Use of protein markers or / and detection reagents thereof in preparation of a chronic obstructive pulmonary disease detection kit, protein markers, detection kit and detection system

Abstract

This application belongs to the field of biomedical technology, and specifically relates to the application of protein biomarkers and / or their detection reagents in the preparation of chronic obstructive pulmonary disease (COPD) diagnostic kits, the protein biomarkers, the diagnostic kits, and the detection system. The application of protein biomarkers and / or their detection reagents in the preparation of COPD diagnostic kits; the protein biomarkers include RSPH1 and TGM2. Compared with conventional technologies, this application has the following advantages: This application uses RSPH1 and TGM2 as a biomarker combination, and prepares a COPD diagnostic kit using this biomarker combination and / or its detection reagents. Using this diagnostic kit, high specificity and high sensitivity diagnosis of COPD can be achieved, effectively distinguishing COPD patients from healthy individuals.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and specifically relates to the application of protein biomarkers and / or their detection reagents in the preparation of chronic obstructive pulmonary disease detection kits, protein biomarkers, detection kits and detection systems. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a common, preventable, and treatable chronic inflammatory airway disease. Its diagnosis and severity assessment primarily rely on pulmonary function tests (such as FEV1 / FVC), combined with clinical symptoms, imaging findings, and medical history. However, pulmonary function tests have limitations, including insufficient sensitivity and low specificity. Therefore, developing COPD testing products with high sensitivity and specificity is crucial for effectively distinguishing COPD patients from healthy individuals.

[0003] Currently, reported biomarkers such as inflammation-related proteins (including TGM2) and miRNAs have improved the detection efficacy of COPD clinical assessment compared to pulmonary function tests, but their diagnostic efficacy still needs to be improved. Summary of the Invention

[0004] Based on this, one or more embodiments of this application provide the application of protein biomarkers and / or their detection reagents in the preparation of chronic obstructive pulmonary disease (COPD) detection kits, protein biomarkers, detection kits, and detection systems. The technical solutions include the following:

[0005] One or more embodiments of this application provide the application of a protein biomarker and / or its detection reagent in the preparation of a detection kit for chronic obstructive pulmonary disease;

[0006] The protein biomarkers include RSPH1 and TGM2.

[0007] In some embodiments of this application, the detection reagent detects RSPH1 in ciliated cells and / or RSPH1 in ciliated cell exosomes.

[0008] In some embodiments of this application, the samples detected by the detection reagent include one or more of the following: bronchial brushing material, respiratory fluid samples, airway epithelial cell culture supernatant, and organoid systems differentiated from bronchial stem cells.

[0009] In some embodiments of this application, the respiratory fluid sample includes one or more of sputum and bronchoalveolar lavage fluid.

[0010] In some embodiments of this application, the detection reagent includes a quantitative detection reagent.

[0011] In some embodiments of this application, the detection reagents for RSPH1 and TGM2 are each detected independently by the following methods:

[0012] Enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry, immunofluorescence, mass spectrometry, real-time quantitative PCR, and immunoturbidimetry.

[0013] One or more embodiments of this application provide a protein biomarker, as defined above.

[0014] One or more embodiments of this application provide a detection kit, the detection kit comprising protein biomarkers and / or detection reagents as defined above.

[0015] In some embodiments of this application, the detection kit further includes exosome extraction reagents and / or exosome protein extraction reagents.

[0016] One or more embodiments of this application provide a detection system for chronic obstructive pulmonary disease, the system comprising:

[0017] Detection component: The detection component is used to detect protein biomarkers as defined above at the protein level;

[0018] Result judgment component: The result judgment component is used to output the detection result based on the result of the protein marker detected by the detection component.

[0019] Compared with traditional technologies, this application has the following advantages:

[0020] This application uses RSPH1 and TGM2 as a combination of biomarkers to prepare a diagnostic kit for chronic obstructive pulmonary disease (COPD). This kit enables highly specific and sensitive diagnosis of COPD, effectively distinguishing COPD patients from healthy individuals. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1The results show the analysis using publicly available human lung single-cell transcriptome data (Integrated Single-Cell Atlas of Endothelial Cells of the Human Lung, Circulation, 2021, PMID: 34850931), and the RSPH1 expression in different lung cell types obtained through the Lung Endothelial Cell Atlas website (https: / / www.lungendothelialcellatlas.com / ).

[0023] Figure 2 The image shows the differences in RSPH1 expression among basal cells (BC), goblet cells (GC), and ciliated cells (CC) from healthy non-smokers, as well as the differences in RSPH1 expression among basal cell exosomes (BC-EV), goblet cell exosomes (GC-EV), and ciliated cell exosomes (CC-EV) derived from healthy non-smokers.

