Construction and evaluation of chronic obstructive pulmonary epithelial cell aging model

Abstract

The invention belongs to the technical field of medical biological research, and particularly relates to construction and evaluation of a chronic obstructive pulmonary epithelial cell senescence model. A method for constructing a chronic obstructive pulmonary epithelial cell senescence model comprises the following steps: exposing tobacco smoke after 6-8 weeks of Muc1 gene whole-body knockout mice and / or exposing tobacco smoke after 6-8 weeks of Muc1 whole-body knockout rats and / or injecting tamoxifen into abdominal cavities of the mice when the Muc1 type II alveolar epithelial cell knockout mice are 6-8 weeks old, and then exposing tobacco smoke every one week. According to the invention, a series of evaluations are carried out on lung tissue pathological changes, lung epithelial cell changes and other indexes of the constructed model product, and the success of the construction of the chronic obstructive pulmonary pulmonary epithelial cell senescence model is jointly proved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical biological research, and particularly relates to construction and evaluation of a lung epithelial cell aging model of chronic obstructive pulmonary disease. BACKGROUND

[0002] Chronic obstructive pulmonary disease (COPD) is a disease characterized by persistent airflow limitation (airflow obstruction), usually caused by lung parenchyma destruction (emphysema) and / or small airway disease (chronic bronchitis), leading to progressive and irreversible decline in lung function. COPD has become an increasingly serious public health problem, and poses a huge treatment burden worldwide. COPD is considered a chronic disease related to age, and its prevalence increases with age. In naturally aging lungs, there are similar changes in lung structure to COPD, such as decreased lung ventilation function, decreased lung elasticity, and increased airway and alveolar diameter. COPD patients also exhibit senescence of multiple cells, including airway epithelial cells and alveolar epithelial cells. In emphysema (one of the main pathological manifestations of COPD), alveolar abnormalities are mainly manifested as destruction of the alveolar wall and impaired epithelial repair, ultimately leading to the development of emphysema. Traditional animal models of COPD and emphysema are mainly constructed by exposing to cigarette smoke (CS), PM2.5, bacterial lipopolysaccharide airway instillation, or elastase airway instillation, which can simulate the functional and pathological changes in humans with COPD and emphysema, such as decreased lung function (indicated by decreased vital capacity and forced expiratory volume), enhanced lung and systemic inflammatory responses, and emphysema-like lesions (such as enlarged alveolar space, alveolar wall rupture, and lung bullae formation). Cigarette smoke extract (CSE), PM2.5 extract, and lipopolysaccharide are commonly used to simulate cell damage caused by COPD risk factors. Mucin 1 (MUC1 / Muc1) is a transmembrane O-glycoprotein with a size of approximately 200 kDa, mainly expressed in AT2 and airway epithelial cells in lung tissue. Previous studies have found that in pulmonary infectious diseases, MUC1 expression in airway epithelial cells increases, which can play an anti-inflammatory role; in pulmonary malignant tumor diseases, MUC1 expression in epithelial cells is abnormally increased, inhibiting cell apoptosis and inducing excessive proliferation, thereby promoting the occurrence of epithelial-mesenchymal transition (EMT); in idiopathic pulmonary fibrosis, the KL-6 domain of the extracellular segment of MUC1 is increased in serum and alveolar lavage fluid, and forms a MUC1-CT / Smad3 / β-catenin complex in the nucleus under the synergistic action of transforming growth factor β1 (TGF-β1), promoting the occurrence of EMT.These research results show that MUC1 plays an important role in maintaining lung homeostasis, and also suggest that abnormal reduction of MUC1 expression can destroy the balance of lung homeostasis. In our previous study, it has been found that Muc1 knockout mice naturally age to 18 months and develop severe spontaneous emphysema. Therefore, the intervention of CS / CSE after MUC1 / Muc1 knockout is expected to construct a new experimental model of lung epithelial cell aging of chronic obstructive pulmonary disease. SUMMARY

