Construction method and application of human-derived far-end airway organ-like COPD model

Abstract

The invention discloses a construction method and application of a human-derived far-end airway organ-like COPD model, the constructed human-derived far-end airway organ-like COPD model is highly similar to COPD far-end airway tissue on the transcriptional level, and the pathological characteristics of COPD are well restored on the protein expression level and cilia function level. Therefore, the human-derived far-end airway organ-like COPD model constructed in vitro can simulate the COPD lesion of the far-end airway, and an available model is provided for researching the COPD pathological mechanism of the far-end airway. In addition, the requirements on time, temperature and quality inspection indexes of CSE extraction in operation are clear, and the batch-to-batch extraction quality and experiment repeatability of CSE can be well ensured. The cilia function is used as a detection index, the improvement effect of the medicine on the lung function can be better reflected, and data support of the function level can be provided for COPD medicine research.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for constructing a human-derived distal airway organoid COPD model and its application. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a highly heterogeneous chronic respiratory disease characterized by chronic respiratory symptoms (dyspnea, cough, sputum) due to progressive and irreversible airflow obstruction caused by airway remodeling, inflammation and / or alveolar abnormalities.

[0003] Since spirometry is currently the gold standard for diagnosing COPD, animal models are still the main vehicle for studying COPD. However, there are species differences between different species, and the research results obtained in animal models are inconsistent with the occurrence and development mechanisms of COPD in humans. At the same time, since COPD patients usually lack the necessary surgical indications, the scope of surgical application is small, and relevant clinical samples are difficult to obtain, research progress is greatly limited. It is urgent to develop a model that can reflect the pathological characteristics of human COPD to conduct related research. Studies have shown that the distal airway is the site of early and major lesions of COPD. However, the existing human airway organoids are mainly bronchial organoids of the proximal large airways or have no clear positioning, which is not suitable for in vitro studies of COPD lesions and disease occurrence and development mechanisms in the distal airways.

[0004] In addition, the prevalence of COPD is directly related to the smoking rate, and smoking is the main risk factor for COPD. Cigarette smoke extract (CSE) is used for in vitro modeling of COPD, and a COPD organoid model can be constructed in vitro to simulate COPD lesions, providing a usable model for related research. However, previous CSE in vitro modeling has mainly focused on 2D cells or alveolar organoids, and has been less used in airway organoids. How to use CSE to construct a human-derived distal airway organoid COPD model to simulate COPD lesions in the distal airways is a technical problem that technicians in this field need to solve. Summary of the invention

[0005] The present invention provides a method for constructing a human-derived distal airway organoid COPD model and its application, so as to construct a human-derived distal airway organoid COPD model by using CSE to better simulate COPD lesions in the distal airway.

[0006] One aspect of the present invention provides a method for constructing a human distal airway organoid COPD model, comprising: Step S1, immersing human distal lung tissue in digestion solution, digesting at 37°C for 30-45 minutes, using a mild sample buffer to terminate the digestion, cutting the tissue into pieces, repeatedly blowing to obtain a single cell suspension, and obtaining human lung tissue single cell a after centrifugation; Step S2, filtering the single cell a with a cell mesh to collect the human lung tissue single cell b, removing the supernatant after centrifugation, and washing 1-2 times with a washing solution to obtain the human lung tissue single cell c; Step S3, observe whether the single cell c has red clumps. If no red clumps are seen, directly proceed to step S4; if red clumps are seen, add 1 mL of red blood cell lysis solution to lyse at room temperature for 3 minutes, then separate at a speed of 300g, discard the supernatant, and obtain human lung tissue single cells d; Step S4, mixing single cell c or single cell d with matrix gel and then seeding the plate, inverting the plate and letting it stand in a cell culture incubator, adding organoid expansion medium after the gel solidifies, and continuing to culture in the cell culture incubator, replacing the medium every 3-4 days to obtain human lung organoids of the first generation; Step S5, after enzymatic hydrolysis of the Yuan generation human lung organoids, mixing with matrix gel and then seeding, inverting in a cell culture incubator and letting it stand, adding organoid expansion medium after the gel solidifies, continuing to culture in the cell culture incubator, replacing the organoid differentiation medium after 7-10 days of culture, replacing the medium every 3-4 days, and culturing for 11-14 days to obtain human distal airway organoids; Step S6, cut off the filter of a cigarette, connect the end near the filter to one end of the connecting tube, connect the other end of the connecting tube to the syringe needle connector containing 5 mL of organoid differentiation medium, light the cigarette, and draw the cigarette smoke into the syringe. When the cigarette has a preset length remaining, use the needle cap and sealing glue to seal the syringe, and then put the syringe containing the cigarette smoke and organoid differentiation medium into a 37°C shaker and shake to mix for 10 minutes; Step S7, when the sealing glue and the needle cap are removed, no cigarette smoke escapes, indicating that the cigarette smoke and the culture medium have been fully mixed, and the cigarette smoke extract e is obtained. The cigarette smoke extract e is filtered using a 0.22 μm filter, 100 μL is drawn into a 96-well plate, and the absorbance value A at 320 nm is detected by an ELISA instrument. 320 and the absorbance at 540 nm A 540 , when A 320 -A 540 =1.2, the cigarette smoke extract e is defined as 100% CSE; Step S8, dilute 100% CSE into 20% CSE using organoid differentiation medium, and use 20% CSE to continuously culture human-derived distal airway organoids for 72 hours, replacing fresh 20% CSE every 24 hours to obtain a human-derived distal airway organoid COPD model.

