin solution, and incubate in the dark at room temperature (about 25 °C) for 20 min.

[0075] Use an ELISA reader to measure the absorbance value at 492 nm. Use any wavelength of 600 nm or greater than 600 nm as the reference wavelength for dual-wavelength measurement.

[0076] The calculation formula for cytotoxicity or mortality rate (%) is as follows:

[0077] Cytotoxicity or mortality rate (%) = ((Absorbance of treated sample - Absorbance of sample control well)) / ((Absorbance of maximum enzyme activity of cells - Absorbance of sample control well)) × 100%.

[0078] 4. The results, as Figure 4 and Figure 5 shown, were used to determine the IC 50 values of 1R6F cigarette smoke condensate and HTP aerosol condensate on murine airway lung organoids by the CCK-8 cytotoxicity assay method, which were 348.9 μg / mL and 8033 μg / mL respectively; the IC 50 values of 1R6F cigarette smoke condensate and HTP aerosol condensate on murine airway lung organoids determined by the LDH cytotoxicity assay method were 330 μg / mL and 7165 μg / mL respectively.

[0079] In subsequent experiments, according to the IC 50 of the tobacco products, multiple concentration gradients were set.

[0080] Among them, the exposure concentration gradients of 1R6F cigarette smoke condensate were: 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL; the exposure concentration gradients of HTP aerosol condensate were: 4000 μg / mL, 6000 μg / mL, 8000 μg / mL, 10000 μg / mL, and 12000 μg / mL.

[0081] The murine lung organoids constructed in step S2 were respectively exposed to 1R6F cigarette smoke condensate and HTP aerosol condensate. For each concentration gradient, 3 technical replicates were set for each test sample, and the exposure time was 48 h.

[0082] S4. The detection of reactive oxygen species in tobacco products based on murine lung organoids is specifically as follows:

[0083] The Reactive Oxygen Species Assay Kit (also known as the ROS Assay Kit) is a kit for detecting reactive oxygen species using the fluorescent probe DCFH-DA. DCFH-DA itself has no fluorescence and can freely cross the cell membrane. After entering the cell, it can be hydrolyzed by intracellular esterase to generate DCFH, and DCFH cannot penetrate the cell membrane, so that the probe can be easily loaded into the cell. Reactive oxygen species in the cell can oxidize non-fluorescent DCFH to generate fluorescent DCF, and by detecting the fluorescence of DCF, the level of reactive oxygen species in the cell can be known.

[0084] (1) Within the first 6 hours before the end of the above-mentioned exposure time, add the hydrogen peroxide positive control reagent provided in the reactive oxygen species detection kit to each well of a 48-well cell culture plate.

[0085] (2) After the end of the exposure time, add 100 μL of the ROS detection solution provided in the reactive oxygen species detection kit to each well, and incubate with shaking at room temperature for 20 min.

[0086] (3) After incubation, discard the supernatant, wash 1 - 2 times with 100 - 200 μL of 1×PBS, add 100 μL of nuclear stain to each well, and incubate at room temperature for nuclear staining for 5 - 6 min.

[0087] (4) After the end of the nuclear staining incubation at room temperature, wash 1 - 2 times with 100 - 200 μL of 1×PBS, and then store with 100 - 200 μL of 1×PBS.

[0088] (5) Use a high-content cell imaging system to collect and analyze images.

[0089] The results are as Figure 6 、 Figure 7 shown. In mouse lung organoid cells, the generation and elimination of ROS are in dynamic balance. When the cells are in an oxidative stress state, a large amount of ROS is produced in the mitochondria. Excessive ROS will cause oxidative damage to proteins, and then cause cell damage and apoptosis.

[0090] By detecting the ROS content in mouse lung organoid cells using the above fluorescent probe, the fluorescence analysis results show that both the 1R6F cigarette smoke condensate and the HTP aerosol condensate can induce an increase in ROS levels in the mouse lung organoid model, and induce the production of reactive oxygen species in a dose-dependent manner. The increase in ROS levels will lead to the disruption of the redox balance. Among them, the increase in ROS levels induced by the 1R6F cigarette smoke condensate is limited. Compared with the 1R6F cigarette smoke condensate, the effect of the HTP aerosol condensate is more significant and dose-dependent, indicating that there are certain differences in the induction of oxidative stress by different tobacco products.

[0091] S5. HE staining of mouse lung organoids after exposure to tobacco products. The specific reference steps are as follows:

[0092] 1. After mouse lung organoids are exposed to the 1R6F cigarette smoke condensate and the HTP aerosol condensate in tobacco products for 48 h, collect tissue samples and fix the collected tissue samples with 4% paraformaldehyde for 1 h.

