Method for in-vitro early diagnosis of small airway lesion

Abstract

The invention provides a method for in-vitro early diagnosis of small airway lesions, and particularly provides application of a functional cell or a detection reagent thereof to preparation of a diagnostic reagent or kit, and the diagnostic reagent or kit is used for (a) early diagnosis of small airway lesions; and / or (b) judging the prognosis of the patient with the small airway lesion. It is found for the first time that by detecting the proportion of functional cells in epithelial cells (namely, the number percentage of goblet cells obtained after differentiation culture of bronchial epithelial stem / progenitor cells and / or cilia cells in the epithelial cells), the application is used for (a) early diagnosis of small airway lesions; and / or (b) judging the prognosis of the patient with small airway lesion, and having very high diagnosis / judgment accuracy.

Description

Technical Field

[0001] The present invention relates to the field of medical diagnosis, and particularly to a method for early diagnosis of small airway lesions in vitro. Background Art

[0002] Small airways refer to airways with a diameter less than 2 mm. At present, it is considered that in the initial stage of the development of respiratory diseases, small airway obstruction has little effect on the change of airway resistance, so it is also called the silent area of lung diseases. However, in the advanced stage of the disease, small airway obstruction will significantly affect lung function.

[0003] According to the research results, among the population aged 20 and above in China, 43.5% of people have small airway dysfunction, with a total number of about 426 million. Among the population with normal forced expiratory volume in the first second (FEV1) and the ratio of forced expiratory volume in the first second to forced vital capacity (FEV1 / FVC) before inhaling bronchodilator, 25.5% have small airway dysfunction, with a total number of about 253 million; among the population with normal FEV1 and FEV1 / FVC before and after inhaling bronchodilator, 11.3% have small airway dysfunction, with a total number of about 111 million. In addition, among the research population, almost all patients with chronic obstructive pulmonary disease (96.15%) have small airway dysfunction, and 68.97% of asthma patients have small airway dysfunction.

[0004] Even among the population without chronic obstructive pulmonary disease or asthma, 41.3% have small airway dysfunction. These figures suggest that small airway dysfunction may exist before the appearance of chronic obstructive pulmonary disease and asthma, which requires more attention from health workers and clinicians.

[0005] Studies in the past decade have shown that in patients with chronic obstructive pulmonary disease (COPD), there are generally pathological changes such as inflammatory cell infiltration characterized by thickening of the small airway wall and epithelial changes, mucus obstruction of the lumen, and hyperplasia of airway smooth muscle and peribronchial fibrosis. Not only in COPD, thickening of the small airway wall is also common in asthma and is related to the frequency and severity of dyspnea and asthma exacerbation. Another prospective study believes that small airway dysfunction may precede the development of asthma, so small airway dysfunction is also considered a precursor of COPD and asthma.

[0006] The diagnosis of small airway dysfunction needs to meet 2 of the following 3 criteria:

[0007] Lung function is lower than 65% of the predicted value of maximum mid-expiratory flow (MMF) / forced expiratory flow at 50% of vital capacity (FEF50) / forced expiratory flow at 75% of vital capacity (FEF75).

[0008] On this basis, small airway dysfunction is further divided into two subtypes: pre-small airway dysfunction (the ratio of forced expiratory volume in the first second (FEV1) and FEV1 / forced vital capacity (FVC) is normal before inhaling bronchodilator) and post-small airway dysfunction (the ratios of FEV1 and FEV1 / FVC are normal before and after inhaling bronchodilator). The National Health Commission promotes the inclusion of lung function in the regular physical examinations for people aged 40 and above, with lung function checked once a year.

[0009] Some researchers believe that the risk of small airway dysfunction in the population is related to age, gender, urbanization, education level, smoking, passive smoking, and exposure to high concentrations of particulate matter with a diameter less than 2.5 μm (PM2.5). In addition, a history of chronic cough, pneumonia, or bronchitis in childhood, a family history of respiratory diseases in parents, and a high body mass index (BMI) are also significantly associated with an increased risk of small airway dysfunction. Currently, the disease burden of small airway dysfunction in China is relatively heavy: 40% of adults aged 20 and above have small airway dysfunction defined by lung function; among all the risk factors leading to small airway dysfunction, smoking is the most important risk factor that can be prevented and controlled. In addition, high exposure to PM2.5 and an increase in BMI will also increase the risk of small airway dysfunction.

[0010] The severity of small airway disease can be evaluated by the following methods: 1. Pulmonary function tests: This is one of the most commonly used testing techniques, including parameters such as forced expiratory volume in the first second (FEV1), forced expiratory flow at 25% to 75% of the vital capacity (FEF25%~75%), and the percentage of FEV1 to forced vital capacity (FEV1 / FVC). Among them, FEF25%~75% is the most commonly used indicator in small airway disease, but it is not sensitive to early and mild parameter changes in the disease. 2. Multiple breath nitrogen washout (MBWT): This is a non-invasive method that can identify the sites of small airway disease causing ventilation inhomogeneity and detect indicators of conductive ventilation inhomogeneity and alveolar ventilation inhomogeneity. 3. Chest high-resolution CT (HRCT): HRCT can perform non-invasive and intuitive imaging evaluations of the wall thickness and lumen diameter of medium and large airways, and indirectly evaluate small airways through changes in regional air trapping. The manifestations of small airway disease in HRCT include direct signs and indirect signs, such as thickening of the bronchiole wall, bronchiole dilation, and mucus impaction in the lumen (centrilobular nodules, tree-in-bud sign, etc.); as well as "mosaic" perfusion and air trapping. 4. Plethysmography: This is a method of evaluating lung function by measuring changes in thoracic and lung volumes. 5. Impulse oscillometry (IOS): This method can directly generate and transmit regular pressure square waves and detect the mechanical activity of the respiratory tract through spectral analysis. 6. Exhaled nitric oxide measurement: This is a non-invasive method for evaluating airway inflammation. 7. Examination methods of nuclear magnetic resonance and nuclear medicine: These methods can also be used for the evaluation of small airway function. 8. Optical coherence tomography (OCT): This is a new technology that can accurately measure the structures of large, medium, and small airways in vivo and can be used to detect small airway abnormalities.

[0011] Early small airway disease is relatively hidden. In the initial stage of the development of respiratory diseases, small airway obstruction has little impact on changes in airway resistance, and pulmonary function appears normal, so it is also known as the "silent area" of lung diseases. In the pre-emphysematous stage of COPD patients, small airway disorders and airway remodeling have already been shown in the lungs, but pulmonary function tests may be normal.

[0012] Currently, early small airway disease cannot be detected by pulmonary function tests: Early small airway disease is relatively hidden. In the initial stage of the development of respiratory diseases, small airway obstruction has little impact on changes in airway resistance, and pulmonary function appears normal. Pulmonary function tests are not sensitive to early and mild parameter changes in small airway disease and are not specific indicators of small airway dysfunction. For example, FEV1 largely reflects large airway obstruction, and FEV1 will only show abnormalities when small airway disease accumulates to a certain extent. Therefore, early small airway disease cannot be detected by pulmonary function tests.

[0013] The detection of small airway lesions by impulse oscillation method is limited in clinical practice because the measuring equipment is bulky and expensive, the popularization range is small, and there is no fixed reference value for different respiratory diseases.

[0014] The inert gas dilution method requires special equipment and is currently difficult to apply clinically. At present, it is only limited to research applications.

[0015] The specificity of the exhaled nitric oxide measurement method is relatively poor and requires extrapolation calculation. Its role in the examination of patients with chronic obstructive pulmonary disease is still unclear.

[0016] Imaging examination methods can only detect small airway lesions that have caused structural changes and cannot observe small airway lesions at an early stage. Before the small airways undergo structural changes, the stem cells of the small airways have already undergone abnormal changes. Such abnormal changes cannot be detected by current examination methods. In short, there is currently a lack of effective early non-invasive detection methods for small airway lesions.

[0017] Therefore, there is an urgent need in this field to develop new methods for the early diagnosis of small airway lesions. Summary of the Invention

[0018] The purpose of the present invention is to provide a new method for the early diagnosis of small airway lesions.

[0019] In the first aspect of the present invention, there is provided the use of a functional cell or its detection reagent for preparing a diagnostic reagent or kit, which is used for (a) the early diagnosis of small airway lesions; and / or (b) judging the prognosis of patients with small airway lesions, and the functional cell is selected from the group consisting of goblet cells, ciliated cells, or a combination thereof.

[0020] In another preferred example, the small airway lesions include small airway abnormality-related diseases.

[0021] In another preferred example, the small airway abnormality includes structural or pathological abnormalities of the small airways.

[0022] In another preferred example, the inner diameter of the small airway is less than 2 mm.

[0023] In another preferred example, the small airways include terminal bronchioles and respiratory bronchioles.

[0024] In another preferred example, the small airway abnormality includes small airway inflammation, small airway fibrosis, small airway stenosis or obstruction, and small airway collapse.

[0025] In another preferred embodiment, the small airway abnormality-related diseases include: chronic obstructive pulmonary disease (COPD), asthma, interstitial pneumonia, idiopathic pulmonary fibrosis, small airway inflammation, bronchiolitis, bronchiolitis obliterans, small airway dysfunction, bronchiectasis, bronchiolitis obliterans organizing pneumonia syndrome, or a combination thereof.

[0026] In another preferred embodiment, the reagent includes a substance for detecting functional cells by using histochemical staining, immunoprecipitation, flow cytometry, Western blotting, ELISA, ELISPOT, antibody microarray, immunohistology, dot blotting, protein microarray, tissue microarray coupled with immunohistochemistry, or other well-known conventional immunological detection / analysis techniques.

[0027] In another preferred embodiment, the detection reagent includes a reagent for detecting functional cells.

[0028] In another preferred embodiment, the detection of functional cells refers to the determination of the proportion of functional cells.

[0029] In another preferred embodiment, the determination of the proportion of functional cells refers to the determination of the percentage of the number of functional cells in the differentiated bronchial epithelial cells.

[0030] In another preferred embodiment, the differentiated bronchial epithelial cells refer to bronchial epithelial cells obtained by differentiating and culturing bronchial epithelial stem / progenitor cells.

[0031] In another preferred embodiment, the functional cells are obtained by differentiating and culturing bronchial epithelial stem / progenitor cells.

[0032] In another preferred embodiment, the proportion of the functional cells is determined by the following method:

[0033] (a) Provide an in vitro bronchial epithelial sample and trophoblast cells, which are obtained by irradiating fibroblasts;

[0034] (b) Centrifuge, wash, and digest the sample to obtain a cell suspension containing bronchial epithelial stem / progenitor cells;

[0035] (c) Inoculate the cell suspension containing bronchial epithelial stem / progenitor cells onto the trophoblast cells and culture in the presence of a medium for culturing bronchial epithelial stem / progenitor cells to obtain the bronchial epithelial stem / progenitor cells;

[0036] (d) Mix the bronchial epithelial stem / progenitor cells obtained in step (c) with the medium for culturing bronchial epithelial stem / progenitor cells to obtain a suspension of bronchial epithelial stem / progenitor cells;

[0037] (e) Under appropriate culture conditions, add the medium for culturing bronchial epithelial stem / progenitor cells and the suspension of the bronchial epithelial stem / progenitor cells into the cell culture insert for culturing;

[0038] (f) On the 3rd - 5th day of culture (preferably the 4th day), remove the medium for culturing bronchial epithelial stem / progenitor cells, add the differentiation medium for bronchial epithelial stem / progenitor cells for differentiation culture, fix and paraffin-embed the bronchial epithelial cells obtained by the differentiation culture to obtain a paraffin block;

[0039] (g) Section the paraffin block and perform HE staining to determine the proportion of functional cells, that is, the percentage of the number of functional cells in the bronchial epithelial cells.

[0040] In another preferred example, the medium for culturing bronchial epithelial stem / progenitor cells comprises a basal medium and additives; wherein the basal medium is selected from the group consisting of: DMEM, Ham's F-12, DMEM / F-12, or a combination thereof, and the additives include: fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, E-Cadherin.

[0041] In another preferred example, the basal medium comprises DMEM and Ham's F-12.

[0042] In another preferred example, based on the total volume of the medium, the concentration (volume percentage) of the basal medium is 80 - 90%.

[0043] In another preferred example, based on the total volume of the medium, the volume ratio of DMEM to Ham's F-12 is 0.5 - 2:0.5 - 2, preferably 1:1.

[0044] In another preferred example, based on the total volume of the medium, the concentration (volume percentage) of fetal bovine serum is 10 - 20%.

[0045] In another preferred example, based on the total volume of the medium, the concentration of glucose is 1000 - 5000 mg / L, preferably, 1500 - 4500 mg / L, more preferably, 2000 - 4000 mg / L, more preferably, 2500 - 3500 mg / L.

[0046] In another preferred example, based on the total volume of the medium, the concentration of insulin is 0.1 - 100 μg / mL, preferably, 0.3 - 80 μg / mL, more preferably, 0.5 - 50 μg / mL, more preferably, 0.8 - 30 μg / mL, more preferably, 1 - 10 μg / mL, more preferably, 3 - 10 μg / mL.

[0047] In another preferred example, based on the total volume of the culture medium, the concentration of epidermal growth factor is 0.1 - 100 ng / mL, preferably 0.5 - 80 ng / mL, more preferably 0.7 - 50 ng / mL, more preferably 0.8 - 30 ng / mL, more preferably 1 - 20 ng / mL, more preferably 2 - 10 ng / ml.

[0048] In another preferred example, based on the total volume of the culture medium, the concentration of hydrocortisone is 0.1 - 100 μg / mL, preferably 0.3 - 50 μg / mL, more preferably 0.8 - 30 μg / mL, more preferably 1 - 10 μg / mL, more preferably 1 - 6 μg / mL.

[0049] In another preferred example, based on the total volume of the culture medium, the concentration of Y-27632 is 0.1 - 100 μM, preferably 0.3 - 50 μM, more preferably 0.8 - 30 μM, more preferably 1 - 10 μM, more preferably 1 - 6 μM.