[0024] Figure 3 The following figures show the expression levels of RSPH1 in exosomes of different airway epithelial cells (ciliated cells, basal cells, and goblet cells); A. Nanoflow cytometry results; B. Western blot results.

[0025] Figure 4 The image shows the differential expression of TGM2 protein in ciliated cells and their exosomes in the healthy non-smoker group, the smoker group, the mild-moderate COPD group (Mild. COPD), and the severe COPD group (Severe COPD).

[0026] Figure 5 The image shows immunofluorescence staining of TGM2 and α-tubulin in bronchial tissues from healthy non-smokers, smokers, mild to moderate COPD patients, and severe COPD patients.

[0027] Figure 6 The image shows a comparison of TGM2 and α-tubulin immunofluorescence staining in ciliated cells derived from healthy controls and patients with mild to moderate COPD.

[0028] Figure 7The image shows the difference in TGM2 expression in exosomes derived from bronchial brushing cells from healthy controls and patients with mild to moderate COPD, as detected by Western blot.

[0029] Figure 8 The image shows CD63 in exosomes derived from bronchoalveolar lavage fluid of healthy controls and patients with mild to moderate COPD. + TGM2 + Exosome analysis; A. Schematic diagram of nanoflow cytometry detection; B. CD63 + TGM2 + C. Statistical analysis of exosome proportions; D. ROC curve analysis of diagnostic efficacy.

[0030] Figure 9 The image shows RSPH1 in exosomes derived from bronchoalveolar lavage fluid (BALF) in healthy controls and patients with mild to moderate COPD (Mild COPD). + TGM2 + Exosome analysis; A. Schematic diagram of nanoflow cytometry detection; B. RSPH1 + TGM2 + C. Statistical analysis of exosome proportions; D. ROC curve analysis of diagnostic efficacy. Detailed Implementation

[0031] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0033] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0034] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0035] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0036] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0037] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0038] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0039] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0040] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0041] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0042] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0043] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0044] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0045] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0046] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0047] Exosomes, as nanoscale vesicles secreted by cells, carry specific molecular information such as proteins and nucleic acids from their originating cells, and have become novel liquid biopsy targets for disease diagnosis. In the respiratory field, exosomes in bodily fluids such as sputum and bronchoalveolar lavage fluid (BALF) have been shown to contain biomarkers associated with lung disease states. However, existing research mainly focuses on exosomes derived from blood or the total exosome population, lacking precise identification and analysis of exosomes derived from specific airway cells. Airway epithelial cells, especially ciliated cells, goblet cells, and basal cells, play a crucial role in the core pathological mechanisms of COPD, such as mucus hypersecretion and ciliary dysfunction. If specific biomarkers of exosomes derived from specific epithelial cells (especially key disease cells) can be found, and disease-related molecules can be discovered from them, the specificity and accuracy of exosome diagnosis will be greatly improved.

[0048] Currently, research on exosome diagnosis for COPD is still in the exploratory stage. The reported biomarkers, such as inflammation-related proteins and miRNAs, have weak cell-derived specificity and their diagnostic efficacy needs to be improved. Moreover, few studies have been able to combine the capture of exosomes from specific cell sources with the detection of disease-specific biomarkers to build a high-precision joint diagnostic platform.

[0049] Based on this, one or more embodiments of this application provide the application of protein biomarkers and / or their detection reagents in the preparation of chronic obstructive pulmonary disease (COPD) detection kits, protein biomarkers, detection kits, and detection systems. The technical solutions include the following:

[0050] A first aspect of this application is to provide the application of a protein biomarker and / or its detection reagent in the preparation of a detection kit for chronic obstructive pulmonary disease;

[0051] The protein biomarkers include RSPH1 and TGM2.

[0052] Compared with traditional technologies, the beneficial effects of this application are mainly reflected in the following aspects:

[0053] Significantly improved diagnostic specificity and sensitivity: By combining RSPH1, a specific marker of ciliated cell-derived exosomes, with TGM2, a marker of COPD disease status, the precise identification and detection of disease-related exosomes released by key disease cells were achieved, thereby improving the specificity and accuracy of diagnosis at the source.

[0054] Highly clinically applicable and convenient for dynamic monitoring: This invention uses readily available respiratory fluids such as bronchoalveolar lavage fluid or sputum as test samples, avoiding the invasiveness of traditional lung biopsies, and is clinically feasible. Since exosomes can be repeatedly obtained, this method provides a convenient dynamic tracking tool for assessing COPD disease progression and monitoring treatment effectiveness.

[0055] In some examples of this application, the detection reagent detects RSPH1 in ciliated cells and / or RSPH1 in ciliated cell exosomes.

[0056] This application does not specifically limit the types of samples that can be detected by the test reagents, and may include, but is not limited to: bronchial brushing material, respiratory fluid samples, airway epithelial cell culture supernatant, and organoid systems differentiated from bronchial stem cells. This application also does not specifically limit the types of respiratory fluid samples, and may include, but is not limited to: sputum and bronchoalveolar lavage fluid.