[0007] Another aspect of the present invention provides the use of the human-derived distal airway organoid COPD model obtained by the above-mentioned construction method in the detection of ciliary beat function for non-therapeutic purposes.

[0008] Specifically, in the successfully constructed human distal airway organoid COPD model, 20% CSE containing drugs at preset concentrations was added and administered continuously for 3 days. During this period, new 20% CSE containing drugs at preset concentrations was replaced every 24 hours, and then ciliary beat function testing was performed.

[0009] Beneficial effects: (1) The human distal airway organoid COPD model constructed by the method of the present invention is highly similar to the COPD distal airway tissue at the transcriptional level, and well restores the pathological characteristics of COPD at the protein expression level and ciliary function level. This indicates that the human distal airway organoid COPD model constructed in vitro by the present invention can simulate COPD lesions in the distal airway and provide a usable model for studying the pathological mechanism of COPD in the distal airway.

[0010] (2) The present invention has clear requirements on the time, temperature and quality inspection indexes of CSE extraction in operation, which can well ensure the extraction quality and experimental repeatability of CSE between batches.

[0011] (3) The present invention conducts drug screening research by detecting ciliary beating function. Compared with previous studies that judge the drug treatment effect based on cell morphology and size, transcription level or protein level, the present invention uses ciliary function as a detection indicator to better reflect the drug's improvement on lung function, and can provide functional level data support for COPD drug research. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a diagram showing the immunofluorescence staining results of human distal airway organoids obtained in Example 1; Figure 2 It is the bright field image before and after modeling in Example 2; Figure 3 It is the bright field image before and after modeling in Example 3; Figure 4 It is the bright field image before and after modeling in Example 4; Figure 5The results of immunofluorescence staining of MUC5AC and ACCTUB of the human-derived distal airway organoid COPD model obtained in Example 2 and the human-derived distal airway organoid obtained in Example 1; Figure 6 This is a comparison result of the ciliated cell ratio and goblet cell ratio of the human-derived distal airway organoid COPD model obtained in Examples 2-4 and the human-derived distal airway organoid obtained in Example 1; Figure 7 This is a comparison chart of the ciliary beat frequencies obtained in Examples 1-4. DETAILED DESCRIPTION

[0013] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0014] Embodiment 1: A method for constructing a human distal airway organoid, comprising: Step S1, immersing the human distal lung tissue in a digestion solution and digesting it at 37°C for 30 minutes, wherein the human distal lung tissue is obtained from a patient with lung nodules (not suffering from COPD), and the site source is the tissue 2-3 cm away from the lung nodules. The digestion solution is TrypLE digestion solution containing 50mM EDTA, and the digestion is terminated using a mild sample buffer. The mild sample buffer is: PBS buffer containing 2% BSA and not containing calcium and magnesium ions. The tissue is cut into pieces, and a single cell suspension is obtained by repeated blowing. After centrifugation, a human lung tissue single cell a is obtained. The centrifugation conditions are: centrifugal speed 300g, temperature 4°C, and centrifugation time 10min.