[0050] In another preferred example, based on the total volume of the culture medium, the concentration of SB431542 is 1 - 100 μM, preferably 5 - 60 μM, more preferably 8 - 20 μM, more preferably 8 - 15 μM.

[0051] In another preferred example, based on the total volume of the culture medium, the concentration of Noggin is 1 - 500 ng / mL, preferably 5 - 300 ng / mL, more preferably 8 - 200 ng / mL, more preferably 10 - 100 ng / mL, more preferably 20 - 50 ng / mL.

[0052] In another preferred example, based on the total volume of the culture medium, the concentration of R-spondin 1 is 1 - 500 ng / mL, preferably 5 - 300 ng / mL, more preferably 8 - 200 ng / mL, more preferably 10 - 100 ng / mL, more preferably 30 - 80 ng / mL.

[0053] In another preferred example, based on the total volume of the culture medium, the concentration of Fibronectin is 0.1 - 100 μg / mL, preferably 0.5 - 50 μg / mL, more preferably 08 - 30 μg / mL, more preferably 1 - 10 μg / mL, more preferably 1 - 6 μg / mL.

[0054] In another preferred embodiment, the concentration of E-Cadherin is 0.01-10 μg / mL, preferably 0.05-5 μg / mL, more preferably 0.08-3 μg / mL, more preferably 0.1-2 μg / mL, more preferably 0.6-2 μg / mL, based on the total volume of the culture medium.

[0055] In another preferred embodiment, the bronchial epithelial sample is a normal bronchial epithelial sample or a pathological bronchial epithelial sample.

[0056] In another preferred embodiment, the bronchial epithelial sample is from a bronchiole, preferably a bronchiole of grade 5 or higher.

[0057] In another preferred embodiment, the bronchial epithelial sample is selected from the group consisting of: a tissue sample, a non-invasive liquid sample, a tissue biopsy forceps sample, a biopsy brush sample, a surgical resection sample, or a combination thereof.

[0058] In another preferred embodiment, the centrifugation conditions in step (b) are: 300-1000 g.

[0059] In another preferred embodiment, in step (b), the sample is washed with a sample preservation solution.

[0060] In another preferred embodiment, the sample preservation solution comprises: a basal medium containing 5-50 mg / mL (preferably 6-30 mg / mL, more preferably 8-20 mg / mL) BSA, Penicillin (20-800 units / mL, preferably 50-500 units / mL, more preferably 80-200 units / mL)-Streptomycin (20-800 μg / mL, preferably 50-500 μg / mL, more preferably 80-200 μg / mL), based on the total volume of the sample preservation solution.

[0061] In another preferred embodiment, in step (b), digestion is carried out using the digestive enzyme TrypLE (purchased from Thermo Fisher Scientific, catalog number: 12604013).

[0062] In another preferred embodiment, in step (a), the irradiation dose is 20-80 Gy, preferably 30-60 Gy.

[0063] In another preferred embodiment, in step (c), the inoculation amount of the cell suspension containing bronchial epithelial stem / progenitor cells is 8000-50000 cells / cm 2 2, preferably 10000-20000 cells / cm 2 .

[0064] In another preferred embodiment, in step (c), the steps further include: at 37 °C, 5%-10% concentration of CO 2Cultured under the conditions of.

[0065] In another preferred example, in step (c), the culture medium is changed 2 - 3 times a week.

[0066] In another preferred example, step (c) further includes: a step of detecting endotoxin and mycoplasma in the cells.

[0067] In another preferred example, step (c) further includes the steps of:

[0068] (c-i) Inoculate the cell suspension containing bronchial epithelial stem / progenitor cells onto the feeder layer cells, and perform primary culture for 5 - 10 days in the presence of the culture medium for culturing bronchial epithelial stem / progenitor cells;

[0069] (c-ii) Digest the cell clones obtained from the primary culture, inoculate the cell suspension obtained after digestion onto the feeder layer cells, and perform passage;

[0070] (c-iii) Perform 2 - 3 passages in the presence of the culture medium for culturing bronchial epithelial stem / progenitor cells, thereby obtaining the bronchial epithelial stem / progenitor cells.

[0071] In another preferred example, in step (c-iii), the number of the bronchial epithelial stem / progenitor cells obtained by culturing and amplification is ≥ 1x10 6 cells, preferably, 1x10 6 -10x10 6 cells, more preferably, 2x10 6 -8x10 6 cells, more preferably, 4x10 6 -6x10 6 cells.

[0072] In another preferred example, in step (c-iii), it further includes a step of analyzing and / or identifying the bronchial epithelial stem / progenitor cells.

[0073] In another preferred example, the analysis and / or identification includes: identification of P63 and / or Krt5 markers.

[0074] In another preferred example, in step (d), in the bronchial epithelial stem / progenitor cell suspension, the concentration of bronchial epithelial stem / progenitor cells is ≥ 0.5x10 6 cells / mL, preferably, 0.8x10 6 -5x10 6 cells / mL, more preferably, 1x10 6 -2x10 6 cells / mL.

[0075] In another preferred example, in step (e), the cell culture chamber includes an upper chamber and a lower chamber of the cell culture chamber. The culture medium for bronchial epithelial stem / progenitor cells is added to the lower chamber of the cell culture chamber, and the suspension of bronchial epithelial stem / progenitor cells is added to the upper chamber of the cell culture chamber.

[0076] In another preferred example, in step (e), the addition amount of the culture medium for bronchial epithelial stem / progenitor cells is ≥700 μL / well, preferably 700 - 800 μL / well.

[0077] In another preferred example, in step (e), the number of bronchial epithelial stem / progenitor cells in the suspension of bronchial epithelial stem / progenitor cells is 1×10 5 -5×10 5 cells, more preferably 2×10 5 -4×10 5 cells.

[0078] In another preferred example, in step (e), it further includes the step of culturing under the conditions of 37 °C and a CO 2 concentration of 5% - 8%.

[0079] In another preferred example, in step (e), it further includes the step of replacing the culture medium for bronchial epithelial stem / progenitor cells every 1 - 2 days (preferably 2 days).

[0080] In another preferred example, in step (f), the differentiation medium for bronchial epithelial stem / progenitor cells is added to the lower chamber of the cell culture chamber for differentiation culture.

[0081] In another preferred example, in step (f), it further includes the step of replacing the differentiation medium for bronchial epithelial stem / progenitor cells every 1 - 2 days (preferably 2 days).

[0082] In another preferred example, the differentiation medium for bronchial epithelial stem / progenitor cells includes a basal medium and additives; wherein the basal medium is selected from the group consisting of: DMEM, Ham's F - 12, DMEM / F - 12, or a combination thereof, and the additives include: glucose, insulin, EGF, RA, hydrocortisone, fetal bovine serum.

[0083] In another preferred example, the basal medium includes DMEM and Ham's F - 12.

[0084] In another preferred example, based on the total volume of the culture medium, the concentration (volume percentage) of the basal medium is 90 - 99%.

[0085] In another preferred example, based on the total volume of the culture medium, the volume ratio of the DMEM to Ham's F-12 is 40-60:45-55, preferably 50:49.

[0086] In another preferred example, based on the total volume of the culture medium, the concentration (volume percentage) of fetal bovine serum is 0.5-4%, preferably 0.8-2%.

[0087] In another preferred example, based on the total volume of the culture medium, the concentration of glucose is 1000-6000 mg / L, preferably 1500-5000 mg / L, more preferably 3000-4500 mg / L.

[0088] In another preferred example, based on the total volume of the culture medium, the concentration of insulin is 0.05-10 μg / mL, preferably 0.08-8 μg / mL, more preferably 0.1-5 μg / mL.

[0089] In another preferred example, based on the total volume of the culture medium, the concentration of EGF is 0.05-5 μg / mL, preferably 0.08-1 μg / mL, more preferably 0.1-0.5 μg / mL.

[0090] In another preferred example, based on the total volume of the culture medium, the concentration of hydrocortisone is 0.1-20 μg / mL, preferably 0.2-10 μg / mL, more preferably 0.5-5 μg / mL.

[0091] In another preferred example, based on the total volume of the culture medium, the concentration of RA is 0.001-10 μM, preferably 0.01-8 μM, more preferably 0.08-5 μM.

[0092] In another preferred example, the addition amount of the bronchial epithelial stem / progenitor cell differentiation culture medium is ≥700 μL / well, preferably 700-800 μL / well.

[0093] In another preferred example, the step (f) further includes steps of washing, membrane separation, and membrane fixation of the bronchial epithelial cells obtained by differentiation culture.

[0094] In another preferred example, the step (f) further includes a step of adding a buffer solution to wash the bronchial epithelial cells obtained by differentiation culture on the 14th-24th day (preferably the 19th day) of culture.

[0095] In another preferred example, the buffer solution includes PBS.

[0096] In another preferred example, the method is an in vitro method.

[0097] In another preferred embodiment, the method is for non-diagnostic and non-therapeutic purposes.

[0098] In another preferred embodiment, the diagnosis includes auxiliary diagnosis and / or confirmatory diagnosis.

[0099] In another preferred embodiment, the functional cells are derived from mammals, preferably from humans.

[0100] In another preferred embodiment, the diagnosis includes the following steps:

[0101] (1) Provide a sample from a subject to be tested, and detect the functional cells in the sample, including detecting the proportion of functional cells, including the proportion of goblet cells and / or the proportion of ciliated cells;

[0102] (2) If the proportion value of goblet cells measured in step (1) is higher than the reference value C1, it indicates that the subject to be tested has small airway lesions; and / or

[0103] If the proportion value of ciliated cells measured in step (1) is lower than the reference value C2, it indicates that the subject to be tested has small airway lesions.

[0104] In another preferred embodiment, the reference value C1 is 22.5%, that is, the percentage of the number of goblet cells in the differentiated bronchial epithelial cells.

[0105] In another preferred embodiment, the reference value C2 is 41.15%, that is, the percentage of the number of ciliated cells in the differentiated bronchial epithelial cells.

[0106] In another preferred embodiment, the subject to be tested includes a subject suspected of having small airway lesions.

[0107] In another preferred embodiment, the prognosis judgment includes the following steps:

[0108] (1) Provide a sample from a subject to be tested, and detect the functional cells in the sample, including detecting the proportion of functional cells, including the proportion of goblet cells and / or the proportion of ciliated cells;

[0109] (2) If the proportion value of goblet cells measured in step (1) is lower than the reference value C1 or conforms to the reference value C0 of the healthy population, it indicates that the prognosis of the small airway lesions of the subject to be tested is good; and / or

[0110] If the proportion value of ciliated cells measured in step (1) is higher than the reference value C2 or conforms to the reference value C0' of the healthy population, it indicates that the prognosis of the small airway lesions of the subject to be tested is good.

[0111] In another preferred embodiment, the subject to be tested includes a subject having small airway lesions.

[0112] In another preferred example, the reference value C0 is the percentage of the number of goblet cells in the bronchial epithelial cells in the same tissue of the healthy control population.

[0113] In another preferred example, the reference value C0' is the percentage of the number of ciliated cells in the bronchial epithelial cells in the same tissue of the healthy control population.

[0114] In another preferred example, the reference value C1 is 22.5%, that is, the percentage of the number of goblet cells in the differentiated bronchial epithelial cells.

[0115] In another preferred example, the reference value C2 is 41.15%, that is, the percentage of the number of ciliated cells in the differentiated bronchial epithelial cells.

[0116] In another preferred example, the sample is selected from the following group: bronchial epithelial samples of patients with small airway lesions.

[0117] In another preferred example, the bronchial epithelial sample is from a bronchiole, preferably a bronchiole of grade 5 or above.

[0118] In another preferred example, the bronchial epithelial sample is selected from the following group: tissue samples, non-invasive liquid samples, tissue biopsy samples, biopsy brush samples, surgical resection samples, or combinations thereof.

[0119] The second aspect of the present invention provides a method for early diagnosis of small airway lesions, comprising the steps of:

[0120] (1) Providing a sample from a subject to be tested, and detecting functional cells in the sample, including detecting the proportion of functional cells, including the proportion of goblet cells and / or the proportion of ciliated cells;

[0121] (2) If the proportion value of goblet cells measured in step (1) is higher than the reference value C1, it indicates that the subject to be tested has small airway lesions; and / or

[0122] If the proportion value of ciliated cells measured in step (1) is lower than the reference value C2, it indicates that the subject to be tested has small airway lesions.

[0123] In another preferred example, the reference value C1 is 22.5%, that is, the percentage of the number of goblet cells in the differentiated bronchial epithelial cells.

[0124] In another preferred example, the reference value C2 is 41.15%, that is, the percentage of the number of ciliated cells in the differentiated bronchial epithelial cells.

[0125] In another preferred example, the small airway lesions include small airway abnormality-related diseases.

[0126] In another preferred example, the small airway abnormality includes structural or pathological abnormalities of the small airways.

[0127] In another preferred example, the inner diameter of the small airways is less than 2 mm.

[0128] In another preferred example, the small airways include terminal bronchioles and respiratory bronchioles.

[0129] In another preferred example, the small airway abnormality includes small airway inflammation, small airway fibrosis, small airway stenosis or obstruction, and small airway collapse.

[0130] In another preferred example, the diseases associated with small airway abnormalities include: chronic obstructive pulmonary disease (COPD), asthma, interstitial pneumonia, idiopathic pulmonary fibrosis, small airway inflammation, bronchiolitis, bronchiolitis obliterans, small airway dysfunction, bronchiectasis, obliterative bronchiolitis syndrome, or a combination thereof.

[0131] In another preferred example, the subject to be tested includes a subject suspected of having a small airway lesion.

[0132] In another preferred example, the sample is selected from the following group: bronchial epithelial samples.

[0133] In another preferred example, the bronchial epithelial sample is from a bronchiole, preferably a bronchiole of grade 5 or above.

[0134] In another preferred example, the bronchial epithelial sample is selected from the following group: tissue samples, non-invasive liquid samples, tissue biopsy samples, biopsy brush samples, surgical resection samples, or a combination thereof.

[0135] In another preferred example, the method is a non-diagnostic and non-therapeutic method.