[0057] The detection reagents in this application can be qualitative or quantitative; in some examples of this application, they are quantitative.

[0058] In the detection reagents of this application, the detection mechanisms / methods of the RSPH1 detection reagent and the TGM2 detection reagent may be the same or different. In some examples of this application, the RSPH1 and TGM2 detection reagents are each independently detected by the following methods: enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry, immunofluorescence, mass spectrometry, real-time quantitative PCR, and immunoturbidimetry.

[0059] A second aspect of this application provides a protein biomarker, as defined in the first aspect above. This protein biomarker can be used to prepare standards, quality control samples, etc.

[0060] A third aspect of this application provides a detection kit, the detection kit comprising protein biomarkers and / or detection reagents as defined in the first aspect above.

[0061] It is understood that the detection kit of this application may also include other detection reagents, including but not limited to: exosome extraction reagents and exosome protein extraction reagents.

[0062] A fourth aspect of the embodiments of this application provides a detection system for chronic obstructive pulmonary disease, the system comprising:

[0063] Detection component: The detection component is used to detect protein biomarkers as described in the second aspect above at the protein level;

[0064] Result judgment component: The result judgment component is used to output the detection result based on the result of the protein marker detected by the detection component.

[0065] Compared with traditional technologies, this application has the following advantages:

[0066] This application uses RSPH1 and TGM2 as a combination of biomarkers to prepare a diagnostic kit for chronic obstructive pulmonary disease (COPD). This kit enables highly specific and sensitive diagnosis of COPD, effectively distinguishing COPD patients from healthy individuals.

[0067] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0068] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0069] Example 1: Single-cell transcriptome analysis of RSPH1 expression in different human lung cells

[0070] The expression levels of RSPH1 in different cells of human control lungs (non-disease lung tissue, including both smokers and non-smokers; both smokers and non-smokers are considered controls in the application) were analyzed using Lung Endothelial Cell Atlas. Single-cell transcriptome data (scRNAseq data) were obtained from 68 control lung tissues from 5 cohorts: Vanderbilt / Translational Genomics Research Institute [TGen] (GSE135893, n=10), Northwestern (phs001750.v1.p1, n=8), Wellcome Sanger Institute [WSI] / Groningen10 (EGAD00001005064, EGAD00001005065, n=10), Leuven Vlaams Institute for Biotechnology [VIB] (E-MTAB-6149 and E-MTAB-6653, n=6), and Yale / Baylor (GSE136831, GSE133747, GSE164829, n=34).

[0071] Lung cell abbreviations: AM indicates alveolar macrophage; AT1 / 2, alveolar cell type 1 / 2; B, B cell; Basal, basal cell; cDC1 / 2, classical dendritic cell type 1 / 2; Cell Cycle; Ciliated; Club, Club cell (bronchiolar exocrine cells); cMono, classical monocyte; DC Langerhans; DC mature; EC Aerocyte; EC arterial; EC generalcapillary; EC lymphatic; EC pulmonary-venous; EC systemic-venous; Fibroblast adventitial. Alveolar fibroblasts; Goblet cells; ILC, innate lymphoid cells; Ionocytes; M, macrophages; Mast cells; Mesothelial cells; ncMono, nonclassical monocytes; NK, natural killer cells; pDC, plasmacytoid dendritic cells; Pericyte cells; Plasma cells; PNEC, pulmonary neuroendocrine cells; SMC, smooth muscle cells; Suprabasal cells; Tcytotoxic T cells; T helper T cells; T regulatory T cells.

[0072] Results: Single-cell transcriptome analysis and violin plot results showed that RSPH1 was highly expressed mainly in ciliated cells, while its expression was very low in other types of lung cells. Figure 1 ).

[0073] Example 2: Screening and identification of RSPH1, a marker of ciliated cell exosomes