[0093] 2. Sequentially place the fixed tissue samples into 70% ethanol for 2 h, 80% ethanol for 2 h, 95% ethanol for 2 h, and absolute ethanol for 2 h for dehydration, and then place them into xylene for clearing.

[0094] 3. Immerse the transparent tissue sample in molten paraffin for wax infiltration, and then place the tissue sample in an embedding cassette and wait for the paraffin to cool and solidify.

[0095] 4. Dewax the paraffin section to water: Fix the embedded paraffin block on a microtome, cut it into thin slices with a thickness of 4 - 6 μm, attach them to glass slides, and sequentially place the sections into xylene I for 20 min - xylene II for 20 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - 75% alcohol for 5 min, and finally wash with tap water to make the sections completely in an aqueous environment.

[0096] 5. Hematoxylin staining: Immerse the section in hematoxylin staining solution for 3 - 5 min, wash with tap water, differentiate with a differentiating solution (1% hydrochloric acid alcohol solution), wash with tap water, blue with a blueing solution (saturated lithium carbonate solution), and rinse with running water until the water becomes clear.

[0097] 6. Eosin staining: Immerse the section in 85% and 95% gradient alcohols for dehydration for 5 min each, and then immerse it in eosin staining solution for 5 min.

[0098] 7. Dehydration and mounting: Sequentially place the section into absolute ethanol I for 5 min - absolute ethanol II for 5 min - absolute ethanol III for 5 min - xylene I for 5 min - xylene II for 5 min for clearing, and mount with neutral balsam.

[0099] 8. Microscopic examination and image acquisition and analysis.

[0100] The results are as Figure 8 shown. After exposing mouse lung organoids to the trap extracts of two tobacco products, it can be seen from the HE staining results that both the 1R6F cigarette smoke trap extract and the HTP aerosol trap extract can cause tissue damage to mouse lung organoids under high - dose exposure, resulting in irregular tissue structure, loose arrangement, and nuclear rupture.

[0101] In summary, both the 1R6F cigarette smoke trap extract and the HTP aerosol trap extract can cause tissue damage in lung organoids, and it is positively correlated with the dose.

[0102] Therefore, it can be concluded that this patent provides a method for evaluating lung injury of tobacco products using mouse lung organoids, which can simulate the impact of tobacco product exposure on lung injury. The exposure injury effects include changes in the reactive oxygen species level and tissue structure of lung organoids. The effect differences between different tobacco products are reflected through the exposure injury effects, providing a more accurate evaluation method for the toxicological research of tobacco products.

[0103] The terms and expressions used herein are for descriptive purposes only, and the present invention should not be limited to these terms and expressions. The use of these terms and expressions does not mean excluding any equivalent features of the illustration and description (or parts thereof), and it should be recognized that various modifications that may exist should also be included within the scope of the claims. Other modifications, variations, and substitutions may also exist. Accordingly, the claims should be regarded as covering all such equivalents.

[0104] Similarly, it should be noted that although the present invention has been described with reference to the current specific embodiments, those of ordinary skill in the art should recognize that the above embodiments are only used to illustrate the present invention, and various equivalent changes or substitutions can be made without departing from the spirit of the present invention. Therefore, as long as the changes and modifications of the above embodiments are within the scope of the spirit of the present invention, they will fall within the scope of the claims of the present invention.

Claims

1. A method for evaluating lung injury of tobacco products based on murine lung organoids, characterized in that, It includes the following steps: Step A: Using a tobacco product to prepare a trap, obtaining a tobacco product trap; Step B: Using a MAOs medium to induce the culture of mouse lung tissue, obtaining mouse lung organoids; Step C: Pre-expose the mouse lung organoids to the tobacco product trap to determine the IC 50 and exposure time of the tobacco product trap; Step D: Based on the IC of the tobacco product trap 50 Set a concentration gradient, expose the mouse lung organoids to the toxin, and evaluate the lung injury caused by the tobacco product trap according to the changes in the physiological indicators of the mouse lung organoids.

2. The method for evaluating lung injury of tobacco products based on murine lung organoids according to claim 1, wherein The said Step A includes: Step A-1: Equilibrating the said tobacco product for 72 h, and using a smoking machine to aspirate the equilibrated tobacco product through a deep aspiration mode, obtaining the mainstream smoke or aerosol of the tobacco product; Step A-2: Using a Cambridge filter to collect the mainstream smoke, and extracting the mainstream smoke rich on the Cambridge filter through a DMSO extraction method, obtaining the tobacco product trap, and the tobacco product trap is a traditional cigarette smoke trap; or, Collecting the aerosol through a solvent-free trapping method to prepare the tobacco product trap, and the tobacco product trap is a heated cigarette aerosol trap.

