Application of BMP4 gene regulation product in preparation of chronic obstructive pulmonary disease prevention and treatment product

Abstract

The invention discloses an application of a BMP4 gene and a regulation product thereof in preparation of a product for preventing and treating chronic obstructive pulmonary disease, and belongs to the technical field of biological medicines. The invention finds that the expression of BMP4 in lung tissues of a patient with chronic obstructive pulmonary disease is reduced; animal experiments prove that after CS exposure, the body weight of a BMP4 < + / -> mouse is reduced, the lung function is obviously reduced, emphysema and inflammation infiltration are more obvious, the average alveolar intercept is obviously increased, inflammatory cells of alveolar lavage fluid are obviously increased, and neutrophils are mainly used; cS exposure and airway instillation of AAV9-BMP4 show that airway instillation of AAV9-BMP4 (1 * 10 < 11 > PFU / mouse) can obviously improve lung function decline, emphysema change, inflammation infiltration, increase of average alveolar intercept and increase of the number of inflammatory cells in alveolar lavage fluid after CS exposure of the mouse. Therefore, the BMP4 can become a potential target for treating the chronic obstructive pulmonary disease and can be applied to the fields of new drugs for treating the chronic obstructive pulmonary disease, new technology research and development and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of BMP4 gene and its regulatory products in the preparation of products for preventing and treating chronic obstructive pulmonary disease (COPD). Background Art

[0002] Chronic Obstructive Pulmonary Disease (COPD) is a heterogeneous pulmonary condition characterized by persistent and progressive airflow limitation, usually accompanied by symptoms of chronic bronchitis and emphysema, where emphysema is a key pathological feature. The morbidity and mortality rates of COPD are among the highest worldwide, and cigarette smoke (CS) exposure is the main risk factor for COPD. The lung tissue has a certain regenerative capacity and can repair itself by activating and differentiating stem / progenitor cells when damaged. Alveoli are mainly composed of flat type I (alveolar type Ⅰ epithelial cells, AT1) and cuboidal type II (alveolar type Ⅱ epithelial cells, AT2) alveolar epithelial cells. AT2 cells are the main progenitor cells in the alveoli and have the ability to self-renew and differentiate into AT1 cells to repair the alveolar structure. However, there is an impairment in the repair of lung tissue in COPD patients. Bone Morphogenetic Protein 4 (BMP4) is a member of the TGF-β superfamily and consists of two polypeptide chains covalently linked by disulfide bonds, participating in the regulation of cell proliferation, differentiation, and maturation. During lung development, BMP4 regulates the formation and maturation of airways and alveoli, promoting normal lung development. Some studies have shown that in emphysema, the abnormal BMP signaling pathway may lead to a decrease in the lung tissue repair ability, affecting the regeneration and differentiation of lung epithelial cells. However, the exact role of BMP4 in the repair of cigarette smoke (CS)-induced emphysema is still not fully clear. Summary of the Invention

[0003] To address the above technical problems, the present invention first provides the application of BMP4 gene and its regulatory products in the preparation of products for preventing and treating COPD. The present invention detected the expression of BMP4 protein in the lung tissues of 17 COPD patients and 17 control groups by Western Blots (WB) experiments and found that the expression of BMP4 was reduced in the lung tissues of COPD patients. Through animal experiments, it was demonstrated that after cigarette smoke exposure, BMP4 + / -The body weight of the mice decreased, and the pulmonary function significantly declined. The lung pathological results showed that emphysema and inflammatory infiltration were more obvious, the mean alveolar intercept significantly increased. At the same time, the inflammatory cells in the bronchoalveolar lavage fluid also significantly increased, mainly neutrophils. After 3 months of exposure to tobacco smoke, AAV9-BMP4 (1×10 11 PFU / mouse) was instilled into the airway, and then the mice continued to be exposed to tobacco smoke for 3 months. It was found that instilling AAV9-BMP4 into the airway could significantly improve the decline in pulmonary function, the changes of emphysema, inflammatory infiltration, the increase in mean alveolar intercept and the increase in the number of inflammatory cells in the bronchoalveolar lavage fluid after tobacco smoke exposure in mice. This result indicates that BMP4 may be a potential target for the treatment of chronic obstructive pulmonary disease. The present invention can be applied to the fields such as the research and development of new drugs and new technologies for the treatment of chronic obstructive pulmonary disease.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] One object of the present invention is to provide the application of a BMP4 gene regulation product in the preparation of a product for preventing and treating chronic obstructive pulmonary disease, wherein the regulation is positive regulation.