[0015] Step S2, filter the single cell a with a 70 μm cell mesh to collect the human lung tissue single cell b, remove the supernatant after centrifugation, the centrifugation conditions are: centrifugal speed 300g, temperature 4°C, centrifugation time 10min, and wash twice with a cleaning solution to obtain the human lung tissue single cell c, the composition and content of the cleaning solution are: amphotericin B, 3ug / mL; Primocin, 1000μg / mL; gentamicin, 500μg / mL.

[0016] Step S3: upon observation, red clumps are visible in the single cell c, and 1 mL of red blood cell lysis solution is added to lyse the single cell c at room temperature for 3 minutes, and then the single cell c is separated at a speed of 300 g, and the supernatant is discarded to obtain the single cell d of human lung tissue.

[0017] Step S4, the single cell d is mixed with the extracellular matrix gel and plated, and the plate is inverted and placed in a cell culture incubator. After the gel solidifies, an organoid expansion medium is added, and the composition and content of the organoid expansion medium are as follows: Advanced DMEM / F12, 1X; R-Spondin 1, 500 ng / mL; FGF 7, 25 ng / mL; FGF 10, 100 ng / mL; Noggin, 100 ng / mL; A83-01, 500 nM; Y-27632, 5 μM; SB202190, 500 μM; B27 supplement, 1X; N-Acetylcysteine, 1.25 mM; Nicotinamide, 5 mM; GlutaMax 100x, 1X; HEPES, 10 mM; Penicillin / Streptomycin, 100μg / mL; Primocin, 50μg / mL, continue to culture in the cell culture incubator, replace the culture medium every 4 days, and obtain Yuan generation human lung organoids.

[0018] Step S5, after enzymatic hydrolysis of the human lung organoids from Yuan Dynasty, mix them with matrix gel and then plate them, turn them upside down in a cell culture incubator and let them stand. After the gel solidifies, add organoid expansion medium and continue to culture in a cell culture incubator. After 7 days of culture, replace the organoid differentiation medium. The composition and content of the organoid differentiation medium are as follows: Advanced DMEM / F12, 1X; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Primocin, 50μg / mL; DAPT, 10μM, the culture medium was replaced every 4 days, and human distal airway organoids were obtained after 14 days of culture.

[0019] Based on the human distal airway organoids obtained above, ciliary beat function was tested after 3 days. Ciliary beat frequency (CBF) is an important indicator for evaluating ciliary beat function and mucociliary clearance rate, and can reflect the functional level of the pulmonary mucociliary clearance system to a certain extent. Mucociliary clearance system disorders are common in chronic respiratory diseases such as chronic obstructive pulmonary disease, primary ciliary dysfunction, cystic pulmonary fibrosis and asthma.

[0020] Embodiment 2: A method for constructing a human distal airway organoid COPD model, comprising: Step S1, immersing the human distal lung tissue in a digestion solution and digesting it at 37°C for 45 minutes, wherein the human distal lung tissue is obtained from a patient with lung nodules (not suffering from COPD), and the site source is tissue 2-3 cm away from the lung nodules. The digestion solution is TrypLE digestion solution containing 50mM EDTA, and the digestion is terminated using a mild sample buffer. The mild sample buffer is: PBS buffer containing 1% BSA and not containing calcium and magnesium ions. The tissue is cut into pieces, and a single cell suspension is obtained by repeated blowing. After centrifugation, a human lung tissue single cell a is obtained. The centrifugation conditions are: centrifugal speed 300g, temperature 4°C, and centrifugation time 10min.

[0021] Step S2, filter the single cell a with a 70 μm cell mesh to collect the human lung tissue single cell b, remove the supernatant after centrifugation, the centrifugation conditions are: centrifugal speed 300g, temperature 4°C, centrifugation time 10min, and wash twice with a cleaning solution to obtain the human lung tissue single cell c, the composition and content of the cleaning solution are: amphotericin B, 3ug / mL; Primocin, 1000μg / mL; gentamicin, 500μg / mL.

[0022] Step S3: upon observation, red clumps are visible in the single cell c, and 1 mL of red blood cell lysis solution is added to lyse the single cell c at room temperature for 3 minutes, and then the single cell c is separated at a speed of 300 g, and the supernatant is discarded to obtain the single cell d of human lung tissue.