[0003] In order to construct the above-mentioned new experimental model, the present application first uses Muc1 knockout mice and rats to construct a lung epithelial cell aging model of chronic obstructive pulmonary disease, wherein the related products include a lung epithelial cell aging model of chronic obstructive pulmonary disease generated by Muc1 knockout mice / rats combined with CS exposure and a new model of chronic obstructive pulmonary disease generated by Muc1 type II alveolar epithelial cell knockout mice combined with CS exposure. - / - ) combined with CS exposure and a new model of chronic obstructive pulmonary disease generated by Muc1 type II alveolar epithelial cell knockout (Muc1 AT2- / - ) mice combined with CS exposure, and application transformation, etc. The present application evaluates a series of indexes of lung tissue pathological changes and lung epithelial cell changes of the constructed model products, which jointly prove the success of the construction of the lung epithelial cell aging model of chronic obstructive pulmonary disease.

[0004] The present application provides a method for constructing a lung epithelial cell aging model of chronic obstructive pulmonary disease, which comprises tobacco smoke exposure of Muc1 knockout mice at 6-8 weeks after whole-body knockout and / or tobacco smoke exposure of Muc1 knockout rats at 6-8 weeks after whole-body knockout and / or tobacco smoke exposure of Muc1 type II alveolar epithelial cell knockout mice at 6-8 weeks after intraperitoneal injection of tamoxifen to the mice.

[0005] Further, the injection dose of tamoxifen is 75 mg / kg.

[0006] Further, the injection time of tamoxifen is 4 days.

[0007] Further, the injection frequency of tamoxifen is 2 days / time.

[0008] Further, the modeling time of the lung epithelial cell aging model of chronic obstructive pulmonary disease of the mice / rats is 3-8 months.

[0009] The present application provides a method for constructing a lung epithelial cell aging model of chronic obstructive pulmonary disease.

[0010] The present application provides a lung epithelial cell aging model of chronic obstructive pulmonary disease obtained by the construction method, and the lung epithelial cell aging model is applied to chronic obstructive pulmonary disease and basic research thereof.

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

[0012] (1) The present application finds that, compared with wild-type mice of the same age, 18-month-old Muc1 - / - Mice exhibit more severe airway and alveolar epithelial inflammation, and the expression of aging-related inflammatory markers IL-4, IL-6, KC and TNF-α in bronchoalveolar lavage fluid (BALF) is significantly increased, and the expression of aging-related molecules Lamin B1 and p53 in lung epithelial cells is significantly increased.

[0013] (2) The present application finds that, compared with wild-type smoke-exposed mice of the same age, Muc1 - / - The body weight of the mice further decreases, the airway and alveolar epithelium shows more significant inflammatory response, accompanied by increased airway collagen deposition, increased alveolar septum rupture, and increased secretion of mucin and glycoprotein by epithelial cells.

[0014] (3) The present application finds that, compared with wild-type smoke-exposed mice of the same age, Muc1 AT2- / - Mice also exhibit more significant inflammatory response in the airway and alveolar epithelium, and Muc1 AT2- / - The total number of cells in the BALF of smoke-exposed mice is significantly increased.