[0006] Preferably, the BMP4 gene regulation product is AAV9-BMP4.

[0007] More preferably, the using method of the BMP4 gene regulation product is instillation into the airway.

[0008] Still more preferably, the dosage of the BMP4 gene regulation product is 0.5 - 1.5×10 11 PFU.

[0009] Even more preferably, the dosage of the BMP4 gene regulation product is 1.0×10 11 PFU.

[0010] Another object of the present invention is to provide a product for preventing and treating chronic obstructive pulmonary disease, and the product contains the BMP4 gene regulation product in any of the above applications.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] (1) The present invention finds that the expression level of BMP4 in the lung tissue of patients with chronic obstructive pulmonary disease decreases, and the immunofluorescence results show that the expression of BMP4 in the lung tissue of COPD patients decreases, the fluorescence intensity weakens, and it is mainly expressed in type II alveolar epithelial cells.

[0013] (2) The present invention finds that compared with wild-type littermate mice, BMP4 exposed to CS + / -The body weight of the mice decreased significantly, and the decline in lung function was more severe, manifested as an increase in the functional residual capacity (FRC), resistance index (RI), and a decrease in the dynamic compliance of the lung (Cchord), the forced expiratory volume in 20 milliseconds (FEV20) / FVC value, and the forced expiratory volume in 50 milliseconds (FEV50) / FVC value. In BMP4 + / - mice, under microscopic observation of HE-stained sections, the alveolar structure was disrupted and disordered, the alveolar wall was thinned or ruptured, alveolar fusion occurred, the alveolar lumen was enlarged, and inflammatory cells appeared in the alveolar septum and alveolar lumen. Inflammatory cell infiltration was observed in the submucosa and mucosa of the alveolar septum. In BMP4 gene-deficient mice, there was no significant change in lung function under normal air exposure. Six months of CS exposure led to an increase in the total number of cells and differential cell counts in the BALF of wild-type mice, and a significant increase in BMP4 knockout mice. These results indicate that BMP4 knockout exacerbates emphysema caused by CS exposure.

[0014] (3) The present invention found that compared with mice exposed to simple tobacco smoke, after intratracheal instillation of AAV9-BMP4, the decline in lung function of the mice was improved, manifested as a reduced increase in the functional residual capacity (FRC) and a reduced decrease in the forced expiratory volume in 50 milliseconds (FEV50) / FVC value. After intratracheal instillation of AAV9-BMP4, the alveolar structure caused by CS exposure was relatively intact with less damage, only a few alveolar septa were ruptured, alveolar fusion occurred, and the inflammatory cell infiltration was significantly reduced. The number of inflammatory cells in the alveolar lavage fluid of the CS + AAV9-GFP group increased significantly, and neutrophils were the main type. Overexpression of BMP4 improved the increase in inflammatory cells after CS exposure. These results indicate that overexpression by intratracheal instillation can improve emphysema caused by CS exposure.

[0015] (4) The present invention found that compared with wild-type littermate mice, BMP4 + / - mice exposed to CS showed significantly reduced expression of the surface marker AQP5 of type I alveolar epithelial cells and increased expression of the surface marker SFTPC of type II alveolar epithelial cells in the immunofluorescence results of lung tissue sections. These results indicate that tobacco smoke exposure may lead to a disorder in the transdifferentiation of type II alveolar epithelial cells into type I alveolar epithelial cells.