[0023] Step S4, the single cell d is mixed with the extracellular matrix gel and plated, and the plate is inverted and placed in a cell culture incubator. After the gel solidifies, an organoid expansion medium is added, and the composition and content of the organoid expansion medium are as follows: Advanced DMEM / F12, 1X; R-Spondin 1, 500 ng / mL; FGF 7, 25 ng / mL; FGF 10, 100 ng / mL; Noggin, 100 ng / mL; A83-01, 500 nM; Y-27632, 5 μM; SB202190, 500 μM; B27 supplement, 1X; N-Acetylcysteine, 1.25 mM; Nicotinamide, 5 mM; GlutaMax 100x, 1X; HEPES, 10 mM; Penicillin / Streptomycin, 100μg / mL; Primocin, 50μg / mL, continue to culture in the cell culture incubator, replace the culture medium every 4 days, and obtain Yuan generation human lung organoids.

[0024] Step S5, after enzymatic hydrolysis of the human lung organoids from Yuan Dynasty, mix them with matrix gel and then plate them, turn them upside down in a cell culture incubator and let them stand. After the gel solidifies, add organoid expansion medium and continue to culture in a cell culture incubator. After 7 days of culture, replace the organoid differentiation medium. The composition and content of the organoid differentiation medium are as follows: Advanced DMEM / F12, 1X; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Primocin, 50μg / mL; DAPT, 10μM, the culture medium was replaced every 4 days, and human distal airway organoids were obtained after 11 days of culture.

[0025] Step S6, cut off the filter of a cigarette (length 8.4 cm), connect one end of a connecting tube (length 3 cm) to the end near the filter, connect the other end of the connecting tube to a 50 mL syringe needle connector containing 5 mL of organoid differentiation medium, light the cigarette, and draw cigarette smoke into the syringe. When 2 cm of the cigarette remains, seal the syringe with a needle cap and sealing glue, and then put the syringe containing cigarette smoke and organoid differentiation medium into a 37°C shaker and shake to mix for 10 minutes; Step S7, when the sealing glue and the needle cap are removed, no cigarette smoke escapes, indicating that the cigarette smoke and the culture medium have been fully mixed, and the cigarette smoke extract e is obtained. The cigarette smoke extract e is filtered using a 0.22 μm filter, 100 μL is drawn into a 96-well plate, and the absorbance value A at 320 nm is detected by an ELISA instrument. 320 and the absorbance at 540 nm A 540 , when A 320 -A 540 =1.2, the cigarette smoke extract e is defined as 100% CSE; Step S8, dilute 100% CSE into 20% CSE using organoid differentiation medium, and use 20% CSE to continuously culture human-derived distal airway organoids for 72 hours, replacing fresh 20% CSE every 24 hours to obtain a human-derived distal airway organoid COPD model.

[0026] Based on the human-derived distal airway organoid COPD model constructed above, a method for detecting ciliary beat function for non-therapeutic purposes is performed, the steps comprising: replacing 20% ​​CSE for 3 days (new 20% CSE is replaced every 24 hours) in the successfully constructed human-derived distal airway organoid COPD model as the model group, and performing ciliary beat function detection.

[0027] Embodiment 3: A method for constructing a human distal airway organoid COPD model, comprising: Step S1, immersing the human distal lung tissue in a digestion solution and digesting it at 37°C for 30 minutes, wherein the human distal lung tissue is obtained from a patient with lung nodules (not suffering from COPD), and the site source is the tissue 2-3 cm away from the lung nodules. The digestion solution is TrypLE digestion solution containing 10mM EDTA, and the digestion is terminated using a mild sample buffer. The mild sample buffer is: PBS buffer containing 3% BSA and not containing calcium and magnesium ions. The tissue is cut into pieces, and a single cell suspension is obtained by repeated blowing. After centrifugation, a human lung tissue single cell a is obtained. The centrifugation conditions are: centrifugal speed 500g, temperature 4°C, and centrifugation time 5min.

[0028] Step S2, filter the single cell a with a 100 μm cell mesh to collect the human lung tissue single cell b, remove the supernatant after centrifugation, the centrifugation conditions are: centrifugal speed 500g, temperature 4°C, centrifugation time 5min, and wash once with a cleaning solution to obtain the human lung tissue single cell c, the composition and content of the cleaning solution are: amphotericin B, 1ug / mL; Primocin, 10μg / mL; gentamicin, 100μg / mL.