[0074] (1) Sample collection and cell sorting: Bronchial brushing samples were collected from 28 healthy non-smoker subjects (25-55 years old). After washing and filtering with PBS (containing 2% FBS), the cells were resuspended in PneumaCult-ALI medium. High-purity airway epithelial cell sorting was performed by flow cytometry: the cells were resuspended in ALI medium containing 10 μM Verapamil (MCE, HY-14275) and SiR-Tubulin (Cytoskeleton, CY-SC002) and incubated at 37°C in the dark for 1 h. After centrifugation at 400g for 5 min, the supernatant was discarded and the cells were washed once with 1 mL PBS. After centrifugation again, 500 μL of 5% NGS / PBS (goat serum, Solarbio, S9070) containing human TruStain FcX (Biolegend, 422302) was added and the cells were incubated at room temperature in the dark for 15 min to block non-specific binding. The following antibodies were then added: CD271 / NGFR (PE-Cy7, Biolegend, 345110), CD66c / CEACAM6 (BV510, BD, 742684), and TSPAN8 (AF488, R&D Systems, FAB4734G-100UG). Negative control cells were blocked without antibodies. Cells were incubated at 4°C in the dark for 30 min. After incubation, cells were centrifuged at 400g for 5 min, the supernatant was discarded, and the cells were washed once with 1 mL PBS. After centrifugation, the cells were resuspended in eBioscience Flow Cytometry Staining Buffer (Invitrogen, 00-4222-26), and DAPI was added for staining to determine cell viability. The cell suspension was filtered through a 70 μm filter into flow cytometry tubes. Ciliated cells (CC, SIR-Tubulin) were sorted using a sorting flow cytometer (Sony LE-MA900FP). + / NGFR - ), basal cells (BC, NGFR) + / CEACAM6 - ) and goblet cells (GC, CEACAM6) + / TSPAN8 + ).

[0075] (2) Isolation and proteomics analysis of airway epithelial cells and their exosomes: Three types of airway epithelial cells were separated and centrifuged at 400g for 5 min at room temperature. One portion of the cells was collected and flash-frozen in liquid nitrogen (for cell proteomics), while the other portion was cultured in PneumaCult™-ALI Medium (Stemcell). The culture supernatant was collected every 48 h, centrifuged at 400g for 5 min, and then centrifuged at 2000g for 15 min to remove cell debris. This process was repeated three times. The culture conditions included: PneumaCult-ALI medium; temperature of 37±0.5℃; gas environment of 5% CO2, normoxic (approximately 21% O2); and saturated humidity.

[0076] After removing debris by low-speed centrifugation, exosomes were separated and purified using microfluidic chip-based technology (refer to Example 1 of CN114247489A). Collected cells and exosomes were added to 4 volumes of urea and 1% PMSF and lysed on ice for 30 min. Total protein was obtained by lysing using an ultrasonic cell disruptor (30W, 3s sonication, 5s pause, 2min per sample, on ice). The cells were then centrifuged at 12000g for 15 min at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA (Beyotime, P0009) analyzer. The samples were then transferred to 10kD ultrafiltration tubes and centrifuged at 12000g for 15 min at 4°C. This process was repeated until all samples were transferred to the ultrafiltration tubes. The cells were then rinsed with 8M urea and 50mM TEAB, and centrifuged at 12000g for 15 min at 4°C to remove the lower layer. Replace with a new collection tube, add 120 µL of 50 mM TEAB and 1 µg / µL rLys-C protease (Hualisheng, HLS LYS001C) sequentially, and incubate at 37°C for 1 h. Then add 1 µg / µL rTrypsin enzyme (Hualisheng, HLS rTRY005C) and incubate at a constant temperature for 16 h. Centrifuge at 12000g for 15 min at 4°C, collect the filtered peptide solution, and transfer it to a clean low-profile EP tube. Concentrate the sample using a refrigerated vacuum centrifuge (CV600), and reconstitute the peptides with 100 µL of 0.5% TFA solution. Equilibrate the desalting column with 100 µL of equilibration buffer (5% ACN + 0.5% TFA), centrifuge at 12000g for 1 min at 4°C, and remove the waste liquid. Add the reconstituted peptides to a C18 desalting column, centrifuge at 1500g for 5 min at 4°C, and remove the waste liquid. Wash the desalting column with 100 µL of 0.1% TFA solution, centrifuge at 12000 g for 1 min at 4 °C, and remove the waste liquid. Replace with a new collection tube, add 30 µL of 70% mass spectrometry grade acetonitrile (ACN) solution to the desalting column, centrifuge at 1000 g for 2 min at 4 °C, and elute the peptides. Then perform LC-MS / MS and data-independent acquisition (DIA) proteomics sequencing.

[0077] (3) Immunofluorescence verification: Paraffin sections of bronchial tissue (4 μm thick, n=6) from non-smoker donors were dewaxed to water using standard procedures. The sections were then soaked sequentially in xylene I and xylene II for 15 min each, followed by anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 85% ethanol, and 75% ethanol for 5 min each, and rinsed twice with distilled water (3 min each time). Antigen retrieval was performed using microwave-induced antigen retrieval: the sections were placed in a retrieval chamber containing 0.01 M sodium citrate buffer (pH 6.0), heated to boiling on high heat, then immediately reduced to low heat for 10 min, and allowed to cool naturally to room temperature. The sections were washed three times with PBS (3 min each time). Immunohistochemical pen was then used to draw circles around the tissue, and 0.2% Triton X-100 permeabilization buffer was added for 15 min at room temperature. After washing three times with PBS, 5% BSA blocking solution (containing 5% goat serum) was added, and the sections were blocked at room temperature for 30 min. Discard the blocking solution and add a mixed primary antibody working solution (diluted with 3% BSA / PBS) of anti-α-Tubulin antibody (cilia marker, abcam, ab289863) and anti-RSPH1 antibody (Sigma, HPA017382), respectively, and incubate overnight in a humidified chamber at 4°C in the dark. The next day, after washing three times with PBS, add a mixed working solution of Alexa Fluor 488-labeled goat anti-mouse IgG secondary antibody (Invitrogen, A32723) and Alexa Fluor 555-labeled goat anti-rabbit IgG secondary antibody (Invitrogen, A32732), and incubate at room temperature in the dark for 1 h. After washing three times with PBS, mount the slides with DAPI-containing anti-fluorescence quenching mounting medium and observe using a confocal microscope.