[0016] (5) The present invention found that after stimulating hAT2 cells with 2% CSE for 36 hours, the WB results showed that the expressions of AQP5, p-SMAD159, and β-catenin were all decreased, while after giving 20 ng BMP4 while stimulating with 2% CSE, the expressions of AQP5, p-SMAD159, and β-catenin were all increased.

[0017] In summary, the present invention has for the first time discovered that overexpression of BMP4 can improve emphysema caused by CS exposure. This may become a potential target for the treatment of COPD. The present invention can be applied to the research and development of new drugs and new technologies for the treatment of COPD and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It shows the expression and expression distribution of BMP4 in human lung tissue samples of COPD patients and normal controls in Example 1 of the present invention; wherein: (A) BMP4 protein expression level in human lung tissue; (B) Gray value of protein expression level; (C) Representative immunofluorescence diagram of BMP4 in human lung tissue sections; (D) Representative immunofluorescence co-staining diagram of BMP4 and SFTPC in human lung tissue sections; (E) Representative immunofluorescence co-staining diagram of BMP4 and FN in human lung tissue sections; N = 17 for each group in WB experiment; N = 5 for immunofluorescence experiment; *P < 0.05, **P < 0.01, ***P < 0.001.

[0019] Figures 2 to 3 It shows the deterioration of lung function decline and alveolar cavity enlargement in mice exposed to cigarette smoke due to BMP4 gene deletion in Example 1 of the present invention, wherein: ( Figure 2 A) FRC value; ( Figure 2 B) IC value; ( Figure 2 C) Cydn value; ( Figure 2 D) RI value; ( Figure 2 E) FEV20 / FVC value; ( Figure 2 F) FEV50 / FVC value; ( Figure 2 G) HE staining of lung sections; ( Figure 2 H) Statistical chart of mean linear intercept of lung sections; ( Figure 3 A) HE staining of bronchoalveolar lavage fluid; ( Figure 3 B) Total number of cells in bronchoalveolar lavage fluid; ( Figure 3 C) Number of macrophages; ( Figure 3 D) Number of lymphocytes; ( Figure 3 E) Number of neutrophils; *P < 0.05, **P < 0.01, ***P < 0.001.

[0020] Figure 4 It shows the BMP4 protein expression level in lung tissue after BMP4 knockout and tobacco smoke exposure in Example 1 of the present invention, wherein: (A) BMP4 protein expression level in lung tissue; (B) Gray value of protein expression level; *P < 0.05, **P < 0.01, ***P < 0.001.

[0021] Figures 5 to 6 It shows the changes in lung function and alveolar cavity in mice exposed to cigarette smoke after airway overexpression of BMP4 in Example 1 of the present invention; wherein: ( Figure 5 A) FRC value; (Figure 5 B) IC value; ( Figure 5 C) Cydn value; ( Figure 5 D) RI value; ( Figure 5 E) FEV50 / FVC value; ( Figure 5 F) FEV100 / FVC value; ( Figure 5 G) Expression level of BMP4 protein in lung tissue; ( Figure 5 H) Gray value of protein expression level; ( Figure 5 I) HE staining of lung sections; ( Figure 5 J) Statistical chart of mean linear intercept of lung sections; ( Figure 6 A) HE staining of bronchoalveolar lavage fluid; ( Figure 6 B) Total number of cells in bronchoalveolar lavage fluid; ( Figure 6 C) Number of macrophages; ( Figure 6 D) Number of lymphocytes; ( Figure 6 E) Number of neutrophils; *P<0.05, **P<0.01, ***P<0.001.

[0022] Figure 7 This is an immunofluorescence image of GFP expression in mouse lung tissue after overexpression of BMP4 in the airway in Example 1 of the present invention.

[0023] Figures 8 to 9 This is the immunofluorescence staining of lung tissue sections after knocking out BMP4 and exposing to tobacco smoke in Example 1 of the present invention; ( Figure 8 ) Representative image of co-immunofluorescence staining of BMP4 and AQP5 in mouse lung tissue sections; ( Figure 9 ) Representative image of co-immunofluorescence staining of BMP4 and SFTPC in mouse lung tissue sections.