[0029] Step S3: upon observation, red clumps are visible in the single cell c, and 1 mL of red blood cell lysis solution is added to lyse the single cell c at room temperature for 3 minutes, and then the single cell c is separated at a speed of 300 g, and the supernatant is discarded to obtain the single cell d of human lung tissue.

[0030] Step S4, the single cell d is mixed with the extracellular matrix gel and plated, and the plate is inverted and placed in a cell culture incubator. After the gel solidifies, an organoid expansion medium is added, and the composition and content of the organoid expansion medium are as follows: Advanced DMEM / F12, 1X; R-Spondin 1, 500 ng / mL; FGF 7, 25 ng / mL; FGF 10, 100 ng / mL; Noggin, 100 ng / mL; A83-01, 500 nM; Y-27632, 5 μM; SB202190, 500 μM; B27 supplement, 1X; N-Acetylcysteine, 1.25 mM; Nicotinamide, 5 mM; GlutaMax 100x, 1X; HEPES, 10 mM; Penicillin / Streptomycin, 100μg / mL; Primocin, 50μg / mL, continue to culture in the cell culture incubator, replace the culture medium every 3 days, and obtain Yuan generation human lung organoids.

[0031] Step S5, after enzymatic hydrolysis of the Yuan generation human lung organoids, mix with matrix gel and then plate, invert and let stand in a cell culture incubator, add organoid expansion medium after the gel solidifies, continue to culture in the cell culture incubator, and replace the organoid differentiation medium after 10 days of culture. The composition and content of the organoid differentiation medium are as follows: Advanced DMEM / F12, 1X; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Primocin, 50μg / mL; DAPT, 10μM, the culture medium was replaced every 3 days, and human distal airway organoids were obtained after 14 days of culture.

[0032] Step S6, cut off the filter of a cigarette (length 8.4 cm), connect one end of a connecting tube (length 3 cm) to the end near the filter, connect the other end of the connecting tube to a 50 mL syringe needle connector containing 5 mL of organoid differentiation medium, light the cigarette, and draw cigarette smoke into the syringe. When 2 cm of the cigarette remains, seal the syringe with a needle cap and sealing glue, and then put the syringe containing cigarette smoke and organoid differentiation medium into a 37°C shaker and shake to mix for 10 minutes; Step S7, when the sealing glue and the needle cap are removed, no cigarette smoke escapes, indicating that the cigarette smoke and the culture medium have been fully mixed, and the cigarette smoke extract e is obtained. The cigarette smoke extract e is filtered using a 0.22 μm filter, 100 μL is drawn into a 96-well plate, and the absorbance value A at 320 nm is detected by an ELISA instrument. 320 and the absorbance at 540 nm A 540 , when A 320 -A 540 =1.2, the cigarette smoke extract e is defined as 100% CSE; Step S8, dilute 100% CSE into 20% CSE using organoid differentiation medium, and use 20% CSE to continuously culture human-derived distal airway organoids for 72 hours, replacing fresh 20% CSE every 24 hours to obtain a human-derived distal airway organoid COPD model.

[0033] Based on the above-mentioned cultured human distal airway organoid COPD model, a method for detecting ciliary beat function for non-therapeutic purposes is performed, and the steps include: adding 20% ​​CSE containing 0.1 μM formoterol to the above-mentioned successfully constructed human distal airway organoid COPD model, and administering the drug continuously for 3 days (new 20% CSE containing 0.1 μM formoterol is replaced every 24 hours), and then performing ciliary beat function detection.

[0034] Embodiment 4: A method for constructing a human distal airway organoid COPD model, comprising: Step S1, immersing the human distal lung tissue in a digestion solution and digesting it at 37°C for 40 minutes, wherein the human distal lung tissue is obtained from a patient with lung nodules (not suffering from COPD), and the site source is the tissue 2-3 cm away from the lung nodules. The digestion solution is a TrypLE digestion solution containing 30mM EDTA, and the digestion is terminated using a mild sample buffer. The mild sample buffer is: a PBS buffer containing 2% BSA and not containing calcium and magnesium ions. The tissue is cut into pieces, and a single cell suspension is obtained by repeated blowing. After centrifugation, a human lung tissue single cell a is obtained. The centrifugation conditions are: a centrifugal speed of 400g, a temperature of 4°C, and a centrifugal time of 8min.