[0136] The third aspect of the present invention provides a method for judging the prognosis of a patient with a small airway lesion, comprising the steps of:

[0137] (1) Providing a sample from a subject to be tested, and detecting functional cells in the sample, including detecting the proportion of functional cells, including the proportion of goblet cells and / or the proportion of ciliated cells;

[0138] (2) If the value of the proportion of goblet cells measured in step (1) is lower than the reference value C1 or conforms to the reference value C0 of the healthy population, it indicates that the prognosis of the small airway lesion of the subject to be tested is better; and / or

[0139] If the value of the proportion of ciliated cells measured in step (1) is higher than the reference value C2 or conforms to the reference value C0' of the healthy population, it indicates that the prognosis of the small airway lesion of the subject to be tested is better.

[0140] In another preferred example, the small airway lesion includes small airway abnormality-related diseases.

[0141] In another preferred example, the small airway abnormality includes structural or pathological abnormalities of the small airway.

[0142] In another preferred example, the inner diameter of the small airway is less than 2 mm.

[0143] In another preferred example, the small airway includes terminal bronchioles and respiratory bronchioles.

[0144] In another preferred example, the small airway abnormality includes small airway inflammation, small airway fibrosis, small airway stenosis or obstruction, and small airway collapse.

[0145] In another preferred example, the small airway abnormality-related diseases include: chronic obstructive pulmonary disease (COPD), asthma, interstitial pneumonia, idiopathic pulmonary fibrosis, small airway inflammation, bronchiolitis, bronchiolitis obliterans, small airway dysfunction, bronchiectasis, obliterative bronchiolitis syndrome, or a combination thereof.

[0146] In another preferred example, the reference value C0 is the percentage of the number of goblet cells in the bronchial epithelial cells in the same tissue of healthy control subjects.

[0147] In another preferred example, the reference value C0' is the percentage of the number of ciliated cells in the bronchial epithelial cells in the same tissue of healthy control subjects.

[0148] In another preferred example, the reference value C1 is 22.5%, that is, the percentage of the number of goblet cells in the differentiated bronchial epithelial cells.

[0149] In another preferred example, the reference value C2 is 41.15%, that is, the percentage of the number of ciliated cells in the differentiated bronchial epithelial cells.

[0150] In another preferred example, the subject to be tested includes a subject to be tested with a small airway lesion.

[0151] In another preferred example, the sample is selected from the following group: a bronchial epithelial sample of a patient with a small airway lesion.

[0152] In another preferred example, the bronchial epithelial sample is from a bronchiole, preferably a bronchiole of grade 5 or above.

[0153] In another preferred example, the bronchial epithelial sample is selected from the following group: a tissue sample, a non-invasive liquid sample, a tissue biopsy sample, a biopsy brush sample, a surgical resection sample, or a combination thereof.

[0154] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0155] A fourth aspect of the present invention provides a method for establishing a model for (a) early diagnosis of small airway lesions; and / or (b) predicting the prognosis of patients with small airway lesions, the method comprising the step of identifying differential biomarkers in a biological sample between patients with small airway lesions and healthy controls or patients with non-small airway lesions, wherein the differential biomarkers include the proportion of functional cells, and the functional cells are selected from the group consisting of goblet cells, ciliated cells, or a combination thereof.

[0156] In another preferred embodiment, the small airway lesions include small airway abnormality-related diseases.

[0157] In another preferred embodiment, the small airway abnormality includes an abnormality in the structure or pathology of the small airway.

[0158] In another preferred embodiment, the inner diameter of the small airway is less than 2 mm.

[0159] In another preferred embodiment, the small airway includes terminal bronchioles and respiratory bronchioles.

[0160] In another preferred embodiment, the small airway abnormality includes small airway inflammation, small airway fibrosis, small airway stenosis or obstruction, and small airway collapse.

[0161] In another preferred embodiment, the small airway abnormality-related diseases include: chronic obstructive pulmonary disease (COPD), asthma, interstitial pneumonia, idiopathic pulmonary fibrosis, small airway inflammation, bronchiolitis, bronchiolitis obliterans, small airway dysfunction, bronchiectasis, obliterative bronchiolitis syndrome, or a combination thereof.

[0162] In another preferred embodiment, the proportion of functional cells refers to the percentage of the number of functional cells in bronchial epithelial cells.

[0163] In another preferred embodiment, the functional cells are obtained by differentiating and culturing bronchial epithelial stem / progenitor cells.

[0164] A fifth aspect of the present invention provides a system for early diagnosis of small airway lesions, the system comprising:

[0165] (a) A feature receiving module, which is used to receive feature data from a sample of a subject to be tested; the feature data includes: the proportion values of functional cells in the sample of the subject to be tested, including the proportion value of goblet cells and / or the proportion value of ciliated cells;

[0166] (b) Discrimination processing module, which compares the received feature data with a reference value to obtain a diagnosis or evaluation result. Among them, when the proportion value of goblet cells in the feature data is higher than the reference value C1, it is prompted that the subject has small airway lesions; and / or

[0167] when the proportion value of ciliated cells in the feature data is lower than the reference value C2, it is prompted that the subject has small airway lesions; and

[0168] (c) Result output module, which is used to receive and output the evaluation result.

[0169] In another preferred example, the proportion value of functional cells refers to the percentage of the number of functional cells in the differentiated bronchial epithelial cells.

[0170] In another preferred example, the reference value C1 is 22.5%, that is, the percentage of the number of goblet cells in the differentiated bronchial epithelial cells.

[0171] In another preferred example, the reference value C2 is 41.15%, that is, the percentage of the number of ciliated cells in the differentiated bronchial epithelial cells.

[0172] In another preferred example, the small airway lesions include small airway abnormality-related diseases.

[0173] In another preferred example, the small airway abnormalities include structural or pathological abnormalities of the small airways.

[0174] In another preferred example, the inner diameter of the small airways is less than 2 mm.

[0175] In another preferred example, the small airways include terminal bronchioles and respiratory bronchioles.

[0176] In another preferred example, the small airway abnormalities include small airway inflammation, small airway fibrosis, small airway stenosis or obstruction, and small airway collapse.

[0177] In another preferred example, the small airway abnormality-related diseases include: chronic obstructive pulmonary disease (COPD), asthma, interstitial pneumonia, idiopathic pulmonary fibrosis, small airway inflammation, bronchiolitis, bronchiolitis obliterans, small airway dysfunction, bronchiectasis, obliterative bronchiolitis syndrome, or a combination thereof.

[0178] In another preferred example, the subject includes a subject suspected of having small airway lesions.

[0179] In another preferred example, the subject is a human.

[0180] In another preferred example, the subject includes men and women.

[0181] In another preferred example, the objects include infants, adolescents or adults.

[0182] In another preferred example, the method for detecting the proportion value of the functional cells includes: using histochemical staining, immunoprecipitation, flow cytometry, Western blotting, ELISA, ELISPOT, antibody microarray, immunohistology, dot blotting, protein microarray, tissue microarray coupled with immunohistochemistry, or other well-known conventional immunological detection / analysis techniques.

[0183] In another preferred example, the feature receiving module includes a sample collector and a feature signal input terminal.

[0184] In another preferred example, the calculation and processing module includes a processor and a storage device, wherein the storage device stores data on the percentage of the number of functional cells in the differentiated bronchial epithelial cells.

[0185] In another preferred example, the output module includes any terminal, preferably a display, a printer, a tablet computer (PAD), or a smart phone.

[0186] In another preferred example, the modules are connected by wired or wireless means.

[0187] The sixth aspect of the present invention provides a system for judging the prognosis of patients with small airway lesions, including:

[0188] (a) A feature receiving module, which is used to receive feature data from a sample of a to-be-tested object suffering from small airway lesions; the feature data includes: the proportion value of functional cells in the sample of the to-be-tested object, including the proportion value of goblet cells and / or the proportion value of ciliated cells;

[0189] (b) A discrimination and processing module, which compares the received feature data with a reference value to obtain a diagnosis or evaluation result. Among them, when the proportion value of goblet cells in the feature data is lower than the reference value C1 or conforms to the reference value C0 of the healthy population, it indicates that the prognosis of the object is better; and / or

[0190] When the proportion value of ciliated cells in the feature data is higher than the reference value C2 or conforms to the reference value C0' of the healthy population, it indicates that the prognosis of the object is better; and

[0191] (c) A result output module, which is used to receive and output the evaluation result.

[0192] In another preferred example, the proportion value of the functional cells refers to the percentage of the number of functional cells in the differentiated bronchial epithelial cells.

[0193] In another preferred example, the reference value C0 is the percentage of the number of goblet cells in the same tissue of the healthy control population in the bronchial epithelial cells.

[0194] In another preferred example, the reference value C0' is the percentage of the number of ciliated cells in the same tissue of the healthy control population in the bronchial epithelial cells.

[0195] In another preferred example, the reference value C1 is 22.5%, that is, the percentage of the number of goblet cells in the differentiated bronchial epithelial cells.

[0196] In another preferred example, the reference value C2 is 41.15%, that is, the percentage of the number of ciliated cells in the differentiated bronchial epithelial cells.

[0197] In another preferred example, the small airway lesion includes small airway-related diseases.

[0198] In another preferred example, the small airway abnormality includes the abnormality that occurs in the structure or pathology of the small airway.

[0199] In another preferred example, the inner diameter of the small airway is less than 2 mm.

[0200] In another preferred example, the small airway includes the terminal bronchiole and the respiratory bronchiole.

[0201] In another preferred example, the small airway abnormality includes small airway inflammation, small airway fibrosis, small airway stenosis or obstruction, and small airway collapse.

[0202] In another preferred example, the small airway-related diseases include: chronic obstructive pulmonary disease (COPD), asthma, interstitial pneumonia, idiopathic pulmonary fibrosis, small airway inflammation, bronchiolitis, bronchiolitis obliterans, small airway dysfunction, bronchiectasis, obliterative bronchiolitis syndrome, or a combination thereof.

[0203] In another preferred example, the subject includes a subject suffering from a small airway lesion.

[0204] In another preferred example, the subject is a human.

[0205] In another preferred example, the subject includes men and women.

[0206] In another preferred example, the subject includes infants, adolescents or adults.

[0207] In another preferred embodiment, the method for detecting the proportion value of the functional cells includes: using histochemical staining, immunoprecipitation, flow cytometry, Western blotting, ELISA, ELISPOT, antibody microarray, immunohistology, dot blotting, protein microarray, tissue microarray coupled with immunohistochemistry, or other well-known conventional immunological detection / analysis techniques.

[0208] In another preferred embodiment, the feature receiving module includes a sample collector and a feature signal input terminal.

[0209] In another preferred embodiment, the calculation and processing module includes a processor and a memory, wherein the memory stores data on the percentage of the number of functional cells in the differentiated bronchial epithelial cells.

[0210] In another preferred embodiment, the output module includes any terminal, preferably a display, a printer, a tablet computer (PAD), or a smart phone.

[0211] In another preferred embodiment, the modules are connected by wired or wireless means.

[0212] The seventh aspect of the present invention provides a method of using functional cells in a subject to be tested as an indicator for (a) early diagnosis of small airway lesions; and / or (b) judging the prognosis of patients with small airway lesions, wherein the functional cells are selected from the group consisting of goblet cells, ciliated cells, or a combination thereof;

[0213] The method includes measuring the proportion value of the functional cells in a sample from the subject to be tested.

[0214] In another preferred embodiment, the small airway lesions include small airway abnormality-related diseases.

[0215] In another preferred embodiment, the small airway abnormality includes structural or pathological abnormalities of the small airway.

[0216] In another preferred embodiment, the inner diameter of the small airway is less than 2 mm.

[0217] In another preferred embodiment, the small airway includes terminal bronchioles and respiratory bronchioles.

[0218] In another preferred embodiment, the small airway abnormality includes small airway inflammation, small airway fibrosis, small airway stenosis or obstruction, and small airway collapse.

[0219] In another preferred embodiment, the small airway abnormality-related diseases include: chronic obstructive pulmonary disease (COPD), asthma, interstitial pneumonia, idiopathic pulmonary fibrosis, small airway inflammation, bronchiolitis, bronchiolitis obliterans, small airway dysfunction, bronchiectasis, obliterative bronchiolitis syndrome, or a combination thereof.

[0220] In another preferred example, the comparison of the proportion value of functional cells with a reference value is used as an indicator for (a) early diagnosis of small airway lesions; and / or (b) judging the prognosis of patients with small airway lesions.

[0221] In another preferred example, the proportion value of functional cells refers to the percentage of the number of functional cells in the differentiated bronchial epithelial cells.

[0222] In another preferred example, the object to be tested includes an object to be tested with small airway lesions or an object to be tested suspected of having small airway lesions.

[0223] In another preferred example, if the proportion value of goblet cells is higher than the reference value C1, it indicates that the object to be tested has small airway lesions.

[0224] In another preferred example, if the proportion value of ciliated cells is lower than the reference value C2, it indicates that the object to be tested has small airway lesions.

[0225] In another preferred example, if the proportion value of goblet cells is lower than the reference value C1 or conforms to the reference value C0 of the healthy population, it indicates that the prognosis of the object to be tested is better.

[0226] In another preferred example, if the proportion value of ciliated cells is higher than the reference value C2 or conforms to the reference value C0' of the healthy population, it indicates that the prognosis of the object to be tested is better.

[0227] In another preferred example, the reference value C0 is the percentage of the number of goblet cells in the bronchial epithelial cells in the same tissue of the healthy control population.

[0228] In another preferred example, the reference value C0' is the percentage of the number of ciliated cells in the bronchial epithelial cells in the same tissue of the healthy control population.

[0229] In another preferred example, the reference value C1 is 22.5%, that is, the percentage of the number of goblet cells in the differentiated bronchial epithelial cells.

[0230] In another preferred example, the reference value C2 is 41.15%, that is, the percentage of the number of ciliated cells in the differentiated bronchial epithelial cells.

[0231] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0232] Figure 1Shows the process of bronchoscope sampling-----bronchial trunk / progenitor cell culture-----bronchial trunk / progenitor cell amplification-----bronchial trunk / progenitor cell seeding into the chamber-----completion of differentiation.