[0078] Results: Proteomics data showed that RSPH1 expression was significantly higher in ciliated cells and their exosomes than in basal cells and goblet cells and their exosomes. Figure 2 Immunofluorescence results showed that RSPH1 signaling and α-Tubulin-positive ciliated structures were highly colocalized in bronchial tissue. Figure 2 (B). The above indicates that RSPH1 is a specific marker protein of ciliated cells and their exosomes.

[0079] Example 3: Validation of RSPH1 as a marker of ciliated exosomes

[0080] (1) Using the airway epithelial cell sorting method described in Example 2, three major types of airway epithelial cells were sorted and exosomes were isolated from 20 independent healthy non-smoking subjects (different from the samples in Example 2).

[0081] (2) Nanoflow cytometry detection: The exosome sample was adjusted to a concentration of 1×10⁻⁶ using PBS. 8Particles / mL were permeabilized with 0.1% Triton X-100, then mixed with anti-RSPH1 antibody (Abcam, ab224386) and Alexa Fluor 488-labeled goat anti-rabbit IgG fluorescent secondary antibody (Abcam, ab150077), and incubated at 37°C in the dark for 30 min. After incubation, the sample was washed once with PBS and centrifuged at 120,000×g for 70 min. The supernatant was carefully removed, leaving a small amount of liquid at the bottom, and the precipitate was resuspended with an appropriate amount of PBS. Detection was performed using a nanoflow cytometer (Micro Plus, Apogee). Results ( Figure 3 A) shows that RSPH1 positive signals are mainly found on exosomes derived from ciliated cells.

[0082] (3) Western Blot Validation: Total protein was extracted from exosomes derived from ciliated cells, basal cells, and goblet cells using RIPA lysis buffer (containing protease inhibitors), and protein quantification was performed using the BCA method. An equal amount of protein (20 μg) was added to 5×SDS loading buffer, boiled for denaturation for 10 min, and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The protein was then transferred to a PVDF membrane (0.22 μm pore size) using a wet transfer method. After transfer, the membrane was blocked with 5% skim milk (prepared with TBST) at room temperature for 1 h, followed by the addition of anti-RSPH1 antibody (abcam, ab224386) and incubation at 4°C overnight. The next day, the membrane was washed three times with TBST (10 min each time), and HRP-labeled goat anti-rabbit IgG secondary antibody (Servicebio, GB23303) was added and incubated at room temperature for 1 h. After three washes with TBST, enhanced chemiluminescence (ECL) was used for development, and images were acquired using a gel imaging system, with TSG101 (abcam, ab125001) used as an internal control. Results ( Figure 3 (B) Confirmed that RSPH1 protein was clearly detected only in ciliated cell exosomes.

[0083] This embodiment cross-validates, through two independent techniques, that RSPH1 can serve as a specific surface marker for ciliated cell-derived exosomes.

[0084] Example 4: Discovery of TGM2, a COPD-related exosomal biomarker

[0085] (1) Construction of ALI organoid models: This study included healthy non-smokers (Non-smokers, n=30), healthy smokers (Smokers, n=32), patients with mild-moderate COPD (N=26), and patients with severe COPD (N=29). Inclusion criteria: Healthy non-smokers had no smoking history and normal lung function (FEV1 / FVC ≥ 0.7, FEV1%pred ≥ 80%); healthy smokers had smoked for ≥10 pack-years and normal lung function; patients with mild-moderate COPD (FEV1 / FVC < 0.7, FEV1%pred ≥ 50%); and patients with severe COPD (FEV1 / FVC < 0.7, FEV1%pred < 50%). All subjects were excluded if they had asthma, lung cancer, or other serious cardiopulmonary diseases. Bronchial mucosal tissue was obtained by brushing with fiberoptic bronchoscopy and transported in pre-cooled basal culture medium. The tissue was minced and digested with 0.25% trypsin-EDTA for 30 min, then filtered through a 100 μm filter to obtain bronchial basal stem cells. The cells were seeded in type I collagen-coated culture flasks and cultured using PneumaCult. TM Expand and culture in Ex Plus Medium (Stemcell), changing the medium every 3 days. When the cells reach 80% confluence, increase the culture temperature to 2.5 × 10⁻⁶. 4 Seeded at a density of cells / well in Transwell inserts (0.4 μm pore size, Corning), with PneumaCult added on top. TM Add Ex Plus Medium to the lower layer, and after the cells have completely covered the membrane (approximately 3-5 days), remove the upper layer of medium and replace the lower layer with PneumaCult. TM -Ex Plus Medium (Stemcell) was used to establish an air-liquid interface culture, with the lower culture medium being replaced every 2-3 days. After 24±2 days of culture, a large number of waving cilia were visible under an optical microscope. Immunofluorescence staining was used to detect the ciliary marker α-Tubulin (abcam, ab289863), confirming that the epithelial cells had differentiated into fully functional pseudostratified ciliated epithelium.