[0015] (4) The present application first finds that, after knocking out Muc1 in mice and knocking out Muc1 in type II alveolar epithelial cells, giving tobacco smoke exposure for 6 months can form a more severe chronic obstructive pulmonary disease model, and the lung epithelial cells in this model have undergone significant senescent-like changes. Compared with traditional chronic obstructive pulmonary disease models, the present application introduces the genetic knockout of Muc1 to produce a more severe chronic obstructive pulmonary disease phenotype in the same smoke exposure time, and verifies the senescent-like changes in lung epithelial cells, solving the defects of traditional chronic obstructive pulmonary disease animal models. The senescent-like changes in lung epithelial cells established by knocking out Muc1 after CS exposure not only have important economic value, but also have far-reaching significance for basic and clinical research, providing an efficient and reliable modeling method for the study of chronic obstructive pulmonary disease. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 For the pathological changes and senescent-like changes of lung epithelial cells in Muc1 knockout mice in Example 3: (A): Hematoxylin-eosin (HE) staining results of lung tissue of 3-month-old, 6-month-old and 18-month-old wild-type and Muc1 - / - Mice showed no significant pathological differences in airway and alveolar epithelium compared with wild-type mice, while 18-month-old Muc1 - / - Mice showed no significant pathological differences in airway and alveolar epithelium compared with wild-type mice, while 18-month-old Muc1 - / -Mice showed more severe airway inflammation and significant alveolar septal damage compared to age-matched wild-type mice (n=8 per group); (BE): Changes in IL-6, KC, IL-4, and TNF-α levels in the bronchoalveolar lavage fluid (BALF) of mice in the two age groups, with 18-month-old Muc1... - / - Mice showed significantly higher levels of the above indicators compared to age-matched wild-type mice, with n=6 in each group; (F): Immunofluorescence results showed that 18-month-old Muc1 mice had significantly higher levels of the above indicators. - / - Lamin B1 expression was significantly increased in lung tissue of mice, with n=5 per group; (G): Immunofluorescence results showed that 18-month-old Muc1... - / - Mouse Sftpc + p53 + The proportion of cells increased significantly, with n=3 in each group. A p < 0.05 was considered statistically significant.

[0017] Figure 2 Muc1 in Example 3 - / - Changes in severity of CS exposure in mice after 6 months compared to wild-type mice under the same treatment conditions: (A): Wild-type vs. Muc1 - / - The changes in body weight of mice after 6 months of normal environment feeding and 6 months of CS exposure compared to wild-type smoke-exposed mice were studied. - / - Mice showed a significant decrease in body weight after smoking, with n=10 in each group; (B): RNA extracted from the lung tissue of the above mice was reverse transcribed and then subjected to qPCR experiments. The results showed that Muc1 - / - The transcriptional level of Muc1 in mice was significantly decreased, indicating a significant knockout effect; (CD): Western blot results of the above mouse lung tissue showed that Muc1 knockout was successful, and under CS exposure conditions, Muc1 expression in wild-type mouse lung tissue was increased compared to the air control group, with n=6 in each group; (EH): HE staining results of paraffin sections of the above mouse lung tissue showed changes in airway and alveolar morphology, indicating that after CS exposure, the expression of Muc1 in wild-type and air-controlled mice was significantly reduced. - / - Compared to their respective air control groups, mice showed significant airway inflammation and alveolar septal rupture, and Muc1 - / - Mice exposed to CS showed more severe airway inflammation and a further increase in mean alveolar intercept compared to wild-type CS-exposed mice (n=10 per group). (IJ): Masson staining results of paraffin sections of mouse lung tissue after CS exposure; Muc1... - / - Compared to wild-type mice, mice showed significantly more airway collagen deposition, with n=10 in each group; (KL): PAS staining results of paraffin sections of the airways of the mice above, after CS exposure, Muc1 - / - Mice produced more glycoproteins through airway secretion compared to wild-type mice, with n=10 per group. A p < 0.05 was considered statistically significant.

[0018] Figure 3 Muc1 - / - The severity of the changes in the rats after 6 months of CS exposure compared to wild-type rats under the same treatment conditions: (A) Wild-type and Muc1 - / - The changes in body weight in rats after 6 months of normal environment feeding and 6 months of CS exposure, compared to wild-type smoke-exposed rats, Muc1 - / - The body weight of the rats significantly decreased after smoke exposure, n = 10 in each group; (B) The changes in the expression of Muc1 in the lung cells of the rats were observed by immunohistochemical staining of the paraffin sections of the lung tissues of the rats, and the results showed that Muc1 - / - The lung epithelial Muc1 of the rats was significantly knocked out; (C) The Western blot results of the lung tissues of the rats showed that the knockout of Muc1 was successful, and the expression of Muc1 in the lung tissues of the wild-type rats was increased compared to the control group under the CS exposure condition, n = 6 in each group; (D-E) The changes in the airway and alveolar morphology in the HE staining results of the paraffin sections of the lung tissues of the rats, and the results showed that after CS exposure, the wild-type and Muc1 - / - The rats showed obvious airway inflammatory response and alveolar septum rupture compared to the air control group of each rat, and Muc1 - / - The rats showed more severe airway inflammatory response compared to the wild-type CS-exposed rats, and the average alveolar intercept was further increased, n = 10 in each group. (F) The Masson staining results of the paraffin sections of the lung tissues of the rats, Muc1 - / - The rats showed more significant airway collagen deposition compared to the wild-type rats, n = 10 in each group; (G) The PAS staining results of the paraffin sections of the airways of the rats, Muc1 - / - The rats showed more significant airway collagen deposition compared to the wild-type rats, n = 10 in each group; (G) The PAS staining results of the paraffin sections of the airways of the rats, Muc1 - / - The rats showed more significant airway collagen deposition compared to the wild-type rats, n = 10 in each group; (G) The PAS staining results of the paraffin sections of the airways of the rats, Muc1 - / - The rats showed more significant airway collagen deposition compared to the wild-type rats, n = 10 in each group; (G) The PAS staining results of the paraffin sections of the airways of the rats, Muc1