[0233] Figure 2 Shows the cloning rate of stem cells on trophoblast cells at different irradiation doses, and a dose of 30 - 60 Gy is more optimal.

[0234] Figure 3 Shows good cloning of bronchial epithelial stem / progenitor cells, with a regular and smooth cloning contour, clear cloning boundaries, uniform cell sizes within the clone, and tight arrangement.

[0235] Figure 4 Shows the results of P63 staining of markers of bronchial epithelial stem / progenitor cell clones, Ki67 staining of proliferation markers, DAPI staining of cell nuclei, and the merging of the above stainings.

[0236] Figure 5 Shows good cloning of bronchial epithelial stem / progenitor cells, with a regular and smooth cloning contour, clear cloning boundaries, uniform cell sizes within the clone, and tight arrangement.

[0237] Figure 6 Shows a HE staining image of bronchial epithelial cells formed after differentiation of cultured and amplified bronchial epithelial stem / progenitor cells in one case.

[0238] Figure 7 Shows a HE staining image of bronchial epithelial cells formed after differentiation of cultured and amplified bronchial epithelial stem / progenitor cells in another case.

[0239] Figure 8 Shows an ROC curve graph made by analyzing the goblet cell proportion data of 400 samples with Graphpad.

[0240] Figure 9 Shows the sensitivity and specificity for early diagnosis of small airway lesions with different goblet cell proportions as cut-off values, and the sensitivity and specificity for diagnosing small airway lesions are optimal when it is greater than 22.5%.

[0241] Figure 10 Shows an ROC curve graph made by analyzing the ciliated cell proportion data of 400 samples with Graphpad.

[0242] Figure 11 Shows the sensitivity and specificity for early diagnosis of small airway lesions with different ciliated cell proportions as cut-off values, and the sensitivity and specificity for diagnosing small airway lesions are optimal when it is less than 41.15%.

[0243] Figure 12Shows the proportions of ciliated cells and goblet cells in bronchial epithelial cells of patients with small airway lesions before infusion of bronchial trunk / progenitor cells.

[0244] Figure 13 Shows the proportions of ciliated cells and goblet cells in bronchial epithelial cells of patients with small airway lesions after infusion of bronchial trunk / progenitor cells. Detailed implementation manners

[0245] Through extensive and in-depth research, the present inventors have for the first time discovered that by detecting the proportion of functional cells (i.e., the percentage of functional cells (including goblet cells and / or ciliated cells) obtained after differentiation culture of bronchial epithelial trunk / progenitor cells in bronchial epithelial cells), it can be used for (a) early diagnosis of small airway lesions; and / or (b) judging the prognosis of patients with small airway lesions, and has a very high diagnostic / judging accuracy. On this basis, the inventors have completed the present invention.

[0246] Terms

[0247] The terms used in the present invention have the meanings commonly understood by those of ordinary skill in the relevant art. However, for a better understanding of the present invention, the explanations of some definitions and related terms are as follows. It should be noted that the explanations of the terms provided herein are only for those skilled in the art to better understand the present invention and do not limit the present invention.

[0248] According to the present invention, the term "individual" refers to an animal, particularly a mammal, such as a primate, preferably a human.

[0249] According to the present invention, terms such as "a", "an" and "the" not only refer to a single individual, but also include the general category that can be used to illustrate a specific embodiment.

[0250] As used herein, when referring to a specifically listed numerical value, the term "about" means that the value can vary by no more than 1% from the listed value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0251] As used herein, the term "comprising" or "including (containing)" can be open-ended, semi-closed and closed. In other words, the term also includes "consisting essentially of...", or "consisting of...".

[0252] Early diagnosis of small airway lesions

[0253] Early diagnosis of small airway disease refers to the diagnosis of small airway disease that cannot be detected by conventional examination methods such as pulmonary function tests and imaging examinations. Currently, early small airway disease cannot be detected by pulmonary function tests. Small airway disease is relatively concealed in the early stage. In the initial stage of the development of respiratory diseases, small airway obstruction has little impact on the change of airway resistance, and the pulmonary function shows normal. Pulmonary function tests are not sensitive to the early and mild parameter changes of small airway disease and are not specific indicators of small airway dysfunction. For example, FEV1 largely reflects large airway obstruction, and abnormal FEV1 will only appear when small airway disease accumulates to a certain extent. Therefore, small airway disease cannot be detected early by pulmonary function examination methods. Imaging examination methods can only detect small airway disease that has caused structural changes and cannot observe small airway disease early.

[0254] Bronchial epithelial stem / progenitor cells

[0255] Bronchial epithelial stem / progenitor cells are a type of cells with self-renewal and differentiation abilities that exist in the bronchial epithelium. They play an important role in maintaining the homeostasis of the bronchial epithelium and the repair process after injury. The commonly used markers are P63 and Krt5.

[0256] Bronchial epithelial cells

[0257] Bronchial epithelial cells are the main cell type that constitutes the inner wall of the bronchus. Bronchial epithelial cells mainly include the following types of cells: ciliated cells, goblet cells, club cells, basal cells, and neuroendocrine cells.

[0258] Small airway disease and small airway dysfunction

[0259] Small airway disease (small airway disease) refers to the abnormalities in structure or pathology of small airways (airways with an inner diameter less than 2 mm, including terminal bronchioles and respiratory bronchioles), such as inflammation, fibrosis, stenosis, or obstruction, etc. It is usually related to chronic inflammation, infection, smoking, or other injury factors. Small airway dysfunction (Small Airway Dysfunction, SAD) refers to the abnormalities in function of small airways (airways with an inner diameter less than 2 mm), resulting in airflow limitation or decreased ventilation function, but not yet reaching the diagnostic criteria for obstructive ventilatory dysfunction. It reflects the abnormal manifestations of small airways in gas exchange and air flow conduction. Small airway disease may lead to small airway dysfunction, but dysfunction may also occur when the disease is not obvious. Small airway dysfunction may be an early manifestation of small airway disease, indicating potential pathological changes.

[0260] Small airway disease includes abnormalities in structure or pathology of small airways (airways with an inner diameter less than 2 mm, including terminal bronchioles and respiratory bronchioles), such as inflammation, fibrosis, stenosis, or obstruction, etc.

[0261] Indicators reflecting small airway lesions in pathological examinations:

[0262] 1. Indicators related to bronchiolitis

[0263] Cellular bronchiolitis: It is mainly manifested by a large number of inflammatory cell infiltrations around the bronchioles, such as lymphocytes, plasma cells, etc. The aggregation degree and distribution range of these cells can reflect the severity and scope of inflammation.

[0264] Constrictive bronchiolitis: The pathological feature is fibrosis of the bronchiolar wall, resulting in lumen stenosis or occlusion. During the examination, it can be seen that the bronchiolar wall is significantly thickened, the lumen becomes narrower, or even completely occluded. The thickness of this wall and the degree of lumen stenosis are important indicators for evaluating constrictive bronchiolitis.

[0265] 2. Indicators related to small airway inflammation

[0266] Epithelial cell changes: Necrosis and exfoliation of small airway epithelial cells are common pathological manifestations. In pathological sections, the observation of a large number of epithelial cell fragments and exfoliated epithelial cells indicates an acute inflammatory reaction in the small airways.

[0267] Mucus secretion and goblet cell hyperplasia: During small airway inflammation, goblet cells will hyperplasia and mucus secretion will increase. In pathological examinations, it can be seen that the bronchiolar lumen is filled with mucus and inflammatory exudates. These changes reflect the hyperreactivity and inflammatory state of the small airways.

[0268] 3. Indicators related to small airway stenosis

[0269] Wall thickening: The thickening of the small airway wall is an important cause of airway stenosis. In pathological examinations, by measuring the wall thickness, the degree of small airway stenosis can be evaluated. The degree of wall thickening is directly related to the increase in airflow resistance.

[0270] Lumen stenosis: By observing the change in the lumen diameter, the stenosis of the small airways can be evaluated. The degree of lumen stenosis can be quantified by measuring the minimum diameter of the lumen. The stenotic lumen will cause airflow limitation, which is an important pathological feature of small airway lesions.

[0271] 4. Other indicators

[0272] Alveolar structure changes: Small airway lesions may also be accompanied by the destruction of the alveolar structure, such as the rupture of the alveolar wall and the formation of pulmonary emphysema. In pathological examinations, observing the destruction of the alveolar structure and the scope of pulmonary emphysema can reflect the extensive impact of small airway lesions on lung tissue.

[0273] Inflammatory cell infiltration: A large number of inflammatory cells, such as eosinophils and neutrophils, can be seen around small airways and in the alveolar septum. The degree of aggregation of these inflammatory cells can reflect the activity of inflammation.

[0274] These pathological indicators are of great significance in the diagnosis of small airway diseases. By comprehensively evaluating these indicators, the type and severity of small airway diseases can be diagnosed more accurately.

[0275] Definition and function of goblet cells

[0276] · Definition: Goblet cells are a special type of epithelial cells, mainly present on the mucosal surfaces of the respiratory tract, digestive tract, etc. Their nuclei and most organelles are concentrated in the long-stalked base, and there is a well-developed Golgi apparatus at the apex, which stores a large number of mucin-containing granules, making the apex bulge from the edge part.

[0277] · Function:

[0278] Lubrication: The secreted mucus can wrap and protect the mucosal surface, prevent harmful substances from directly contacting cells, reduce the risk of mucosal damage. Its lubricating property helps reduce the friction on the mucosal surface and helps substances such as food and gas pass smoothly on the mucosa.

[0279] Microbial clearance: The mucin in the produced mucus can adsorb and fix foreign particles, bacteria, viruses and other microorganisms, form a mucus layer, and promote the clearance of microorganisms on the mucosal surface, thus keeping the mucosa clean and healthy.

[0280] Immune function: Participate in immune responses and can secrete some immune-related substances, such as antimicrobial peptides, immunoglobulins, etc., to resist invading pathogenic microorganisms.

[0281] Relationship between goblet cells and small airway diseases

[0282] Normal situation: Normal small airway epithelium lacks goblet cells and hardly secretes mucin5AC (mucin5AC, MUC5AC), but mainly secretes MUC5B to maintain mucosal defense. The proportion of goblet cells in human bronchial epithelial cells varies depending on the specific location and physiological state. The proportion of goblet cells in normal human bronchial epithelial cells is relatively low, usually between 10% and 20%.

[0283] Ciliated cells

[0284] Ciliated cells are a type of bronchial epithelial cells with numerous cilia on their surface. These cilia help to clear foreign substances and secretions in the respiratory tract through coordinated beating. Ciliary structure: There are hundreds of cilia on the cell surface, and each cilium is composed of microtubules and has the ability to move. Distribution: Mainly distributed in the epithelial layer of bronchi and bronchioles. Cell morphology: Columnar or cubic, with cilia at the top and connected to the basement membrane at the bottom. Clearance function: The cilia move regularly to push the mucus layer upward, clearing inhaled particulate matter, pathogens, and secretions. Defense barrier: The ciliary movement and the mucus layer together form the first line of defense in the respiratory tract, preventing pathogens and foreign substances from entering the lungs. Immune regulation: Participate in immune responses and reduce the risk of infection by clearing pathogens. Related diseases include: Ciliary dysfunction: such as primary ciliary dyskinesia (PCD), abnormal ciliary movement leads to mucus clearance disorders and is prone to respiratory tract infections. Chronic respiratory diseases: such as chronic bronchitis and COPD, ciliated cells are damaged or reduced, affecting mucus clearance function.

[0285] Sample

[0286] As used herein, the term "sample" or "specimen" refers to a material specifically associated with a subject from which specific information related to the subject can be determined, calculated, or inferred. A sample can be wholly or in part composed of biological material from the subject. A sample can also be a material that has been in contact with the subject in such a way that testing the sample can provide information related to the subject. A sample can also be a material that has been in contact with other material that is not the subject, but which enables the first material to be subsequently tested to determine information related to the subject, e.g., a sample can be a cleaning solution for a probe or scalpel. A sample can be a source of biological material outside of contact with the subject, provided that a person skilled in the art can still determine information related to the subject from the sample.

[0287] Samples are selected from biological samples such as, for example: tissue samples, such as bronchial epithelial samples.

[0288] In all of its embodiments, the methods as described above are applied to tissue samples. The tissue samples can be, for example, purified bronchial epithelial cell samples.

[0289] Reference value

[0290] As used herein, the term "reference value" or "reference quantity" or "reference level" refers to the value (or quantity, or level) of a parameter or biomarker that indicates the status of a subject with respect to a particular disease (or minor ailment, or condition). The appropriate reference level of a parameter or biomarker can be quantified, determined, or measured by detecting the parameter / biomarker in a number of suitable reference subjects. Such reference levels can be adjusted according to a particular subject population. A reference value or reference level can be an absolute value; a relative value; a value with an upper or lower limit; a series of values; an average value; a median, mean, or value compared to a particular control or baseline value. A reference value can be based on the value of an individual sample, such as a value obtained from a sample from a tested subject but at an earlier time point. A reference level can be based on a large number of samples, such as a population of subjects in a chronological age-matched group, or on a pool of samples that includes or excludes the sample to be tested.

[0291] In the present invention, the reference value C1 is obtained by screening a large number of proportional values higher than the proportion (10 - 20%) of goblet cells (or goblet cells obtained by differentiating and culturing bronchial epithelial stem / progenitor cells) in bronchial epithelial cells (or differentiated bronchial epithelial cells) in the healthy population, and finally obtaining a proportional value that can more accurately, sensitively, and specifically diagnose early small airway lesions, which is 22.5%.

[0292] In the present invention, the reference value C2 is obtained by screening a large number of proportional values lower than the proportion (50 - 70%) of ciliated cells (or ciliated cells obtained by differentiating and culturing bronchial epithelial stem / progenitor cells) in bronchial epithelial cells (or differentiated bronchial epithelial cells) in the healthy population, and finally obtaining a proportional value that can more accurately, sensitively, and specifically diagnose early small airway lesions, which is 41.15%.