[0035] Step S2, filter the single cell a with a 70 μm cell mesh to collect the human lung tissue single cell b, remove the supernatant after centrifugation, the centrifugation conditions are: centrifugal speed 400g, temperature 4°C, centrifugation time 8min, and wash twice with a cleaning solution to obtain the human lung tissue single cell c, the composition and content of the cleaning solution are: amphotericin B, 2ug / mL; Primocin, 500μg / mL; Gentomycin, 300μg / mL.

[0036] Step S3, after observation, if no red clumps are seen in the single cell c, the single cell c is mixed with the extracellular matrix gel and plated, and then inverted in a cell culture incubator and left to stand. After the gel solidifies, the organoid expansion medium is added, and the composition and content of the organoid expansion medium are as follows: Advanced DMEM / F12, 1X; R-Spondin 1, 500ng / mL; FGF 7, 25ng / mL; FGF 10, 100ng / mL; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Primocin, 50μg / mL, continue to culture in the cell culture incubator, replace the culture medium every 3 days, and obtain Yuan generation human lung organoids.

[0037] Step S4, after enzymatic hydrolysis of the human lung organoids from Yuan Dynasty, mix them with matrix gel and then plate them, turn them upside down in a cell culture incubator and let them stand. After the gel solidifies, add organoid expansion medium and continue to culture in a cell culture incubator. After 8 days of culture, replace the organoid differentiation medium. The composition and content of the organoid differentiation medium are as follows: Advanced DMEM / F12, 1X; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Primocin, 50μg / mL; DAPT, 10μM, the culture medium was replaced every 3 days, and human distal airway organoids were obtained after 12 days of culture.

[0038] Step S5, cut off the filter of a cigarette (length 8.4 cm), connect one end of a connecting tube (length 3 cm) to the end near the filter, connect the other end of the connecting tube to a 50 mL syringe needle connector filled with 5 mL of organoid differentiation medium, light the cigarette, and draw cigarette smoke into the syringe. When 2 cm of the cigarette remains, seal the syringe with a needle cap and sealing glue, and then put the syringe containing cigarette smoke and organoid differentiation medium into a 37°C shaker and shake to mix for 10 minutes; Step S6, when the sealing glue and the needle cap are removed, no cigarette smoke escapes, indicating that the cigarette smoke and the culture medium have been fully mixed, and the cigarette smoke extract e is obtained. The cigarette smoke extract e is filtered using a 0.22 μm filter, 100 μL is drawn into a 96-well plate, and the absorbance value A at 320 nm is detected by an ELISA instrument. 320 and the absorbance at 540 nm A 540 , when A 320 -A 540 =1.2, the cigarette smoke extract e is defined as 100% CSE; Step S7, dilute 100% CSE into 20% CSE using organoid differentiation medium, and use 20% CSE to continuously culture human-derived distal airway organoids for 72 hours, replacing fresh 20% CSE every 24 hours to obtain a human-derived distal airway organoid COPD model.

[0039] Based on the above-mentioned cultured human distal airway organoid COPD model, a method for detecting ciliary beat function for non-therapeutic purposes is performed, and the steps include: adding 20% ​​CSE containing 0.1 μM theophylline to the above-mentioned successfully constructed human distal airway organoid COPD model, and administering the drug continuously for 3 days (new 20% CSE containing 0.1 μM theophylline is replaced every 24 hours), and then performing ciliary beat function detection.

[0040] Experiment 1 Immunofluorescence staining was performed on the human distal airway organoids obtained in Example 1. The results are as follows: Figure 1 shown.

[0041] Figure 1 The organoids shown in the figure contain four main types of airway cells (ACCTUB: ciliated cell marker, MUC5AC: goblet cell marker, CC10: club cell marker, P63: basal cell marker) and SCGB3A2-positive secretory cells unique to the distal airway, indicating that the human-derived distal airway organoids have the four main airway cell types and secretory cells unique to the distal airway, indicating that the organoids are distal airway organoids.

[0042] Experiment 2 Based on the human distal airway organoid COPD model obtained in Example 2-4, the Pearson coefficient correlation analysis with COPD tissue was performed. The results are shown in Table 1: Table 1

[0043] In Table 1, COPD model refers to the human-derived distal airway organoid COPD model of the present invention, COPD tissue refers to the distal airway tissue from COPD patients, and R is the correlation coefficient value. As can be seen from Table 1, the human-derived distal airway organoid COPD model is highly similar to the COPD tissue at the transcriptional level.