[0086] (2) Biomarker screening: The airway epithelial cell flow cytometry sorting method and exosome extraction method described in Example 2 were used to sort ciliated cells from the above ALI culture model, and the culture supernatant was collected and exosomes were separated. At the same time, the proteomics assay method described in Example 2 was used to perform proteomics (DIA) analysis on ciliated cells and their exosomes from the four groups of people.

[0087] Results: Comparative analysis revealed that, compared with healthy non-smokers and healthy smokers, the expression level of TGM2 protein was significantly upregulated in ciliated cells and exosomes derived from COPD patients (including Mild COPD and Sev. COPD, especially Sev. COPD patients), and its expression level was positively correlated with disease severity. Figure 4 ).

[0088] Examples 5-7: Multi-level validation of TGM2 as a COPD biomarker

[0089] Example 5 (Tissue-level Validation): Paraffin sections of bronchial tissue from healthy non-smoking controls, healthy smokers, and patients with mild, moderate, and severe COPD (as described in Example 4) were routinely dewaxed to water. They were then sequentially immersed in xylene I and xylene II for 15 min each, followed by anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 85% ethanol, and 75% ethanol for 5 min each, and rinsed twice with distilled water (3 min each time). Antigen retrieval was performed using microwave-induced antigen retrieval: the sections were placed in a retrieval chamber containing 0.01 M sodium citrate buffer (pH 6.0), heated to boiling on high, then immediately reduced to low heat and maintained for 10 min, and allowed to cool naturally to room temperature. The sections were washed three times with PBS (3 min each time). Subsequently, an immunohistochemical pen was used to draw circles around the tissue, and 0.2% Triton X-100 permeabilization buffer was added for 15 min at room temperature. After washing three times with PBS, 5% BSA blocking solution (containing 5% goat serum) was added, and the sections were blocked at room temperature for 30 min. Discard the blocking solution and add a mixed primary antibody working solution (diluted with 3% BSA / PBS) of anti-α-Tubulin antibody (abcam, ab289863) and anti-TGM2 antibody (Proteintech, 15100-1-AP), respectively, and incubate overnight in a humidified chamber at 4°C in the dark. The next day, after washing three times with PBS, add a mixed working solution of Alexa Fluor 488-labeled goat anti-mouse IgG secondary antibody (Invitrogen, A32723) and Alexa Fluor 555-labeled goat anti-rabbit IgG secondary antibody (Invitrogen, A32732), and incubate at room temperature in the dark for 1 h. After washing three times with PBS, mount the slides with DAPI-containing anti-fluorescence quenching mounting medium and observe using a confocal microscope. The results showed that TGM2 co-localized with ciliated cells, and its fluorescence intensity significantly increased with the severity of COPD. Figure 5 ).