[0293] In the present invention, the reference value C0 corresponds to the value of a parameter (or biomarker) that is quantified, or determined, or measured on a sample from a healthy reference subject; or corresponds to the average (mean value) of the values of a parameter (or biomarker) that is quantified, or determined, or measured on different samples from the same healthy reference subject (values quantified / determined / measured on samples collected from the same healthy reference subject at various time intervals); or corresponds to the average (mean value) of the values of a parameter / biomarker that is determined / measured on the same sample from a healthy reference subject but at various time intervals; or corresponds to the average (or mean value) of the values of a parameter / biomarker that is quantified / determined / measured on samples from a number of healthy reference subjects (at least two healthy reference subjects).

[0294] ROC-AUC

[0295] ROC-AUC is a method for evaluating the accuracy of a model. The ROC curve is the Receiver Operating Characteristic curve, a coordinate graph with the false positive rate on the horizontal axis and the true positive rate on the vertical axis, which is a comprehensive index reflecting continuous variables of sensitivity and specificity. AUC is the area under the ROC curve. The ROC-AUC value is between 1.0 and 0.5. The closer it is to 1, the better the diagnostic effect. When it is between 0.5 and 0.7, there is low accuracy; when it is between 0.7 and 0.9, there is certain accuracy; when AUC is above 0.9, there is high accuracy. When AUC = 0.5, it means that the diagnostic method is completely ineffective and has no diagnostic value. AUC < 0.5 does not conform to the actual situation and rarely occurs in practice.

[0296] Use of functional cells or their detection reagents

[0297] In the present invention, there is provided a use of functional cells or their detection reagents, characterized in that it is used for preparing a diagnostic reagent or kit, and the diagnostic reagent or kit is used for (a) early diagnosis of small airway lesions; and / or (b) judging the prognosis of patients with small airway lesions, and the functional cells are selected from the group consisting of: goblet cells, ciliated cells, or a combination thereof.

[0298] In one embodiment, the kit includes: a reagent specifically detecting functional cells, and the reagent includes substances for detecting functional cells by using histochemical staining, immunoprecipitation, flow cytometry, Western blotting, ELISA, ELISPOT, antibody microarray, immunohistology, dot blotting, protein microarray, tissue microarray coupled with immunohistochemistry, or other well-known conventional immunological detection / analysis techniques.

[0299] In another preferred embodiment, the functional cells are detected or identified by one or more methods selected from the group consisting of: histochemical staining, immunoprecipitation, flow cytometry, Western blotting, ELISA, ELISPOT, antibody microarray, immunohistology, dot blotting, protein microarray, tissue microarray coupled with immunohistochemistry, or other well-known conventional immunological detection / analysis techniques.

[0300] In a specific embodiment, the functional cells are detected or identified by HE staining characteristics. The characteristics of goblet cells in HE staining are as follows: cell morphology: goblet cells are goblet-shaped, with a bulging top and a narrow bottom; cytoplasm staining: the cytoplasm is rich in mucin particles and is lightly stained, light blue or light pink; nuclear position: the nucleus is located at the bottom of the cell, is oblate or oval, and is stained darker; mucus secretion: a large number of mucus particles are often found in the top cytoplasm, which is lightly stained and sometimes vacuolated; distribution: goblet cells are scattered among airway epithelial cells to identify goblet cells in the epithelium.

[0301] The characteristics of ciliated cells in HE staining are as follows: Cell morphology: columnar or cubic, ciliated cells are usually columnar or cubic, arranged in the surface layer of bronchial epithelium; Presence of cilia: dense cilia can be seen at the top of the cell, which are long and thin hair-like structures facing the lumen; Staining of cilia: cilia are usually light pink or colorless in HE staining because they are mainly composed of protein; Nucleus: The nucleus is located at the base of the cell, usually oval or round; Nuclear staining: The nucleus is stained blue-purple by hematoxylin, and the chromatin is evenly distributed; Cytoplasmic staining: The cytoplasm is stained pink by eosin, which is usually relatively uniform; Epithelial arrangement: Ciliated cells and other epithelial cells (such as goblet cells) together constitute pseudostratified ciliated columnar epithelium, and the nuclei are located at different layers.

[0302] In a specific embodiment, functional cell markers (surface or intracellular) are detected or identified by specific antibodies against them.

[0303] In a preferred embodiment, the present invention provides a method for (a) early diagnosis of small airway disease in a patient / subject, wherein the patient / subject is a patient / subject in a healthcare institution, preferably a patient in a hospital, the method comprising the following steps:

[0304] (1) providing a sample from a subject to be tested, and detecting functional cells in the sample, including detecting the proportion of functional cells, including the proportion of goblet cells, and / or the proportion of ciliated cells;

[0305] (2) If the goblet cell ratio value determined in step (1) is higher than the reference value C1, it indicates that the subject suffers from small airway disease; and / or

[0306] If the ciliated cell ratio value determined in step (1) is lower than the reference value C2, it indicates that the subject to be tested suffers from small airway disease.

[0307] In a preferred embodiment, the present invention provides a method for determining the prognosis of a patient with small airway disease, wherein the patient / subject is a patient / subject in a healthcare institution, preferably a patient in a hospital, and the method comprises the following steps:

[0308] (1) Provide a sample from a subject to be tested, and detect the functional cells in the sample, including detecting the proportion of functional cells, including the proportion of goblet cells and / or the proportion of ciliated cells;

[0309] (2) If the proportion value of goblet cells measured in step (1) is lower than the reference value C1 or conforms to the reference value C0 of the healthy population, it indicates that the prognosis of the small airway lesion of the subject to be tested is better; and / or

[0310] If the proportion value of ciliated cells measured in step (1) is higher than the reference value C2 or conforms to the reference value C0' of the healthy population, it indicates that the prognosis of the small airway lesion of the subject to be tested is better.

[0311] In the present invention, the goblet cells have the marker expression characteristic of expressing Muc5AC protein; among them, the ciliated cells have the marker expression characteristic of expressing FoxJ1 or acetylated α-tubulin.

[0312] Using the antibodies of the cell markers described above to detect the goblet cells and ciliated cells of the present invention is a method well-known to those skilled in the art.

[0313] The method of the present invention is a method carried out in vitro or ex vivo. For example, the present invention has the advantages of being able to easily (a) early diagnose small airway lesions; and / or (b) judge the prognosis of patients with small airway lesions by providing directly measurable markers, especially being more accurate in the early diagnosis of small airway lesions and the judgment of prognosis. The measurement of the markers is completely applicable to be carried out by an automated analysis machine or by a test method called a rapid test.

[0314] The sample for implementing the method of the present invention is also referred to as a test sample in the present invention.

[0315] The test sample is taken from the biological sample of a patient / subject suspected of having a small airway lesion or the biological sample of a patient / subject with a small airway lesion.

[0316] In particular, the test sample is selected from biological samples such as tissue samples, such as bronchial epithelial cell samples.

[0317] The sample from which the reference value can be determined is also called a "control sample". In particular, in order to obtain the reference value, these samples are preferably obtained from subjects or patients / subjects from a healthy control population with the same characteristics or most common characteristics, especially the same gender and / or similar or the same age and / or ethnic origin.

[0318] In the context of the present invention, the terms "detection" or "measurement" or "determination" are used interchangeably and have the same meaning. These terms can represent the detection and quantification of the functional cell proportion value. To this end, any detection and / or quantification method known to those skilled in the art can be used to implement the present invention.

[0319] In particular, the determination of the proportion value of functional cells is carried out using specific tools or reagents that allow the direct or indirect determination of their presence and / or quantification of the functional cell proportion value, including detecting substances of functional cells using histochemical staining, immunoprecipitation, flow cytometry, Western blotting, ELISA, ELISPOT, antibody microarray, immunohistology, dot blotting, protein microarray, tissue microarray coupled with immunohistochemistry, or other well-known immunological conventional detection / analysis techniques.

[0320] In the method of the present invention, well-known analysis techniques can be particularly used to detect and / or quantify the functional cell proportion value, such as histochemical staining, cell membrane staining using biotinylation or other equivalent techniques followed by immunoprecipitation with specific antibodies, flow cytometry, Western blotting, ELISA, ELISPOT, antibody microarray, immunoprecipitation, immunohistology, dot blotting, protein microarray, or tissue microarray coupled with immunohistochemistry. Other suitable techniques include FRET or BRET, single-cell microscopy or histochemical methods using single or multiple excitation wavelengths and applying any suitable optical method, such as electrochemical methods (voltammetry and amperometry techniques), atomic force microscopy, and radiofrequency methods, such as multipolar resonance spectroscopy, confocal and non-confocal, detecting fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, resonant mirror method, grating-coupled waveguide method, or interferometry), cell ELISA, radioisotopes, magnetic resonance imaging, polyacrylamide gel electrophoresis (SDS-PAGE) analysis; HPLC-mass spectrometry; liquid chromatography / mass spectrometry / mass spectrometry (LC-MS / MS).

[0321] In a preferred embodiment, the proportion value of functional cells is determined by the following method:

[0322] (a) Providing an ex vivo bronchial epithelial sample and trophoblast cells obtained by irradiating fibroblasts;

[0323] (b) Centrifuging, washing, and digesting the sample to obtain a cell suspension containing bronchial epithelial stem / progenitor cells;

[0324] (c) Inoculate the cell suspension containing bronchial epithelial stem / progenitor cells onto the feeder layer cells, and culture in the presence of a medium for culturing bronchial epithelial stem / progenitor cells, thereby obtaining the bronchial epithelial stem / progenitor cells;

[0325] (d) Mix the bronchial epithelial stem / progenitor cells obtained in step (c) with the medium for culturing bronchial epithelial stem / progenitor cells, thereby obtaining a bronchial epithelial stem / progenitor cell suspension;

[0326] (e) Under appropriate culture conditions, add a stem cell medium and the bronchial epithelial stem / progenitor cell suspension to the upper chamber of a cell culture insert for culturing;

[0327] (f) On the 3rd - 5th day of culture (preferably the 4th day), remove the bronchial epithelial stem / progenitor cell medium, add a bronchial epithelial stem / progenitor cell differentiation medium for differentiation culture, change the bronchial epithelial stem / progenitor cell differentiation medium every two days, and fix and paraffin-embed the epithelial cells obtained after 14 - 24 days (preferably the 19th day) of differentiation culture to obtain a paraffin block;

[0328] (g) Section the paraffin block and perform HE staining to determine the proportion of functional cells (including goblet cells and / or ciliated cells), that is, the percentage of the number of functional cells (including goblet cells and / or ciliated cells) in bronchial epithelial cells.

[0329] In a preferred embodiment, the specific scheme is as follows:

[0330] The first part is the culture and expansion of bronchial stem / progenitor cells:

[0331] (1) Take exfoliated bronchial epithelial cells by brushing. Place the cell brush in a sample preservation solution and transport it to the production workshop at a temperature of 2 - 8°C.

[0332] (2) Flush the bronchial epithelial cells off the cell brush, centrifuge at 300g - 1000g and wash 3 times with the sample preservation solution, then digest with the digestive solution TrypLE (purchased from Thermo Fisher Scientific, catalog number: 12604013) for 10 - 60 minutes to obtain an epithelial cell suspension containing bronchial epithelial stem / progenitor cells;

[0333] (3) Thaw 10 6 frozen feeder layer cells from liquid nitrogen 24 hours in advance. The feeder layer cells are fibroblasts of the 3T3 - J2 cell line (purchased from Kerafast, product number: EF3003), which are frozen in liquid nitrogen after being irradiated with 20 - 80 Gy. The results show that the feeder layer cells after 20 - 80 Gy irradiation dose have a high stem cell cloning rate, and the effect of 30 - 60 Gy dose is better. For example, Figure 2As shown, it was laid on two wells of a 12-well cell culture plate. All of the cell suspension obtained in step (2) was seeded onto the feeder layer.

[0334] (4) Perform primary culture with bronchial epithelial stem / progenitor cell medium, and culture at 37 °C under a CO₂ concentration of 5% - 10%. After the cells adhered and grew, change the medium 2 - 3 times a week; during the culture process, sample for endotoxin and mycoplasma detection to ensure negative results. 2 Concentration was 5% - 10% condition, and after the cells adhered and grew, the medium was changed 2 - 3 times a week; during the culture process, samples were taken for endotoxin and mycoplasma detection to ensure they were negative.

[0335] (5) After culturing for 5 - 10 days, clones appeared. More than 80% of the clones had regular and smooth outlines, clear clone boundaries, and the cells inside the clones were tightly arranged and uniform in size. As Figure 3 . The clone immunofluorescence staining map is as Figure 4 . When the cell clones were approaching confluence, the clones were digested. The cell suspension obtained after digestion was seeded onto the feeder layer cells at 10,000 - 20,000 cells / cm² to complete passage. After culturing for 5 days, cell clones appeared. The cell clone density was uniform, the clone outlines were regular, the boundaries were clear, and the cells inside the clones were uniform in size, as 2 shown. Figure 5

[0336] (6) Use bronchial epithelial stem / progenitor cell medium to repeat the above clone passage (passage 2 times, transfer the cell order to 3.5 cm and 6 cm culture dishes, and expand the epithelial stem cells to a quantity of 5×10⁶ cells. 6 cells.

[0337] The samples were collected following the Declaration of Helsinki and approved by the Ethics Committee of Zhejiang Xinda Hospital, and were collected using the method of the present invention with the patient's informed consent signature.

[0338] The second part is the air - liquid differentiation and analysis of bronchial stem / progenitor cells.

[0339] (a) Collect the bronchial epithelial stem / progenitor cells amplified in step (6), add an appropriate amount of pre - warmed bronchial epithelial stem / progenitor cell medium, and gently pipette several times with a pipette gun to obtain a single - cell suspension (concentration = 1.5×10⁵ / mL). 6 / mL).

[0340] (b) Add 700 μL of bronchial epithelial stem / progenitor cell medium / well (24 - well plate) to the lower chamber of the cell culture insert.