3. The method for evaluating lung injury of tobacco products based on murine lung organoids according to claim 2, wherein In the said Step A-1, the temperature for equilibrating the tobacco product is 21-23 °C, and the humidity for equilibration is 58-62%.

4. The method for evaluating lung injury of tobacco products based on murine lung organoids according to claim 3, wherein The said traditional cigarette smoke trap includes a 1R6F reference cigarette smoke trap; the said heated cigarette aerosol trap includes a heated cigarette HTP aerosol trap.

5. The method for evaluating lung injury of tobacco products based on murine lung organoids according to claim 1, wherein The said Step B includes: Step B-1: After obtaining normal mouse lung tissue through dissection, using a blade to chop the mouse lung tissue into pieces, obtaining pretreated lung tissue; subjecting the pretreated lung tissue to two digestion reactions, and centrifuging to obtain a cell mass precipitate; Step B-2: Resuspending the cell mass precipitate with the said MAOs medium to obtain a cell suspension, mixing the cell suspension with Matrigel in a ratio of 4:6, adding the mixture to a cell culture plate, and placing it in a constant temperature incubator for induced culture, obtaining the mouse lung organoids.

6. The method for evaluating lung injury of a tobacco product based on mouse-derived lung organoids according to claim 5, wherein The said two digestion reactions are a first digestion reaction and a second digestion reaction; the digestive juice in the first digestion reaction is Collagenase I digestive juice, and the digestion time is 15-30 min; the digestive juice in the second digestion reaction is DNase I, and the digestion time is 10-15 min; The said MAOs medium is based on DMEM medium or F12 medium, and adding a penicillin-streptomycin mixture, a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution, an L-alanyl-glutamine solution, a recombinant human R-Spondin-1 protein, a recombinant human Noggin protein, a human leukocyte antigen B27 additive, a ROCK inhibitor, a TGF-β type I receptor (ALK5 / 4 / 7) inhibitor, a selective p38 MAPK inhibitor, fibroblast growth factor 7, fibroblast growth factor 10, puromycin, human neuregulin-β1, N-acetylcysteine, and nicotinamide to the basic medium.

7. The method for evaluating lung injury of tobacco products based on murine lung organoids according to claim 6, wherein In the MAOs medium, the concentration of the penicillin-streptomycin mixture is 1×, the concentration of the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer is 1×, the concentration of the L-alanyl-glutamine solution is 1×, the final concentration of the recombinant human R-Spondin-1 protein is 200 ng / mL, the final concentration of the recombinant human Noggin protein is 300 ng / mL, the concentration of the human leukocyte antigen B27 additive is 1×, the final concentration of the ROCK inhibitor is 50 μM, the final concentration of the TGF-β type I receptor (ALK5 / 4 / 7) inhibitor is 500 nM, the final concentration of the selective p38MAPK inhibitor is 1 μM, the final concentration of the fibroblast growth factor 7 is 5 ng / mL, the final concentration of the fibroblast growth factor 10 is 20 ng / mL, the final concentration of the puromycin is 100 μg / mL, the final concentration of the human neuregulin-β1 is 5 nM, the final concentration of the N-acetylcysteine is 1 mM, and the final concentration of the nicotinamide is 10 mM.

8. The method for evaluating lung injury of tobacco products based on murine lung organoids according to claim 1, wherein The specific steps of step C are as follows: Use the tobacco product trap to conduct CCK-8 and LDH cytotoxicity tests on the mouse lung organoids to determine the IC of the tobacco product trap 50 .

9. The method for evaluating lung injury of tobacco products based on murine lung organoids according to claim 1, wherein The physiological indexes of the mouse lung organoids include changes in the level of reactive oxygen species and changes in the tissue structure.

10. The method for evaluating lung injury of tobacco products based on murine lung organoids according to claim 9, wherein a high-content cell imaging system is used to detect the reactive oxygen species of the mouse lung organoids after exposure to the toxin, and the change in the level of reactive oxygen species is used to evaluate the oxidative stress level of the tobacco product trap on the mouse lung organoids; a microscope is used to detect the tissue structure of the mouse lung organoids after exposure to the toxin, and HE staining is used to evaluate the effect of the tobacco product trap on the tissue structure in the mouse lung organoids; the physiological indexes further include antibody detection, and different types of antibodies are used to stain different types of cells in the organoids to explore the effects on different cells after exposure to the toxin.

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