[0090] Example 6 (Cellular Validation): Airway epithelial cells were obtained from bronchial brushing samples from healthy non-smoking controls and patients with mild to moderate COPD via bronchoscopy. A cell brush with a protective sheath (Hobbs Medical) was inserted into the target bronchus through a bronchoscope. The brush head was then withdrawn, and the mucosal surface was gently scraped to obtain bronchial epithelial tissue. After removing the brush head, the sample was placed in pre-cooled ALI complete culture medium. The brush head was cut off and transferred to Accutase digestion solution (Stemcell) containing 1 mg / mL collagenase D (Roche) and 0.1 mg / mL DNase I (Roche). The sample was digested at 37°C with shaking for 1 h. After digestion, the sample was filtered through a 70 μm nylon filter, treated with erythrocyte lysis buffer (Stemcell) for 5 min, washed with PBS containing 0.04% BSA, and centrifuged at 400×g for 5 min to obtain a single-cell suspension. Ciliated cells were sorted using the airway epithelial cell sorting method described in Example 2. Take the sorted ciliated cell suspension (approximately 1×10⁻⁶) 5 (100 cells), adjust the volume to 200 μL with PBS, add to the sample chamber of a cell smear centrifuge (Cytospin), centrifuge at 500 rpm for 5 min, and transfer the cells to a detachable slide. After air drying at room temperature, fix with 4% paraformaldehyde for 15 min, and wash 3 times with PBS. Permeabilize with 0.2% Triton X-100 for 10 min, wash with PBS, and block with 5% BSA at room temperature for 30 min. Add mixed primary antibody working solution: mouse anti-α-Tubulin (abcam, ab289863) and rabbit anti-TGM2 (Proteintech, 15100-1-AP), and incubate overnight in a humidified chamber at 4°C. The next day, wash 3 times with PBS, add secondary antibodies of Alexa Fluor 488-labeled goat anti-mouse IgG (Invitrogen, A32723) and Alexa Fluor 555-labeled goat anti-rabbit IgG (Invitrogen, A32732), and incubate at room temperature in the dark for 1 h. After washing with PBS, the slides were mounted with an anti-quenching agent containing DAPI, and images were observed and acquired under a fluorescence microscope. The results showed that the TGM2 signal in ciliated cells of patients with mild to moderate COPD was significantly stronger than that in the control group. Figure 6 ).

[0091] Example 7 (Exosome Level Validation): Bronchial brush samples from healthy non-smoking controls and patients with mild to moderate COPD were centrifuged at 2000g for 15 min at 4°C to remove cells and obtain supernatant. Total exosomes were isolated from the supernatant using microfluidic chip-based technology (refer to Example 1 of CN114247489A). Total exosome protein was quantified using the BCA method with RIPA lysis buffer (containing protease inhibitor). An equal volume of protein (20 μg) was added to 5×SDS loading buffer, boiled for denaturation for 10 min, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a PVDF membrane (0.22 μm pore size) using wet transfer. After transfer, the membrane was blocked with 5% skim milk (prepared by TBST) at room temperature for 1 h, followed by incubation at 4°C overnight with anti-TGM2 antibody (Proteintech, 15100-1-AP). The next day, the sample was washed three times with TBST (10 min each time), and HRP-labeled goat anti-mouse IgG secondary antibody (Servicebio, GB23301) was added. The sample was incubated at room temperature for 1 h. After three TBST washes, enhanced chemiluminescence (ECL) was used for development, and images were acquired using a gel imaging system, with TSG101 (abcam, ab125001) used as an internal control. Results showed that the expression level of TGM2 protein in exosomes from brushing samples from patients with mild to moderate COPD was significantly higher than that in healthy controls. Figure 7 ).

[0092] The three examples above consistently validated that TGM2 is a reliable COPD-related biomarker at the tissue, primary ciliated cell, and exosome levels.

[0093] Example 8: Assessment of the diagnostic potential of COPD based on a single disease biomarker (TGM2)

[0094] (1) Sample processing: Bronchoalveolar lavage fluid (BALF) was collected separately from healthy non-smoking controls (30 cases) and patients with mild to moderate COPD (34 cases), and total exosomes were separated using the microfluidic technology mentioned in Example 2.

[0095] (2) Nanoflow cytometry detection: BALF exosomes were double-stained with antibodies against the universal exosome marker CD63 and the disease marker TGM2, and CD63 was quantitatively detected by nanoflow cytometry. + TGM2 + The proportion of double-positive exosome subsets ( Figure 8 A). The specific steps are as follows:

[0096] The exosome sample was adjusted to a concentration of 1×10⁻⁶ using PBS. 8Particles / mL were permeabilized with 0.1% Triton X-100, then mixed with anti-CD63 antibody (Abcam, ab271286), anti-TGM2 antibody (Proteintech, 15100-1-AP), and Alexa Fluor 488-labeled goat anti-rabbit IgG fluorescent secondary antibody (Abcam, ab150077) and Alexa Fluor 647-labeled goat anti-mouse IgG fluorescent secondary antibody (Biolegend, 405322). The mixture was incubated at 37°C in the dark for 30 min. After incubation, the sample was washed once with PBS and ultracentrifuged at 120,000×g for 70 min. The supernatant was carefully removed, leaving a small amount of liquid at the bottom. The precipitate was resuspended in PBS. Detection was performed using a nanoflow cytometer (Micro Plus, Apogee).

[0097] Diagnostic efficacy analysis: Statistical results showed that CD63 in BALF of patients with mild to moderate COPD + TGM2 + The proportion of exosomes was significantly higher than that of healthy controls. Figure 8 .B). Receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) distinguishing this index from healthy controls and patients with mild to moderate COPD was 0.895 ( ). Figure 8 (.C), showing good diagnostic potential.