[0341] (c) Vertically pipette 200 μL of the cell suspension containing 3×10⁴ cells into the upper chamber of the cell culture insert, and then place the well plate in an incubator at 37 °C and 7.5% CO₂. 5 cells of the cell suspension, and then place the well plate in an incubator at 37 °C and 7.5% CO₂. 2 ​Cultivate in an incubator. Replace the bronchial epithelial stem / progenitor cell medium in the lower chamber every two days, 700 μL each time.

[0342] (d) After 4 days of cultivation, remove the bronchial epithelial stem / progenitor cell medium from the upper and lower chambers.

[0343] (e) Add 700 μL of bronchial epithelial stem / progenitor cell differentiation medium to the lower chamber, and then replace it with fresh 700 μL of bronchial epithelial stem / progenitor cell differentiation medium every two days.

[0344] (f) On the 14th - 24th day of cultivation (preferably the 19th day), aspirate the medium in the lower chamber, and add 700 μL of PBS buffer to the lower chamber for washing;

[0345] (g) After discarding the PBS, use a sterile blade to separate the membrane, transfer the membrane to 5 mL of 4% PFA, and fix it at room temperature for 30 min; after fixation, wash it once with PBS and then transfer it to a 1.5 mL EP tube for subsequent paraffin embedding.

[0346] (h) Cut the paraffin sections with a thickness of 5 - 10 μm. Perform HE staining on the sections, and scan the panoramic images after staining. Analyze the proportions of ciliated cells and goblet cells; among them,

[0347] (i) If the proportion of goblet cells is greater than the proportion of goblet cells in normal epithelial cells by 10 - 20%, preferably greater than 22.5%, it is diagnosed as having small airway lesions; and / or

[0348] (ii) If the proportion of ciliated cells is less than the proportion of ciliated cells in normal epithelial cells by 50 - 70%, preferably less than 41.15%, it is diagnosed as having small airway lesions.

[0349] The bronchial epithelial stem / progenitor cell differentiation medium contains the following components: 50% (v / v) DMEM medium, 49% (v / v) Ham's F - 12 medium, glucose (4000 mg / L), insulin (0.1 - 5 μg / mL), EGF (0.1 - 0.5 μg / mL), RA (0.08 - 5 μM), hydrocortisone (0.5 - 5 μg / mL), 1% fetal bovine serum (v / v)

[0350] Basal medium

[0351] Cell culture basal medium is the basic nutrient medium for cell culture and usually needs to be selected and supplemented according to different cell types and experimental requirements. DMEM medium is a widely used basal medium and is suitable for the culture of various mammalian cells. Ham's F12 medium is a classic cell culture medium developed by Ham in 1965. It contains nutrients similar to DMEM, but its formula is slightly different, especially containing richer trace elements and growth factors. DMEM / F12 medium is composed of DMEM medium and Ham's F-12 medium mixed in a ratio of 1:1, combining the advantages of the two media and providing more comprehensive nutrients, which is suitable for the culture of various cell types.

[0352] Fetal bovine serum

[0353] Fetal bovine serum is a slightly viscous liquid with a light yellowish clear appearance, no hemolysis, and no foreign matter. Fetal bovine serum should be obtained from fetuses delivered by cesarean section; serum is a very complex mixture formed by removing fibrin from plasma. Serum contains various plasma proteins, polypeptides, fats, carbohydrates, growth factors, hormones, inorganic substances, etc.

[0354] Glucose

[0355] Glucose is a colorless monosaccharide that is easily soluble in water, sweet-tasting, and widely distributed in nature, with the molecular formula C 6 H 12 O 6 , and has a very wide range of uses. In the human body and cells, glucose can quickly supplement energy.

[0356] Insulin

[0357] Insulin is a protein hormone secreted by pancreatic islet β cells in the pancreas stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc.

[0358] Epidermal growth factor (EGF)

[0359] Epidermal growth factor (EGF) is a heat-resistant single-chain low-molecular polypeptide composed of 53 amino acid residues. After EGF specifically recognizes and binds to the EGF receptor on the target cell, a series of biochemical reactions occur, and ultimately it can promote the DNA synthesis and mitosis of the target cell.

[0360] Hydrocortisone

[0361] Hydrocortisone, also known as cortisol, is an organic compound with the chemical formula C21H30O5. It is a corticosteroid extracted from the adrenal cortex that has the strongest effect on carbohydrate metabolism, that is, it belongs to a kind of glucocorticoid.

[0362] Y-27632

[0363] Y-27632 is a small molecule inhibitor of Rho-associated protein kinase p160ROCK.

[0364] The structural formula of Y-27632 (CAS.NO: 146986-50-7) is shown below:

[0365]

[0366] SB431542

[0367] SB431542 is a potent and selective inhibitor of the TGF-β signaling pathway.

[0368] Structural formula:

[0369]

[0370] Noggin

[0371] Noggin is a secreted homodimeric glycoprotein and an antagonist of bone morphogenetic proteins (BMPs).

[0372] During skeletal development, Noggin prevents chondrocyte proliferation and thus regulates the normal formation of joints. When culturing human embryonic stem cells (hESCs) or neural stem cells in adult central nervous system and peripheral tissues (such as the lung) under certain conditions, adding Noggin to antagonize BMP activity can enable stem cell proliferation while maintaining their undifferentiated state, or differentiating into dopaminergic neurons. In mice lacking Noggin, enhanced BMP activity causes a series of developmental abnormalities, including failure of neural tube formation, retarded hair follicle development, axial skeletal malformations, and joint lesions, etc.

[0373] Noggin also plays an important role in organoid culture. It is widely used in the long-term culture of various organoids, such as liver organoids, small intestine organoids, and fallopian tube organoids. Noggin binds to bone morphogenetic proteins to coordinate the activation of Wnt signals and promote the proliferation of stem cells. During organoid culture, Noggin maintains the self-renewal ability of stem cells and prevents premature differentiation of stem cells by inhibiting the BMP signaling pathway. For example, in the culture of intestinal organoids, Noggin is added to the culture medium to inhibit the BMP signaling pathway and maintain the self-renewal ability of intestinal stem cells. In the culture of brain organoids, Noggin promotes the self-renewal of neural stem cells and the generation of neurons by inhibiting BMP signals.

[0374] R-spondin 1

[0375] R-spondin 1 (RSPO1) is a secreted activating protein belonging to the R-spondin protein family, which includes four members: R-spondin 1-4. RSPO1 has two cysteine-rich furin-like domains (FU-like CR) and one thrombospondin type 1 domain (TSR). It is a pleiotropic signaling ligand, and its most well-known function is to activate the Wnt / β-catenin signaling pathway. RSPO1 enhances Wnt / β-catenin signaling by acting on the cognate receptors LGR4 / 5 / 6 expressed by stem cells and progenitor cells, especially in proliferative stem cells. Therefore, RSPO1 plays an important role in the regulation of stem cells in multiple organs and is a key factor in the in vitro expansion culture of various adult stem cells, including those in the intestine, stomach, and liver.

[0376] Fibronectin

[0377] Fibronectin is a large extracellular membrane protein present on the surface of various animal cells and is the main non-collagenous glycoprotein in the extracellular matrix and basement membrane. It plays a central role in cell adhesion and can regulate cell polarity, differentiation, and growth.

[0378] E-Cadherin

[0379] E-Cadherin (cadherin E) protein is also known as intercellular adhesion molecule 1 (CDH1). It plays an important role in cell-cell junctions, can maintain cell adhesion and structural integrity between cells, and is involved in cell polarity and cell migration processes. E-Cadherin is mainly expressed in [missing context], can play a key role in cell-cell recognition and adhesion, and due to its important functions, E-Cadherin plays an important role in physiological and pathological processes such as embryonic development, tissue repair, and tumor metastasis.

[0380] RA

[0381] RA (Retinoic acid), also known as vitamin A acid, retinoic acid, tretinoin, retinol, etc., is a metabolic intermediate of vitamin A, which can induce cell differentiation and apoptosis and plays a key role in cell growth, differentiation, and organ formation.

[0382] Structural formula:

[0383]

[0384] Culture medium for culturing bronchial epithelial stem / progenitor cells

[0385] The present invention provides a culture medium for culturing bronchial epithelial stem / progenitor cells, which comprises a basal medium and additives; wherein the basal medium is selected from the following group: DMEM, Ham's F-12, DMEM / F12, or a combination thereof, and the additives include: fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, E-Cadherin.

[0386] The bronchial epithelial stem / progenitor cell clones amplified by the above specially added components have a relatively high cloning rate, can isolate epithelial stem cell clones from extremely trace amounts of cells, and achieve more than 50 passages of amplification. The cell stemness is maintained normally, the normal differentiation potential is maintained, and a sufficient number of bronchial epithelial stem / progenitor cells that can be infused clinically can be cultured for autologous infusion, realizing the repair of small airway injuries in the lungs clinically and significantly improving lung function.

[0387] Preferably, the basal medium is selected from the following group: DMEM, Ham's F-12, DMEM / F12, or a combination thereof; more preferably, the basal medium is DMEM, Ham's F-12, or a combination thereof.

[0388] Preferably, the culture medium for culturing bronchial epithelial stem / progenitor cells further comprises the following components: fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, E-Cadherin.

[0389] The preferred concentrations of the components in the culture medium are as described above, and most preferably, they can be the concentrations of the components in the examples of the present invention.

[0390] Using the culture medium of the present invention and in combination with the culture method of the present invention, the bronchial epithelial stem / progenitor cell clones amplified by the present invention have a relatively high cloning rate, can isolate epithelial stem cell clones from extremely trace amounts of epithelial cells, and achieve more than 50 passages of amplification. The cell stemness is maintained normally, the normal differentiation potential is maintained, and a sufficient number of bronchial epithelial stem / progenitor cells that can be infused clinically can be cultured for autologous infusion, realizing the repair of small airway injuries in the lungs clinically and significantly improving lung function.

[0391] Culture method of bronchial epithelial stem / progenitor cells

[0392] The present invention also provides a culture method of bronchial epithelial stem / progenitor cells, and the method comprises the steps:

[0393] (S1) Provide a bronchial epithelial sample and trophoblast cells, where the trophoblast cells are obtained by irradiating fibroblasts;

[0394] (S2) Centrifuge, wash, and digest the sample to obtain a cell suspension containing bronchial epithelial stem / progenitor cells;

[0395] (S3) Inoculate the cell suspension containing bronchial epithelial stem / progenitor cells onto the trophoblast cells and culture in the presence of the culture medium for culturing bronchial epithelial stem / progenitor cells of the present invention to obtain the bronchial epithelial stem / progenitor cells.

[0396] Preferably, the method further includes step (S4): Mix the bronchial epithelial stem / progenitor cells obtained by culturing and amplifying with a pharmaceutically acceptable carrier to prepare a biological preparation.

[0397] In another preferred example, in step (S4), there is also a step of detecting endotoxin.

[0398] In another preferred example, in step (S4), there is also a step of analyzing and / or identifying the bronchial epithelial stem / progenitor cells.

[0399] In a preferred embodiment, the culturing method of the present invention includes the following steps:

[0400] 1. Use a cell brush to brush a pulmonary bronchial epithelial cell sample from the airway of a COPD patient through a bronchoscope (the sample can also be a non-invasive liquid sample, tissue biopsy forceps sampling, biopsy brush sampling, surgical resection). Wash the cell sample off the cell brush, centrifuge at 300g - 1000g and wash 3 times with the sample preservation solution, and then digest with the digestive enzyme TrypLE (purchased from Thermo Fisher Scientific, catalog number: 12604013) for 10 - 60 minutes to obtain a cell suspension containing bronchial epithelial stem / progenitor cells;

[0401] 2. Irradiate 3T3 mouse fibroblasts at 20 - 80 Gy to prepare trophoblast cells and freeze them in liquid nitrogen for storage;

[0402] 3. Resuscitate the frozen cells in step 2 24 hours in advance to culture trophoblast cells, and seed the cell suspension obtained in step 1 at 10000 - 20000 cells / cm 2 onto the irradiated 3T3 mouse fibroblast trophoblast;

[0403] Perform primary culture with the bronchial epithelial stem / progenitor cell medium and culture at 37°C under the condition of a CO 2 concentration of 5% - 10%. After the cells adhere and grow, change the medium 2 - 3 times a week; During the culture process, sample for endotoxin and mycoplasma detection to ensure negative results;

[0404] 4. After 5 - 10 days of primary culture, when the cell clones are approaching confluence, digest the clones. After digestion, the cell suspension is seeded at 10,000 - 20,000 cells / cm 2 onto an irradiated 3T3 mouse fibroblast feeder layer to complete passage;

[0405] 5. Repeat the above-mentioned clone passage using bronchial epithelial stem / progenitor cell medium for 2 passages. Transfer the cells in sequence to 3.5 cm and 6 cm culture dishes, and expand the epithelial stem cells to more than 5×10 6 cells.

[0406] In the present invention, the sample preservation solution comprises the following components: DMEM medium containing 10 mg / mL BSA, and Penicillin (100 units / ml) - Streptomycin (100 μg / ml) therein.

[0407] In the present invention, the bronchial epithelial stem / progenitor cell medium comprises the following components: 45% (v / v) DMEM medium, 45% (v / v) Ham's F-12 medium, 10% (v / v) fetal bovine serum, 3000 mg / L glucose, 8 μg / mL insulin, 5 ng / mL epidermal growth factor, 3 μg / mL hydrocortisone, 2 μM Y-27632, 10 μM SB431542, 30 ng / mL Noggin, 60 ng / mL R-spondin 1, 2 μg / mL Fibronectin, 1 μg / mL E-Cadherin.

[0408] The main advantages of the present invention include:

[0409] (1) It is first discovered that by detecting the proportion of functional cells (i.e., the percentage of goblet cells and / or ciliated cells in bronchial epithelial cells, preferably the percentage of goblet cells and / or ciliated cells obtained after differentiation culture of bronchial epithelial stem / progenitor cells in differentiated bronchial epithelial cells), it can be used for (a) early diagnosis of small airway lesions; and / or (b) judging the prognosis of patients with small airway lesions, and has a very high diagnostic / judgment accuracy.