[0024] Figure 10 This is the effect of BMP4 on cell transdifferentiation under CSE stimulation in human type II alveolar epithelial cell line (hAT2 cells) in Example 1 of the present invention. Detailed implementation manners

[0025] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention. The reagents and instruments used in the following examples can all be obtained commercially, and the methods used in the examples are the same as the commonly used methods unless otherwise specified.

[0026] The technical solutions of the present invention will be further elaborated in detail below in conjunction with the examples.

[0027] Example 1

[0028] 1 Experimental method

[0029] 1.1 Research subjects

[0030] In this study, patients who underwent surgical treatment for pulmonary nodules or lung transplantation for COPD at the First Affiliated Hospital of Guangzhou Medical University from January 2023 to March 2024 were consecutively enrolled. Twenty normal control patients and 20 COPD patients were collected. The age was between 40 and 80 years old, and there were no statistically significant differences in gender ratio and age between the groups. The lung function of the normal control group was normal, and there were no other underlying lung diseases except for pulmonary nodules. Normal lung tissue adjacent to the pulmonary nodules was taken as the normal control group, and the lung tissue used for normal control showed normal structure, and there was no infiltration of macrophages and neutrophils in the alveoli; the lung function of the COPD group showed obstructive ventilatory dysfunction, and there were no other underlying lung diseases except for COPD. All participants were informed and signed the informed consent form.

[0031] The inclusion criteria were as follows: (1) For COPD patients: 1. Population: Chinese Han population; 2. Age: between 40 and 80 years old; 3. Diagnosed with COPD according to the GOLD guidelines, stable-phase patients; 4. Lung function classification: GOLD I-IV. For normal population: 1. Population: Chinese Han population; 2. Age: between 40 and 80 years old; 3. Normal lung function. Basic information was roughly matched (such as gender). "Chronic" was defined as symptoms lasting more than 3 months or recurring every 3 months. The respiratory symptoms mainly included cough, sputum, wheezing, dyspnea, etc.

[0032] Exclusion criteria: 1. Pregnant women; 2. History of acute exacerbation of COPD within 6 weeks; 3. History of lung diseases: asthma, bronchiectasis, cystic fibrosis, panbronchiolitis, pulmonary fibrosis, pneumonia, lung cancer during treatment or in the advanced stage, suspected lung cancer, history of lung surgery and other parenchymal lung lesions; 3. Acute myocardial infarction, other patients with acute-stage heart diseases; combined with severe dysfunction of other organs such as heart failure, chronic renal insufficiency, liver insufficiency, etc.; 4. Unable to cooperate with the examination and operation or enroll in the study due to any other reasons such as organic neurological diseases and mental disorders.

[0033] 1.2 COPD mouse model

[0034] Bone morphogenetic protein 4 (BMP4) gene knockout (BMP4 + / - ) mice and their wild-type (BMP4 + / +)The littermate mice were provided by the Jackson Laboratory in Bar Harbor, Maine, USA. The animals were housed in a specific pathogen-free facility, and all experimental protocols were approved by the Animal Protection and Use Committee of Guangzhou Medical University. All methods were carried out in accordance with the guidelines and regulations approved by the Animal Protection and Use Committee of Guangzhou Medical University. The cigarettes used to establish the mouse COPD model were Hongmei brand filtered cigarettes (China Tobacco Guangdong Industrial Corporation), each containing 11 mg of tar, 1.0 mg of nicotine, and 13 mg of carbon monoxide. 6- to 8-week-old BMP4 + / - The BMP4 mice and their littermate wild-type mice were exposed to CS using a whole-body exposure system. Briefly, the mice were exposed to CS (12 cigarettes / h, 1 h / time, 4 times / day, 6 days / week) for 24 weeks. The control group was only exposed to filtered room air (RA). After the experiment, all mice were subjected to pulmonary function testing and sacrificed.