[0044] Experiment 3 For Example 2, the bright field images of the human-derived distal airway organoids obtained before modeling (i.e., obtained in step S5) and the human-derived distal airway organoids obtained after modeling (i.e., obtained in step S8) were observed. For the results, please refer to Figure 2 ,from Figure 2 It can be seen that there is no obvious difference in the morphology of organoids before and after modeling.

[0045] For Example 3, the bright field images of the human distal airway organoids obtained before modeling (i.e., obtained in step S5) and the human distal airway organoids obtained after modeling (i.e., obtained in step S8) were observed. For the results, please refer to Figure 3 ,from Figure 3 It can be seen that there is no obvious difference in the morphology of organoids before and after modeling.

[0046] For Example 4, the bright field images of the human distal airway organoids obtained before modeling (i.e., obtained in step S4) and the human distal airway organoids obtained after modeling (i.e., obtained in step S7) were observed. For the results, please refer to Figure 4 ,from Figure 4 It can be seen that there is no obvious difference in the morphology of organoids before and after modeling.

[0047] Experiment 4 The human-derived distal airway organoid COPD model obtained in Example 2 and the human-derived distal airway organoid obtained in Example 1 were subjected to MUC5AC and ACCTUB immunofluorescence staining. The results are as follows: Figure 5 The human-derived distal airway organoid COPD model obtained in Examples 2-4 and the human-derived distal airway organoid obtained in Example 1 were compared in terms of ciliated cell ratio and goblet cell ratio. The results are shown in Figure 6 shown. Figure 5 and Figure 6 In the data, Normal organoids refers to human-derived distal airway organoids, and CSE-induced COPD organoids refers to human-derived distal airway organoid COPD model.

[0048] from Figure 5 and Figure 6It can be seen that the expression of MUC5AC in the organoids was significantly upregulated and the expression of ACCTUB was significantly downregulated after modeling, indicating that the number of goblet cells in the organoids after modeling increased and the number of ciliated cells decreased, showing typical pathological changes of airway tissue in COPD.

[0049] Experiment 5 The ciliary beat frequencies obtained in Examples 1-4 were compared and analyzed, and the results are shown in Figure 7. Figure 7 In the figure, A represents Example 1, B represents Example 2, C represents Example 3, and D represents Example 4.

[0050] from Figure 7 It can be seen that compared with Example 1, the CBF of Example 2 was significantly downregulated, indicating that the ciliary beating function of the organoids decreased after modeling, showing typical pathological changes of COPD.

[0051] In Example 3, the administration of formoterol can significantly reverse the downregulation of CBF in the human-derived distal airway organoid COPD model, indicating that formoterol has an improving effect on ciliary dysfunction in COPD, which is consistent with the trend reported in existing literature that formoterol has an agonistic effect on ciliary function in COPD patients.

[0052] In Example 4, theophylline administration showed an upward trend in CBF in the human-derived distal airway organoid COPD model, but there was no statistical significance, suggesting that theophylline has limited effect in improving ciliary dysfunction in COPD.

[0053] In summary, the method for constructing and using the human distal airway organoid COPD model provided by the present invention has the following beneficial effects: (1) The human distal airway organoid COPD model constructed by the method of the present invention is highly similar to the COPD distal airway tissue at the transcriptional level, and well restores the pathological characteristics of COPD at the protein expression level and ciliary function level. This indicates that the human distal airway organoid COPD model constructed in vitro by the present invention can simulate COPD lesions in the distal airway and provide a usable model for studying the pathological mechanism of COPD in the distal airway.

[0054] (2) The present invention has clear requirements on the time, temperature and quality inspection indexes of CSE extraction in operation, which can well ensure the extraction quality and experimental repeatability of CSE between batches.

[0055] (3) The present invention conducts drug screening research by detecting ciliary beating function. Compared with previous studies that judge the drug treatment effect based on cell morphology and size, transcription level or protein level, the present invention uses ciliary function as a detection indicator to better reflect the drug's improvement on lung function, and can provide functional level data support for COPD drug research.