[0410] (2) The present invention early diagnoses small airway diseases by detecting abnormal changes in bronchial epithelial stem / progenitor cells of patients with small airway diseases. Bronchial epithelial cell samples are extracted through a bronchoscope, and bronchial epithelial stem / progenitor cells are amplified in vitro. The cultured bronchial epithelial stem / progenitor cells are differentiated into bronchial epithelial cells by a conventional air-liquid culture method or the method of the present invention, and the cell composition ratio of the bronchial epithelial cells is consistent with that of the bronchial epithelial cells in vivo. Fixed sections of the bronchial epithelial cells are made, and the cell composition ratio of the bronchial epithelial cells is observed by HE-stained sections, and the proportions of goblet cells and ciliated cells are counted to early diagnose small airway diseases.

[0411] (3) The method of the present invention can accurately detect abnormal changes in bronchial epithelial stem / progenitor cells, with high sensitivity and specificity, and can achieve non-invasive early diagnosis of small airway diseases, so as to achieve earlier intervention and treatment of diseases related to small airway diseases. Reducing the disease burden on individuals and countries.

[0412] (4) The present invention firstly develops a method for early diagnosing small airway diseases by culturing and differentiating bronchial epithelial stem / progenitor cells.

[0413] (5) The present invention firstly develops a culture medium for differentiating bronchial epithelial stem / progenitor cells.

[0414] (6) The method of the present invention can detect abnormal changes in bronchial epithelial stem / progenitor cells of patients with small airway diseases.

[0415] (7) The present invention develops a method for early diagnosing small airway diseases by counting the proportions of goblet cells and / or ciliated cells after the differentiation of bronchial epithelial stem / progenitor cells.

[0416] (8) The present invention develops a highly sensitive and specific method for diagnosing small airway diseases when the proportion of goblet cells is greater than 22.5%.

[0417] (9) The stem cell differentiation culture medium of the present invention can achieve the differentiation of bronchial epithelial stem / progenitor cells, reconstruct bronchial epithelial cells in vitro, and can more conveniently analyze the cell ratio of bronchial epithelial cells and evaluate abnormal changes in the airway in vivo.

[0418] (10) The present invention early diagnoses small airway diseases by analyzing that the proportion of goblet cells is greater than 22.5% and / or the proportion of ciliated cells is less than 41.15%, and the diagnosis is easy to operate.

[0419] The following specific embodiments illustrate the implementation manners of the present invention, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0420] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments, rather than limiting the protection scope of the present invention. The test methods without specific conditions indicated in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by each manufacturer.

[0421] When an embodiment gives a numerical range, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, equipment, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the records of the present invention, any methods, equipment, and materials of the prior art similar to or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0422] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions indicated in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0423] Unless otherwise specified, the reagents and materials used in the embodiments of the present invention are all commercially available products.

[0424] The samples were collected in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Zhejiang Xinda Hospital, and were collected by the method of the present invention with the patient's informed consent signed.

[0425] Example 1: Diagnosis of small airway lesions when the proportion of goblet cells exceeds the normal range after culturing and differentiating bronchial epithelial stem / progenitor cells

[0426] Mr. Xu, male, 51 years old, 76 kg, with a smoking history and frequent pulmonary infections, normal pulmonary function tests. To check for small airway lesions, bronchial epithelial cells were obtained by bronchoscopy brushing, and after culturing and differentiating bronchial epithelial stem / progenitor cells, the proportion of goblet cells was detected to exceed 10-20% of the normal range. At the same time, pathological examination found that he had small airway lesions. Bronchoscopy sampling ----- Bronchial stem / progenitor cell culture ----- Bronchial stem / progenitor cell amplification ----- Bronchial stem / progenitor cell inoculation into the chamber ----- Completion of the differentiation process, the flow chart is as Figure 1 . The specific steps are as follows:

[0427] (1) Mr. Xu underwent a bronchoscopy in November 2023, and a small amount of bronchial epithelial cells was obtained through a bronchial brush. The brush was placed in a sample preservation solution and transported to the production workshop at a temperature of 2-8°C. (2) The bronchial epithelial cells were flushed off the brush, centrifuged at 300g - 1000g, washed 3 times with the sample preservation solution, and then digested with TrypLE (purchased from Thermo Fisher Scientific, catalog number: 12604013) for 10 - 60 minutes to obtain a cell suspension containing bronchial epithelial stem / progenitor cells;

[0428] (3) 10 6 frozen trophoblast cells were thawed from liquid nitrogen 24 hours in advance (the trophoblast cells are fibroblast cell line 3T3-J2 cells (purchased from Kerafast, product number: EF3003), which were frozen in liquid nitrogen after irradiation with 20 - 80 Gy. The results showed that the stem cell cloning efficiency of trophoblast cells after 20 - 80 Gy irradiation was high, and the effect was better at a dose of 30 - 60 Gy, as Figure 2 shown), and seeded on 2 wells of a 12-well cell culture plate. All of the cell suspension obtained in step (2) was seeded on the trophoblast.

[0429] (4) Primary culture was carried out using bronchial epithelial stem / progenitor cell medium, and cultured at 37°C under a CO 2 concentration of 7.5%. After the cells adhered and grew, the medium was changed 2 - 3 times per week; samples were taken during the culture process for endotoxin and mycoplasma detection to ensure negative results.

[0430] (5) After 5 - 10 days of culture, clones appeared. More than 80% of the clones had regular and smooth outlines, clear clone boundaries, tightly arranged cells within the clone, and uniform sizes. As Figure 3 . The clone immunofluorescence staining was as Figure 4 . When the cell clones were approaching confluence, the clones were digested. The cell suspension obtained after digestion was seeded on trophoblast cells at 10000 - 20000 cells / cm 2 to complete passage. After 5 days of culture, cell clones appeared. The cell clone density was uniform, the clone outlines were regular, the boundaries were clear, and the cells within the clones had uniform sizes, as Figure 5 shown.

[0431] (6) Repeat the above clone passage using bronchial epithelial stem / progenitor cell medium (passage 2 times, transfer the cell order to 3.5 cm and 6 cm culture dishes, and expand the epithelial stem cells to a quantity of 5x10 6 cells.

[0432] (7)Collect the amplified bronchial epithelial stem / progenitor cells in (6), add an appropriate amount of pre-warmed bronchial epithelial stem / progenitor cell medium, and gently pipette several times with a pipette gun to obtain a single-cell suspension (concentration = 1.5x10 6 / mL). (8)Add 700 μL of bronchial epithelial stem / progenitor cell medium per well to the lower chamber of the cell culture insert (24-well plate).

[0433] (9)Vertically pipette 200 μL of the cell suspension containing 3*10 5 cells into the upper chamber of the cell culture insert, and then place the well plate in an incubator at 37°C and 7.5% CO 2 for culture.

[0434] (10)Replace the bronchial epithelial stem / progenitor cell medium in the lower chamber every two days, 700 μL each time.

[0435] (11)After 4 days of culture, remove the bronchial epithelial stem / progenitor cell medium from both the upper and lower chambers. Add 700 μL of bronchial epithelial stem / progenitor cell differentiation medium to the lower chamber, and then replace the fresh 700 μL of bronchial epithelial stem / progenitor cell differentiation medium every two days thereafter.

[0436] (12)On the 19th day of culture, aspirate the medium from the insert, add 700 μL of PBS buffer to the lower chamber of the insert for washing;

[0437] (13)After discarding the PBS, use a sterile blade to separate the membrane covered with bronchial epithelial cells, transfer the membrane to 5 mL of 4% PFA, and fix it at room temperature for 30 min; after the fixation is completed, wash it once with PBS and then transfer it to a 1.5 mL EP tube for subsequent paraffin embedding.

[0438] (14)Section the embedded paraffin, with a thickness of 5 - 10 μm. Perform HE staining on the sections, and scan the panoramic images after staining. The local images are as Figure 6 .

[0439] (15)The light-colored ones are goblet cells. Analyze the percentage of goblet cells in the bronchial epithelial cells, and the statistical proportion of goblet cells is 32%, which is greater than the normal range of 10 - 20%.

[0440] (16)Through pathological examination, it was found that there were small airway lesions characterized by thickening of the small airway epithelium and infiltration of inflammatory cells.

[0441] Example 2: Diagnosis of small airway lesions when the proportion of ciliated cells is lower than the normal range after culturing and differentiating bronchial epithelial stem / progenitor cells

[0442] Nie, male, 73 years old, 72kg, has a history of smoking, and is prone to lung infection due to colds. His lung function test is normal. In order to check whether there is small airway lesions, bronchial epithelial cells were brushed by bronchoscopy. After differentiation of bronchial epithelial stem / progenitor cells, the ciliated cells were detected to be lower than the normal range of 50-70%. At the same time, pathological examination revealed that he had small airway lesions. The specific steps are as follows:

[0443] (1) Nie underwent bronchoscopy in November 2023, and obtained a small amount of bronchial epithelial cells by bronchoscopic cell brushing. The cell brush was placed in sample preservation solution and transported to the production workshop at a temperature of 2-8°C. (2) The bronchial epithelial cells were flushed from the cell brush, centrifuged at 300g-1000g, washed three times with sample preservation solution, and then digested in digestion solution TrypLE (purchased from Thermo Fisher Scientific, catalog number: 12604013) for 10-60 minutes to obtain a cell suspension containing bronchial epithelial stem / progenitor cells;

[0444] (3) Resuscitate trophoblast cells frozen in liquid nitrogen 24 hours in advance 6 Cells (trophoblast cells are fibroblast cell line 3T3-J2 cells (purchased from Kerafast, product number: EF3003) are irradiated with 20-80 Gy and then frozen in liquid nitrogen. They are spread on two wells of a 12-well cell culture plate. The cell suspension obtained in step (2) is all planted on the trophoblast.

[0445] (4) Primary culture was performed using bronchial epithelial stem / progenitor cell culture medium at 37°C, CO 2 Culture at a concentration of 7.5%. After the cells adhere to the wall and grow, change the medium 2-3 times a week. During the culture process, take samples for endotoxin and mycoplasma testing to ensure that they are negative.

[0446] (5) After 5-10 days of culture, clones appeared, and more than 80% of the clones had regular and smooth outlines, clear clone boundaries, and cells in the clones were tightly arranged and uniform in size. When the cell clones were close to fusion, the clones were digested.

[0447] The cell suspension obtained after digestion was 10,000-20,000 cells / cm 2 After being implanted on trophoblast cells, the cells were subcultured. After 5 days of culture, cell clones appeared with uniform cell clone density, regular clone outlines, clear boundaries, and uniform cell size within the clone.

[0448] (6) Repeat the above cloning subculture using bronchial epithelial stem / progenitor cell culture medium (subculture twice, sequentially subculture the cells onto 3.5 cm and 6 cm culture dishes, and expand the epithelial stem cells to a number of 5×10 6 cells.

[0449] (7)Collect the amplified bronchial epithelial stem / progenitor cells in (6), add an appropriate amount of pre-warmed bronchial epithelial stem / progenitor cell medium, and gently pipette several times with a pipette gun to obtain a single-cell suspension (concentration = 1.5x10 6 / mL). (8) Add 700 μL of bronchial epithelial stem / progenitor cell medium per well to the lower chamber of the cell culture insert (24-well plate).

[0450] (9) Vertically pipette 200 μL of cell suspension containing 3*10 5 cells into the upper chamber of the cell culture insert, and then place the well plate in an incubator at 37 °C and 7.5% CO 2 for culturing.

[0451] (10) Replace the bronchial epithelial stem / progenitor cell medium in the lower chamber every two days, 700 μL each time.

[0452] (11) After 4 days of culturing, remove the bronchial epithelial stem / progenitor cell medium from the upper and lower chambers. Add 700 μL of bronchial epithelial stem / progenitor cell differentiation medium to the lower chamber, and then replace the fresh 700 μL of bronchial epithelial stem / progenitor cell differentiation medium every two days.

[0453] (12) On the 19th day of culturing, add 700 μL of PBS buffer to the lower chamber of the insert for washing;

[0454] (13) After discarding the PBS, use a sterile blade to separate the membrane covered with bronchial epithelial cells, transfer the membrane to 5 mL of 4% PFA, and fix it at room temperature for 30 min; after fixation, wash it once with PBS and then transfer it to a 1.5 mL EP tube for subsequent paraffin embedding.

[0455] (14) Section the embedded paraffin, with a thickness of 5 - 10 μm. Perform HE staining on the sections, and scan the panoramic images after staining. The local images are as shown in Figure 7 .

[0456] (15) Analyze the percentage of ciliated cells in bronchial epithelial cells, and the statistical result shows that the proportion of ciliated cells is 38%, which is lower than the normal range of 50 - 70%.

[0457] (16) Through pathological examination, it is found that there are small airway lesions characterized by thickening of the small airway epithelium and infiltration of inflammatory cells.

[0458] Example 3: The sensitivity and specificity of early diagnosis of small airway lesions using a goblet cell ratio greater than 22.5% are optimal

[0459] Select 200 people suspected of having lung diseases. After routine examinations such as pulmonary function tests or imaging examinations, no small airway lesions are found. At the same time, select 200 healthy people. Brush the bronchial epithelial cells of the people suspected of having lung diseases and healthy people, and use the method of culturing, amplifying and differentiating bronchial epithelial stem / progenitor cells in Example 1 for differentiation culture. Respectively count the proportion of goblet cells in 400 sample populations (the percentage of the number of goblet cells in the differentiated bronchial epithelial cells). At the same time, perform pathological examinations on these 400 samples to confirm the presence or absence of small airway lesions and divide them into a small airway lesion group and a healthy group. Use Graphpad software to perform ROC curve analysis on the 400 goblet cell proportion data. The ROC curve is as Figure 8 shown. The AUC area is 0.9906. Analyze the sensitivity and specificity of different proportion cut-off values as Figure 9 . It can be concluded from the results that the sensitivity and specificity of diagnosing small airway lesions when the proportion of goblet cells is greater than 22.5% are optimal (in the ROC curve evaluation method, when the area value AUC under the ROC curve is greater than 0.5, the closer it is to 1, the better the diagnostic effect).