[0035] 1.3 Pulmonary function testing of mice

[0036] After the CS exposure ended, pulmonary function was measured on the 181st day. The specific steps were as follows: The mice were anesthetized with avertin (17.5 μl / g). After the mice were anesthetized, their limbs were fixed to a plastic board with medical tape. After alcohol disinfection, the skin in the middle of the mouse's neck was cut open, and the trachea was bluntly dissected with forceps to expose it. A small incision was made at the trachea, and a tracheal cannula was inserted. The mouse tracheal cannula was connected to a pulmonary function instrument (PFT system of BUXCO Company) and mechanical ventilation was performed. The parameter settings of the experimental instrument were: the ventilator pressure was ±10 cm water column pressure, the inspiratory airflow rate was 0.8 ml / s, the expiratory airflow rate was 0.5 ml / s, the maximum inspiratory and expiratory pressures were ±40 cm water column pressure, and the respiratory rate was 150 times / min. After the mouse's respiratory rhythm was consistent with the ventilator, pulmonary function was measured, and the forced vital capacity (FVC), airway resistance index, dynamic lung compliance (Cydn), 20-millisecond rate (20 millisecond FEV / FVC, FEV20%), 50-millisecond rate (50 millisecond FEV / FVC, FEV50%), etc. of the mice were measured.

[0037] 1.4 Bronchoalveolar lavage fluid (BALF) analysis

[0038] After the mouse lung function measurement, the mouse was fixed on the dissection plastic board with its abdomen facing up. After opening the mouse's chest cavity, the left main bronchus was clamped with a vascular clamp. An indwelling needle was inserted through the tracheal incision of the mouse, and a slipknot was tied at the distal end of the opening to fix the indwelling needle. An external 1 mL syringe was connected, and 0.4 mL of normal saline was instilled each time for a total of 6 instillations. The first three instilled lavage fluids were collected in the first 1.5 mL EP tube (the first tube), and the last three instilled lavage fluids were mixed and collected in the second 1.5 mL EP tube (the second tube), and left standing on ice; the collected BALF was centrifuged at 1000 rpm at 4 °C for 15 minutes. The supernatant was aliquoted into two tubes and stored at -80 °C for subsequent experiments. The cell pellet was resuspended with 1 mL of cold PBS, counted, and smeared with a cytocentrifuge to make the number of cells on each cell smear 1.5×10^5 cells. Then, HE staining was performed for differential counting of neutrophils, macrophages, and lymphocytes in the cells.

[0039] 1.5 Histopathology of mouse lung tissue

[0040] Hematoxylin-eosin (HE) staining. The left lung of the mouse was fixed with 10% formaldehyde solution for 24 h, then embedded in paraffin, and the paraffin block was cut into 5-μm thick sections. The deparaffinized tissue sections were examined histologically using an HE staining kit (Saiweier, Wuhan).

[0041] 1.6 Western blotting

[0042] The anti-β-actin antibody (66009-1-Ig) was obtained from Proteintech (Wuhan Sanying, China). The anti-BMP4 antibody (sc12721) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and (A11405, A21065) were obtained from Abclonal (Abclonal, China). The anti-SFTPC antibody (A23181) was obtained from Abclonal (Abclonal, China). The anti-AQP5 antibody (A23292) was obtained from Abclonal (Abclonal, China). The peroxidase-labeled secondary antibodies were purchased as Anti-Mouse IgG (H+L) antibody (5220-0341) and Anti-Rabbit IgG (H+L) antibody (5220-0336) from Seracare (USA). The Immun-Star HRP chemiluminescence kit (Horseradish peroxidase) was used to detect the bound antibody signals. The western blot images were taken from the Tanon 5200 chemiluminescence imaging system (Shanghai Tanon Science & Technology, Shanghai, China). The semi-quantitative analysis of immunoblotting was performed using Image J.