[0098] Example 9: Based on dual targets (RSPH1) + TGM2 + Establishment and evaluation of COPD precision diagnostic methods

[0099] Dual-target detection: Using the same BALF exosome sample as in Example 8, dual staining was performed with antibodies against the ciliated cell exosome-derived marker RSPH1 and the disease marker TGM2. RSPH1 was then quantitatively detected by nanoflow cytometry. + TGM2 + The proportion of double-positive exosome subsets ( Figure 9 A). The specific steps are as follows: Adjust the concentration of the exosome sample to 1×10⁻⁶ using PBS. 8Particles / mL were permeabilized with 0.1% Triton X-100, then mixed with anti-CD63 antibody (Abcam, ab224386), anti-TGM2 antibody (Proteintech, 15100-1-AP), and Alexa Fluor 488-labeled goat anti-rabbit IgG fluorescent secondary antibody (Abcam, ab150077) and Alexa Fluor 647-labeled goat anti-mouse IgG fluorescent secondary antibody (Biolegend, 405322). The mixture was incubated at 37°C in the dark for 30 min. After incubation, the sample was washed once with PBS and ultracentrifuged at 120,000×g for 70 min. The supernatant was carefully removed, leaving a small amount of liquid at the bottom. The precipitate was resuspended in PBS. Detection was performed using a nanoflow cytometer (Micro Plus, Apogee).

[0100] Results and Advantages: RSPH1 in BALF of patients with mild to moderate COPD + TGM2 + The proportion of exosomes increased significantly ( Figure 9 ROC analysis showed that the diagnostic AUC value of this dual-target indicator increased to 0.93 (B). Figure 9 .C), with diagnostic efficacy superior to CD63 alone. + TGM2 + index.

[0101] This application discovered the novel ciliated exosome biomarker RSPH1 and the COPD-related biomarker TGM2 through proteomics, and creatively combined the two to establish a highly specific and sensitive dual-target exosome detection strategy. This detection strategy demonstrates excellent diagnostic and differential diagnostic capabilities for COPD and has broad prospects for clinical application and translation.

[0102] This application is the first to clearly demonstrate the specific high expression of RSPH1 in ciliated cells and their exosomes, providing a novel molecular tool for the precise identification and tracking of exosomes derived from airway ciliated cells. It also reveals for the first time that TGM2 is a biomarker associated with chronic obstructive pulmonary disease (COPD). Based on this, this application creatively proposes a dual-targeting exosome diagnostic strategy combining a cell-derived specific biomarker and a disease state biomarker. This strategy combines the ciliated cell-specific biomarker RSPH1 with the COPD disease biomarker TGM2, detecting exosomes that are positive for both (RSPH1 and TGM2). + TGM2 +The proportion of [missing information] was used to accurately capture the disease signal vesicles released by key diseased cells. This helps to establish a non-invasive / minimally invasive COPD precision diagnosis scheme based on respiratory fluids such as bronchoalveolar lavage fluid (BALF): The dual-target quantitative detection platform established in this application, which uses BALF exosomes as the detection object and nanoflow cytometry as the core, has the advantages of easy sample acquisition, standardized operation, and objective and quantifiable results, providing a brand-new solution for early screening, differential diagnosis, and dynamic monitoring of COPD.

[0103] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0104] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. Application of protein biomarkers and / or their detection reagents in the preparation of diagnostic kits for chronic obstructive pulmonary disease; The protein biomarkers include RSPH1 and TGM2.

2. The application according to claim 1, characterized in that, The detection reagent detects RSPH1 in ciliated cells and / or RSPH1 in ciliated cell exosomes.

3. The application according to claim 1, characterized in that, The test reagent detects one or more of the following samples: bronchial brushing material, respiratory fluid sample, airway epithelial cell culture supernatant, and organoid system differentiated from bronchial stem cells.

4. The application according to claim 3, characterized in that, The respiratory fluid sample includes one or more of sputum and bronchoalveolar lavage fluid.

5. The application according to any one of claims 1 to 4, characterized in that, The detection reagents include quantitative detection reagents.

6. The application according to any one of claims 1 to 4, characterized in that, The detection reagents for RSPH1 and TGM2 are each detected independently using the following methods: Enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry, immunofluorescence, mass spectrometry, real-time quantitative PCR, and immunoturbidimetry.

7. A protein biomarker, characterized in that, The protein biomarker is defined as in any one of claims 1 to 6.

8. A test kit, characterized in that, The detection kit includes the protein biomarkers and / or detection reagents thereof as defined in any one of claims 1 to 6.

9. The detection kit according to claim 8, characterized in that, The detection kit also includes exosome extraction reagents and / or exosome protein extraction reagents.

10. A detection system for chronic obstructive pulmonary disease, characterized in that, The system includes: Detection component: The detection component is used to detect protein biomarkers as defined in any one of claims 1 to 6 at the protein level; Result judgment component: The result judgment component is used to output the detection result based on the result of the protein marker detected by the detection component.

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