[0460] Therefore, select the standard that the proportion of goblet cells is greater than 22.5% to diagnose early small airway lesions, and the diagnosis is the most accurate.

[0461] To further verify the effectiveness of the standard with a proportion greater than 22.5%, select 100 people suspected of having lung diseases. After routine examinations such as pulmonary function tests or imaging examinations, no small airway lesions are found. At the same time, select 100 healthy people (pathologically confirmed to have no small airway lesions) as the verification group. Brush the bronchial epithelial cells, and perform the culture, amplification and differentiation of bronchial epithelial stem / progenitor cells as in Example 1. Count the proportion of goblet cells. The statistical table of the diagnostic results and pathological results of diagnosing small airway lesions with a proportion greater than 22.5% as the standard is shown in Table 1 (unit: case).

[0462] Table 1

[0463] The pathology shows small airway lesions The pathology shows no small airway lesions Total The diagnosis shows small airway lesions 96 8 104 The diagnosis shows no small airway lesions 4 92 96 Total (cases) 100 100

[0464] It is calculated that the sensitivity of early diagnosing small airway lesions with a proportion of goblet cells greater than 22.5% as the standard is 96%, and the specificity is 92%.

[0465] Example 4: The sensitivity and specificity of early diagnosing small airway lesions with the proportion of ciliated cells less than 41.15% are optimal

[0466] Two hundred people suspected of having lung diseases were selected. After routine examinations such as pulmonary function tests or imaging examinations, no small airway lesions were found. At the same time, two hundred healthy people were selected. Bronchial epithelial cells were brushed from the people suspected of having lung diseases and healthy people, and were differentiated and cultured by the method of culturing, amplifying and differentiating bronchial epithelial stem / progenitor cells in Example 2. The proportions of ciliated cells (the percentage of the number of ciliated cells in the differentiated bronchial epithelial cells) in the 400 sampled populations were respectively counted. At the same time, these 400 samples were pathologically examined to confirm the presence or absence of small airway lesions and were divided into a small airway lesion group and a healthy group. The Graphpad software was used to perform ROC curve analysis on the data of the proportions of goblet cells in the 400 samples. The ROC curve is as Figure 10 shown, the AUC area is 0.9964. The sensitivities and specificities of different proportion cut-off values are as Figure 11 . It is concluded from the results that the sensitivity and specificity of diagnosing small airway lesions when the proportion of ciliated cells is less than 41.15% are optimal (in the ROC curve evaluation method, when the area value AUC under the ROC curve is greater than 0.5, the closer it is to 1, the better the diagnostic effect).

[0467] Therefore, the standard of selecting the proportion of goblet cells less than 41.15% for diagnosing early small airway lesions is the most accurate.

[0468] To further verify the effectiveness of diagnosing small airway lesions with a proportion value less than 41.15%, one hundred people suspected of having lung diseases were selected. After routine examinations such as pulmonary function tests or imaging examinations, no small airway lesions were found. At the same time, one hundred healthy people (confirmed by pathological examination to have no small airway lesions) were selected as the verification group. Bronchial epithelial cells were brushed and cultured, amplified and differentiated for bronchial epithelial stem / progenitor cells as in Example 2. The proportion of ciliated cells was counted. The statistical table of the diagnostic results of diagnosing small airway lesions with a proportion less than 41.15% as the standard and the clinical pathological results is shown in Table 2 (unit: case).

[0469] Table 2

[0470] The pathology shows small airway lesions The pathology shows no small airway lesions Total The diagnosis shows small airway lesions 97 5 102 The diagnosis shows no small airway lesions 3 95 98 Total (cases) 100 100

[0471] It is calculated that the sensitivity of early diagnosing small airway lesions with a ciliated cell proportion less than 41.15% as the standard is 97%, and the specificity is 95%.

[0472] Example 5: Early diagnosing small airway lesions with two indicators of a ciliated cell proportion less than 41.15% and a goblet cell proportion greater than 22.5% has high sensitivity and high specificity.

[0473] Two hundred people suspected of having lung diseases were selected. No small airway lesions were found through routine examinations such as pulmonary function examinations or imaging examinations. At the same time, two hundred healthy people were selected. Bronchial epithelial cells of the people suspected of having lung diseases and healthy people were brushed, and the method for culturing, amplifying, and differentiating bronchial epithelial stem / progenitor cells in Example 2 was used for differentiation culture. The proportions of ciliated cells and goblet cells (the percentage of the number of ciliated cells and goblet cells in the differentiated bronchial epithelial cells) and the pathological results of 400 cases of sample populations were respectively counted. Two indicators, that is, the proportion of ciliated cells less than 41.15% and the proportion of goblet cells greater than 22.5%, were used for the early diagnosis of small airway lesions. ROC curve analysis and AUC area calculation were performed using Graphpad software, and the AUC area was 0.9984 (in the ROC curve evaluation method, when the area value AUC under the ROC curve is greater than 0.5, the closer it is to 1, the better the diagnostic effect).

[0474] One hundred people suspected of having lung diseases were selected. No small airway lesions were found through routine examinations such as pulmonary function examinations or imaging examinations. At the same time, one hundred healthy people (confirmed to have no small airway lesions by pathological examinations) were selected. Bronchial epithelial cells were brushed, and the culture, amplification, and differentiation of bronchial epithelial stem / progenitor cells were carried out as in Examples 1 and 2. The proportions of ciliated cells and goblet cells were counted. The statistical table of the diagnostic results of diagnosing small airway lesions using the two indicators that the proportion of ciliated cells is less than 41.15% and the proportion of goblet cells is greater than 22.5% and the clinical pathological results is shown in Table 3 (unit: case).

[0475] Table 3

[0476] The pathology shows small airway lesions The pathology shows no small airway lesions Total The diagnosis shows small airway lesions 98 2 100 The diagnosis shows no small airway lesions 2 98 100 Total (cases) 100 100

[0477] It was calculated that the sensitivity of using the two indicators that the proportion of ciliated cells is less than 41.15% and the proportion of goblet cells is greater than 22.5% for the early diagnosis of small airway lesions was 98%, and the specificity was 98%.

[0478] Example 6: After autologous epithelial stem / progenitor cells were infused into patients with small airway lesions, the proportions of goblet cells and ciliated cells became within the normal range

[0479] For 50 patients with small airway lesions, epithelial cells were brushed, and the culture, amplification, and differentiation of bronchial epithelial stem / progenitor cells were carried out as in Example 1. The proportions of ciliated cells and goblet cells were respectively counted. The results showed that the proportion of ciliated cells in 50 patients with small airway lesions was lower than 40%, and the proportion of goblet cells was greater than 25%. The results were as Figure 12 . The preparation and infusion of autologous epithelial stem / progenitor cells were carried out for these 50 patients. The specific steps are as follows:

[0480] The preparation steps of the autologous bronchial epithelial stem / progenitor cell preparation are as follows:

[0481] 1. The patient undergoes bronchoscopy, and a small amount of bronchial epithelial cells are obtained by brushing with a bronchoscope cell brush. The cell brush is placed in a sample preservation solution and transported to the production workshop at a temperature of 2 - 8°C.

[0482] 2. The bronchial epithelial cells are flushed off the cell brush, centrifuged at 300g - 1000g, washed 3 times with the sample preservation solution, and then digested with the digestive solution TrypLE (purchased from Thermo Fisher Scientific, catalog number: 12604013) for 10 - 60 minutes to obtain a cell suspension containing bronchial epithelial stem / progenitor cells.

[0483] 3. Thaw 10 6 cells of cryopreserved trophoblast cells from liquid nitrogen 24 hours in advance (the trophoblast cells are fibroblasts 3T3 - J2 cells (purchased from Kerafast, product number: EF3003), irradiated with 20 - 80 Gy and then cryopreserved in liquid nitrogen). Seed them on 2 wells of a 12 - well cell culture plate. Seed all the cell suspension obtained in step 2 on the trophoblast layer.

[0484] 4. Perform primary culture with the bronchial epithelial stem / progenitor cell medium shown in the following table, culture at 37°C under a CO 2 concentration of 7.5%. After the cells adhere and grow, change the medium 2 - 3 times a week; during the culture process, take samples for endotoxin and mycoplasma detection to ensure they are negative.

[0485] The bronchial epithelial stem / progenitor cell medium includes a basal medium (such as 45% (v / v) DMEM medium, 45% (v / v) Ham's F - 12 medium) and various components added according to the following formula (calculated based on the total volume of the medium).

[0486]

[0487] 5. After 5 - 10 days of culture, clones appear. When the cell clones are nearly confluent, digest the clones. The cell suspension obtained after digestion is seeded on the trophoblast cells at 10000 - 20000 cells / cm 2 to complete passage. After 5 days of culture, cell clones appear with a uniform cell clone density.

[0488] 6. Use the bronchial epithelial stem / progenitor cell medium to repeat the above cloning passage (passage 4 times, transfer the cells in sequence to culture dishes of 3.5 cm, 6 cm, 10 cm, and 15 cm, and expand the epithelial stem / progenitor cells to a quantity of 2.2x10 8 cells.

[0489] 7. Digest and collect the amplified bronchial epithelial stem / progenitor cells described above, take 2x10 8Prepare 20 ml of cell suspension with PBS.

[0490] 8.Put the cell suspension preparation into a pre-filled syringe and store it at 2-8℃ for transportation. The sample cells were tested for endotoxin, mycoplasma and sterility, and the results were all negative. At the same time, 6x10 6 The cells were tested for ITGA6 by flow cytometry, and the positive rate was 99.2%.

[0491] 9. The cell preparation in the above-mentioned prefilled needle was transported to the hospital, and the cell suspension was infused into the bronchi of the five lobes of Li's lungs through bronchoscope to complete the clinical infusion.

[0492] After 6 months, bronchial epithelial cells were brushed and cultured, expanded and differentiated as in Examples 1 and 2 to culture and differentiate bronchial epithelial stem / progenitor cells. The ratios of goblet cells and ciliated cells were counted. The results showed that the goblet cell ratios of the 50 patients with small airway lesions were less than 20% after infusion of autologous epithelial stem / progenitor cells. The ciliated cell ratio was within the normal range of 50-70%. Figure 13 shown.

[0493] The results showed that autologous bronchial epithelial stem / progenitor cell infusion can treat small airway lesions diagnosed at an early stage and has a better prognosis.

[0494] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

[0495] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A use of a functional cell or a detection reagent thereof, characterized in that: Used to prepare a diagnostic reagent or kit, the diagnostic reagent or kit is used for (a) early diagnosis of small airway lesions; and / or (b) judging the prognosis of patients with small airway lesions, the functional cells are selected from the following group: goblet cells, ciliated cells, or a combination thereof.

2. The use according to claim 1, characterized in that The small airway lesions include diseases related to small airway abnormalities.

3. The use according to claim 2, characterized in that The small airway abnormalities include small airway inflammation, small airway fibrosis, small airway stenosis or obstruction, and small airway collapse.

4. The use according to claim 2, characterized in that The diseases associated with small airway abnormality include chronic obstructive pulmonary disease (COPD), asthma, interstitial pneumonia, idiopathic pulmonary fibrosis, small airway inflammation, bronchiolitis, bronchiolitis, small airway dysfunction, bronchiectasis, bronchial obstructive syndrome, or a combination thereof.

5. The use according to claim 1, characterized in that The detection reagent includes a reagent for detecting functional cells. Preferably, the detection of functional cells refers to determining the proportion of functional cells. Preferably, the determination of the proportion of functional cells refers to determining the percentage of functional cells in differentiated bronchial epithelial cells.

6. The use according to claim 1, characterized in that The functional cells are obtained by differentiation and culture of bronchial epithelial stem / progenitor cells.

7. A method for establishing a model for (a) early diagnosis of small airway lesions; and / or (b) determining the prognosis of patients with small airway lesions, characterized in that: The method includes the step of identifying differential biomarkers in biological samples between patients with small airway lesions and healthy controls or patients without small airway lesions, wherein the differential biomarkers include the proportion of functional cells, and the functional cells are selected from the following group: goblet cells, ciliated cells, or a combination thereof.

8. A system for early diagnosis of small airway lesions, characterized in that: The system comprises: (a) a feature receiving module, the feature receiving module is used to receive feature data from a sample of a subject to be tested; the feature data includes: a ratio value of functional cells in the sample of the subject to be tested, including a ratio value of goblet cells and / or a ratio value of ciliated cells; (b) a discrimination processing module, wherein the processing module compares the received characteristic data with a reference value to obtain a diagnosis or evaluation result, wherein when the ratio of goblet cells in the characteristic data is higher than the reference value C1, it indicates that the subject suffers from small airway disease; and / or When the ratio of ciliated cells in the characteristic data is lower than the reference value C2, it indicates that the subject suffers from small airway lesions; and (c) A result output module, which is used to receive and output the evaluation results.

9. A system for determining the prognosis of a patient with small airway disease, characterized in that: include: (a) a feature receiving module, the feature receiving module is used to receive feature data from a sample of a subject to be tested suffering from small airway lesions; the feature data includes: a ratio value of functional cells in the sample of the subject to be tested, including a ratio value of goblet cells and / or a ratio value of ciliated cells; (b) a discrimination processing module, wherein the processing module compares the received characteristic data with a reference value to obtain a diagnosis or evaluation result, wherein when the ratio of goblet cells in the characteristic data is lower than a reference value C1 or conforms to a reference value C0 of a healthy population, it indicates that the prognosis of the subject is good; and / or When the ratio of ciliated cells in the characteristic data is higher than the reference value C2 or conforms to the reference value C0' of healthy people, it indicates that the prognosis of the subject is good; and (c) A result output module, which is used to receive and output the evaluation results.

10. A method, characterized in that The functional cells in the test object are used as an indicator for (a) early diagnosis of small airway lesions; and / or (b) judging the prognosis of patients with small airway lesions, wherein the functional cells are selected from the following group: goblet cells, ciliated cells, or a combination thereof; The method comprises determining the ratio of the functional cells in a sample from the subject.

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