[0043] 1.7 Immunofluorescence experiment of mouse lung tissue

[0044] The antibody FN (A12977) was obtained from Abclonal (Abclonal, China). The anti-SFTPC antibody (A23181) was obtained from Abclonal (Abclonal, China). The anti-AQP5 antibody (A23292) was obtained from Abclonal (Abclonal, China). The anti-BMP4 antibodies (A11405, A21065) were obtained from Abclonal (Abclonal, China). The TSA multiplex immunofluorescence kit (Aifang Biotech, China).

[0045] 1.8 Preparation of cigarette smoke extract (CSE)

[0046] The CSE was taken from Hongmei brand cigarettes (Guangdong Tobacco Industry Corporation) and prepared within 30 minutes before cell treatment.

[0047] 1.9 Cell culture

[0048] The special cell culture medium (TM0482) was purchased from the Chinese subsidiary, Zhenjiang Aibimeng Biotechnology Co., Ltd. The human alveolar type II epithelial cell line (hAT2) was collected from the Chinese subsidiary of Applied Biomaterials (ABM), Zhenjiang Aibimeng Biotechnology Co., Ltd., Canada, and cultured in a humidified incubator at 37 °C with 95% (v / v) air and 5% (v / v) CO2 using the special culture medium for hAT2 cells.

[0049] 1.10 Treatment with human recombinant protein BMP4

[0050] hAT2 cells were seeded in 6-well culture dishes and cultured for 24 h. After treating the cells with 20 ng BMP4, 50 ng human recombinant protein BMP4, and 2% CSE for 36 h respectively, the cell proteins were collected for WB experiments.

[0051] 1.11 Statistical analysis

[0052] All statistical analyses were performed using GraphPad Prism software (version 10.0, USA). Measurement data that conform to the normal distribution are presented as (Mean ± SD), and if the measurement data are skewed distributions that cannot be corrected, then Median (IQR) is used. The t-test was used for comparing the means between two groups of samples. For multiple groups of samples that conform to the normal distribution and have homogeneous variances, one-way analysis of variance (one way-ANOVA) was used for comparing the overall means, and then the LSD method was used for pairwise comparisons. If the samples do not conform to the normal distribution or have heterogeneous variances, the data were transformed (sqrt, lg, exp, etc.), and then normality test and homogeneity of variance test were performed. If they still do not conform to the homogeneity of variance or the normal distribution, non-parametric tests were used. When P < 0.05, the differences between different groups were considered statistically significant.

[0053] 2 Experimental results

[0054] 2.1 Expression and distribution of BMP4 in the lung tissues of COPD patients

[0055] The results of WB experiments showed that the expression level of BMP4 protein decreased in the lung tissues of COPD patients, and the results of immunofluorescence showed that the expression of BMP4 decreased in the lung tissues of COPD patients, the fluorescence intensity weakened, and it was mainly co-localized with type II alveolar epithelial cells ( Figure 1 ).

[0056] 2.2 Deletion of the BMP4 gene exacerbated CS-induced decline in lung function and emphysema in mice

[0057] To understand the role of BMP4 in the occurrence and development of chronic obstructive pulmonary disease, we established a mouse model of chronic obstructive pulmonary disease by continuous CS exposure for 6 months and compared BMP4 + / -Mice and their wild-type littermates BMP4 + / + The performance of the mice, and BMP4 was detected + / - Mice and their wild-type littermates BMP4 + / + The lung function of the mice was measured, and the lung pathology was evaluated simultaneously. The results are as Figures 2 to 3 shown

[0058] The specific experimental methods are as follows: BMP4 + / - and BMP4 + / + Mice were continuously exposed to CS for 6 months (12 cigarettes per hour, 4 times per group, 1 hour each time, 6 days per week). Lung function parameters ( Figure 2 A- Figure 2 F) were measured using a lung function instrument (PFT system of BUXCO), including FRC, IC, Cydn, RI, FEV20 / FVC, and FEV50 / FVC values, with 6 mice in each group; Figure 2 G-2H are statistical charts of HE staining of lung sections and mean linear intercept; N = 6 mice in each group. HE staining of cell smears of bronchoalveolar lavage fluid, total cell count, and cell differential count ( Figure 3 A-3E), with N = 6 mice in each group.