[0019] Figure 4 Muc1 AT2- / - The severity of the changes in the rats after 6 months of CS exposure compared to wild-type rats under the same treatment conditions: (A-B) Wild-type and Muc1 AT2- / -Mice were raised in normal environment for 6 months, and lung tissues were taken for Western blot to verify the expression changes of Muc1 after 6 months of CS exposure. The results showed that the expression of Muc1 in lung tissues of wild-type mice was also increased under CS exposure compared with the air control group. The expression of Muc1 in air control and smoke groups AT2- / - The expression of Muc1 in mice was significantly decreased, which proved that the knockout was successful, n = 3 in each group; (C-D): The results of HE staining of lung tissue paraffin sections of the above mice showed that after CS exposure, the airway and alveolar morphology of wild-type and Muc1 - / - The mice showed obvious airway inflammatory response and alveolar septum rupture compared with the air control group, which was consistent with the previous Muc1 full knockout mouse model. Muc1 AT2- / - After CS exposure, the mice showed more severe airway inflammatory response than wild-type CS-exposed mice, and the average alveolar intercept was further increased, n = 3 in each group. (E-F): The results of HE staining of BALF after shaking of the above mice showed that the wild-type mice and Muc1 AT2- / - The total number of BALF in mice increased significantly compared with the air control group, and Muc1 AT2- / - The total number of BALF in mice increased further compared with wild-type mice of the same modeling method, n = 3 in each group. Differences were considered statistically significant when p < 0.05.

[0020] Figure 5To verify the success of the new COPD lung epithelial cell aging model constructed in this application, lung epithelial cell experiments in Example 3 were used for supplementation: (A): After transfection of the human airway epithelial cell line 16HBE with MUC1 overexpression plasmid or siRNA silencing of MUC1 expression for 48 h, senescence-associated galactosidase staining was performed. The results showed that after overexpression of MUC1, the galactosidase activity of 16HBE cells decreased, and the senescent cells decreased. After siRNA silencing of MUC1, the galactosidase activity of 16HBE cells increased, and the senescent cells increased. n=6 in each group; (B-D): Western Blot experiment was performed on the proteins extracted from 16HBE cells transfected with siRNA NC and MUC1. The results showed that compared with the control group transfected with si-NC, after transfection of si-MUC1, the expression of senescence-associated proteins p53 and p16 increased significantly. n=6 in each group; (E-F): In the MUC1 knockout A549 cell line, Western blot results showed that MUC1 knockout promoted the expression of p21 protein induced by CSE. n=3 in each group; (G-H): qPCR results further found that MUC1 knockout A549 cells showed a significant increase in the mRNA levels of senescence-associated secretory phenotypes IL6 and IL8 under the basic state and CSE stimulation. n=8 in each group; (I): Cell galactosidase staining of human type II alveolar epithelial cell line further confirmed that whether or not the cells received CSE stimulation, the cell galactosidase activity in the MUC1 silenced group was significantly enhanced. n=3 in each group. Differences were considered statistically significant when p<0.05. DETAILED DESCRIPTION

[0021] The small / rats used in the following examples were raised in a specific pathogen-free barrier environment, and the experimental rats were fed with selected maintenance feed. The anesthetic method used was abdominal cavity injection of Avertin.