[0056] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for constructing a human distal airway organoid COPD model, characterized in that: include: Step S1, immersing human distal lung tissue in digestion solution, digesting at 37°C for 30-45 minutes, using a mild sample buffer to terminate the digestion, cutting the tissue into pieces, repeatedly blowing to obtain a single cell suspension, and obtaining human lung tissue single cell a after centrifugation; Step S2, filtering the single cell a with a cell mesh to collect the human lung tissue single cell b, removing the supernatant after centrifugation, and washing 1-2 times with a washing solution to obtain the human lung tissue single cell c; Step S3, observe whether the single cell c has red clumps. If no red clumps are seen, directly proceed to step S4; if red clumps are seen, add 1 mL of red blood cell lysis solution to lyse at room temperature for 3 minutes, then separate at a speed of 300g, discard the supernatant, and obtain human lung tissue single cells d; Step S4, mixing single cell c or single cell d with matrix gel and then seeding the plate, inverting the plate and letting it stand in a cell culture incubator, adding organoid expansion medium after the gel solidifies, and continuing to culture in the cell culture incubator, replacing the medium every 3-4 days to obtain human lung organoids of the first generation; Step S5, after enzymatic hydrolysis of the Yuan generation human lung organoids, mixing with matrix gel and then seeding, inverting in a cell culture incubator and letting it stand, adding organoid expansion medium after the gel solidifies, continuing to culture in the cell culture incubator, replacing the organoid differentiation medium after 7-10 days of culture, replacing the medium every 3-4 days, and culturing for 11-14 days to obtain human distal airway organoids; Step S6, cut off the filter of a cigarette, connect the end near the filter to one end of the connecting tube, connect the other end of the connecting tube to the syringe needle connector containing 5 mL of organoid differentiation medium, light the cigarette, and draw the cigarette smoke into the syringe. When the cigarette has a preset length remaining, use the needle cap and sealing glue to seal the syringe, and then put the syringe containing the cigarette smoke and organoid differentiation medium into a 37°C shaker and shake to mix for 10 minutes; Step S7, when the sealing glue and the needle cap are removed, no cigarette smoke escapes, indicating that the cigarette smoke and the culture medium have been fully mixed, and the cigarette smoke extract e is obtained. The cigarette smoke extract e is filtered using a 0.22 μm filter, 100 μL is drawn into a 96-well plate, and the absorbance value A at 320 nm is detected by an ELISA instrument. 320 and the absorbance at 540 nm A 540 , when A 320 -A 540 =1.2, the cigarette smoke extract e is defined as 100% CSE; Step S8, dilute 100% CSE into 20% CSE using organoid differentiation medium, and use 20% CSE to continuously culture human-derived distal airway organoids for 72 hours, replacing fresh 20% CSE every 24 hours to obtain a human-derived distal airway organoid COPD model.

2. The method for constructing a human distal airway organoid COPD model according to claim 1, characterized in that: In step S1, the digestion solution is TrypLE digestion solution containing 10-50 mM EDTA.

3. The method for constructing a human distal airway organoid COPD model according to claim 1, characterized in that: In step S1, the mild sample buffer is a PBS buffer containing 1-3% BSA and no calcium or magnesium ions.

4. The method for constructing a human distal airway organoid COPD model according to claim 1, characterized in that: In step S2, the composition and content of the cleaning solution are: amphotericin B, 1-3ug / mL; primocin, 10-1000μg / mL; gentamicin, 100-500μg / mL.

5. The method for constructing a human distal airway organoid COPD model according to claim 1, characterized in that: The centrifugal conditions in step S1 and step S2 are: centrifugal speed 300-500g, temperature 4°C, and centrifugal time 5-10min.

6. The method for constructing a human distal airway organoid COPD model according to claim 1, characterized in that: The composition and content of the organoid expansion medium are as follows: Advanced DMEM / F12, 1X; R-Spondin 1, 500ng / mL; FGF 7, 25ng / mL; FGF 10, 100ng / mL; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Primocin, 50μg / mL.

7. The method for constructing a human distal airway organoid COPD model according to claim 1, characterized in that: The composition and content of the organoid differentiation medium are as follows: Advanced DMEM / F12, 1X; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Primocin, 50μg / mL; DAPT, 10μM.

8. Application of the human distal airway organoid COPD model obtained by the construction method described in any one of claims 1 to 7 in the detection of ciliary beat function for non-therapeutic purposes.

9. The use according to claim 8, characterized in that: In the successfully constructed human distal airway organoid COPD model, 20% CSE containing drugs at preset concentrations was added and administered continuously for 3 days. During this period, new 20% CSE containing drugs at preset concentrations was replaced every 24 hours, and then ciliary beat function testing was performed.

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