[0059] The lung function results showed that compared with wild-type littermate mice, the lung function of Muc1 mice exposed to CS decreased more severely, manifested as an increase in functional residual capacity (FRC) ( - / - A), inspiratory capacity (IC) ( Figure 2 B), resistance index (RI) ( Figure 2 D), and a decrease in dynamic lung compliance (Cydn) ( Figure 2 C), 20-millisecond forced expiratory volume (FEV20) / FVC value ( Figure 2 E), 50-millisecond forced expiratory volume (FEV50) / FVC ( Figure 2 F) value. The HE results showed that in the lung tissue sections of mice in the BMP4 Figure 2 CTL group, normal continuous alveolar structures were visible and no obvious inflammatory infiltration was seen; in the lung tissue sections of mice in the BMP4 + / + CTL group, partial alveolar septal rupture and alveolar fusion were visible but no obvious inflammatory infiltration was seen; while in the BMP4 + / - CTL group, the alveolar structure was disrupted and disordered, the alveolar wall was thinned or ruptured, alveolar fusion occurred, the alveolar lumen was enlarged, the alveoli were of different sizes, inflammatory cells appeared in the alveolar septum and alveolar lumen, the bronchial wall was thickened, the lumen was narrowed, the bronchial mucosal epithelium exfoliated, and inflammatory cells infiltrated the submucosa and mucosa of the alveolar septum. Among them, the BMP4 + / - CS and BMP4 + / + CS group showed more severe manifestations ( + / - Figure 2 ​G); Calculating the mean linear intercept (MLI) showed that BMP4 + / - In the CTL group compared with BMP4 + / + the mean linear intercept of CTL increased, and the mean linear intercept increased significantly after CS. For BMP4 + / - CS relative to BMP4 + / + CS increased significantly ( Figure 2 H); After CS, the inflammatory cells in the bronchoalveolar lavage fluid increased significantly, among which BMP4 + / - In the CS group compared with BMP4 + / + the inflammatory cells in the bronchoalveolar lavage fluid of mice in the CS group increased more significantly, mainly with an increase in neutrophils ( Figure 3 A-3E).

[0060] The expression level of BMP4 protein in lung tissue was detected by WB experiment. The results showed that after CS exposure, the expression level of BMP4 decreased ( Figure 4 A-4B).

[0061] In summary, BMP4 knockout aggravated emphysema caused by CS exposure.

[0062] 2.3 Airway instillation of AAV9-BMP4 improves CS-induced decline in lung function and emphysema in mice

[0063] To understand the role of BMP4 in the occurrence and development of chronic obstructive pulmonary disease, after exposing BMP4 + / + mice to CS for 3 months, after instilling AAV9-BMP4 into the airways of the mice, continue to expose them to CS for 3 months to establish a BMP4 treatment model for chronic obstructive pulmonary disease, and compare the performance of mice in the CS+AAV9-GFP group and the CS+AAV9-BMP4 group, detect the lung function of mice in the CS+AAV9-GFP group and the CS+AAV9-BMP4 group, and simultaneously evaluate the lung pathology. The results are as Figures 5 to 7 shown.

[0064] The specific experimental method is as follows: BMP4 + / + Mice were randomly divided into two groups and continuously exposed to CS for 3 months (12 cigarettes per hour, 4 times per group, 1 hour each time, 6 days per week). After that, one group of mice was instilled with AAV9-BMP4 (1X10 11 PFU / mouse) into the airways, and the control group was instilled with the same amount of AAV9-GFP. At the same time, continue to expose them to CS for 3 months. Pulmonary function parameters were measured using a pulmonary function instrument (PFT system of BUXCO company) ( Figure 5 A- Figure 5 F), including FRC, IC, Cydn, RI, FEV20 / FVC and FEV50 / FVC values, 5 mice in each group; the expression of BMP4 protein in lung tissue was detected by WB experiment ( Figure 5G-5H; N = 6 in each group. Figure 5 I-5J is the statistical chart of HE staining and mean linear intercept of lung sections; N = 6 in each group. HE staining of cell smear of bronchoalveolar lavage fluid, total cell count and differential cell count ( Figure 6 A-6E), N = 6 in each group.

[0065] Lung function results showed that after CS, compared with the CTL group, the lung function in the CS+AAV9-GFP group decreased significantly, and FRC ( Figure 5 A), IC ( Figure 5 B) increased significantly, while FEV20 / FVC ( Figure 5 E), FEV50 / FVC ( Figure 5 F) decreased significantly. Intratracheal instillation of AAV9-BMP4 improved the decreased lung function in mice after CS, and improved the increased FRC and IC and the decreased FEV20 / FVC and FEV50 / FVC after CS; HE results showed that there were no obvious abnormalities in the lung tissue of the CTL group, while obvious destruction and disorder of alveolar structure in mice were seen in CS+AAV9-GFP, with thinning or rupture of alveolar walls, and partial fusion into bullae, and infiltration of inflammatory cells. In the CS+AAV9-BMP4 group, the alveolar structure was relatively intact with less damage, only a few alveolar septa were ruptured, alveoli were fused, and the infiltration of inflammatory cells was significantly reduced (5I); Calculation of the mean linear intercept (MLI) showed that the increase in mean alveolar intercept was significantly reduced after intratracheal instillation of AAV9-BMP4 ( Figure 5 J). The number of inflammatory cells in the bronchoalveolar lavage fluid of the CS+AAV9-GFP group increased significantly, mainly neutrophils, and overexpression of BMP4 improved the increase in inflammatory cells after CS exposure ( Figure 6 A-6E). WB results showed that the expression of BMP4 in the CS+AAV9-GFP group was lower than that in the CTL group, while the expression of BMP4 in the CS+AAV9-BMP4 group was higher than that in the CS+AAV9-GFP group ( Figure 5 G-5H). At the same time, the results of frozen sections of lung tissue showed that GFP in the lung tissue of mice increased significantly 10 weeks after intratracheal instillation of AAV9-GFP vector ( Figure 7 ).

[0066] 2.4 Next, we studied the effect of BMP4 on cell transdifferentiation under CSE stimulation in human type II alveolar epithelial cell line (hAT2 cells). Immunofluorescence co-staining experiment of mouse lung tissue sections showed that BMP4 + / - the expression of AQP5 in mice decreased significantly after CS exposure ( Figure 8 ); the expression of SFTPC increased ( Figure 9); Meanwhile, the WB experimental results after treating hAT2 cells with 2% CSE stimulation and 20 ng BMP4 and 50 ng BMP4 for 36 hours showed that treatment with 20 ng BMP4 could promote the expression of p-SMAD159, β-catenin, and AQP5. This result indicates that the BMP4 recombinant protein can alleviate the limited transdifferentiation of hAT2 cells caused by CSE stimulation. Figure 10 A- Figure 10 B).

[0067] The embodiments described above are only for describing the preferred mode of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. The application of BMP4 gene regulatory products in the preparation of products for preventing and treating chronic obstructive pulmonary disease, characterized in that, The regulation is positive regulation.

2. The application according to claim 1, characterized in that, The BMP4 gene regulation product is AAV9-BMP4.

3. The application according to claim 2, wherein The usage method of the BMP4 gene regulation product is airway instillation.

4. The application according to claim 3, characterized in that The usage amount of the BMP4 gene regulation product is 0.5 to 1.5×10 11 PFU.

5. The application according to claim 4, wherein The usage amount of the BMP4 gene regulation product is 1.0×10 11 PFU.

6. A product for preventing and treating chronic obstructive pulmonary disease, characterized in that, The product contains the BMP4 gene regulation product in any one of the applications described in claims 1 to 5.

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