[0022] Example 1

[0023] 1. Animal grouping

[0024] Wild-type and Muc1 - / - mice (both purchased from Guangzhou Sailaibaimu Biotechnology Co., Ltd.) were grouped according to the experiment (n=8 in each group), so that their mouse age reached 3 months, 6 months and 18 months under air exposure conditions, and their lung tissues were taken for pathological sectioning and protein extraction.

[0025] 2. Muc1 - / - mouse gene identification

[0026] Cut a small amount of mouse toes into a pre-labeled 1.5 ml EP tube, mix 200 μl of mouse tail PCR lysis solution with 10 μl of proteinase K, add 200 μl of mouse tail lysis solution + proteinase K mixture to each EP tube, and place it in a pre-heated 55℃ water bath overnight (about 10-12 h), then raise the water bath temperature to 85℃ and water bath for 1 h to inactivate proteinase K. Use the primer sequences shown in Table 1 for gene identification:

[0027] Table 1

[0028]

[0029] Each sample needs to be prepared with 2 systems, one with F+R primers and the other with F+Z primers, and the system configuration volume is shown in Table 2:

[0030] Table 2

[0031]

[0032] After the system configuration is completed, PCR amplification is performed according to the conditions shown in Table 3:

[0033] Table 3

[0034]

[0035] During PCR, 100 μl of freshly prepared 1x TAE is measured with a graduated cylinder into a conical flask, 2 g of agarose powder is weighed and added, shaken to mix, placed in a microwave oven, medium heat for 3-5 min, boiled until the solution is clear and transparent, when the temperature drops to a temperature that can be touched (also can be washed with cold water to accelerate cooling), add 5 μl of nucleic acid dye, shake the conical flask to mix. Pour into a clean gel plate with a comb inserted, cool for about 30 min. After the PCR reaction is completed, 5 μl of each PCR product and 1 μl of 6x loading buffer are mixed in a plastic wrap and added to the loading well. After loading is completed, electrophoresis is performed at 120v for 30 min. After electrophoresis, immediately remove the agarose gel and place it in a gel imaging system for imaging, and determine the mouse genotype according to the results.

[0036] 3. Tobacco smoke exposure mouse model

[0037] The cigarette used to establish the mouse tobacco smoke exposure model is Hongmei brand filtered cigarette (Guangdong Tobacco Industry Company, China), each cigarette contains 11 mg of tar, 1.0 mg of nicotine and 13 mg of carbon monoxide. 6-8 week old Muc1AT2- / - mice and their littermates were exposed to CS using a whole body exposure system: mice were exposed to CS (9 cigarettes / h, 2 hours / time, 2 times / day, 6 days / week) for 24 weeks (the exposure box size is 60 50 50 cm).

[0038] The control group was only exposed to filtered indoor air (RA). After the end of the experiment, all mice were subjected to lung function tests and sacrificed.

[0039] 4, Mouse lung tissue pathology

[0040] After fixing the left lung of the mouse with 10% formaldehyde solution for 24 h, paraffin embedding was performed, and the paraffin block was cut into 4 μm thick sections and spread onto clean glass slides to prepare paraffin tissue sections. After deparaffinization of the tissue sections, staining was performed using an HE kit, and then histological examination was performed.

[0041] 5, Mouse lung tissue paraffin section immunofluorescence staining

[0042] The mouse lung tissue section was placed in a 60 ℃ oven for 1 h to 2 h in advance, and the paraffin section was deparaffinized with xylene for 10 min twice, anhydrous ethanol for 5 min twice, 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and distilled water for 5 min. Gradient dehydration. Prepare 0.2% Triton-X 100, dropwise infiltration to cover the entire tissue section, permeate at room temperature for 15 min, and wash with PBS for 5 min for 3 times. Place the tissue section in a glass bottle containing citric acid antigen repair solution, make sure that the repair solution covers the tissue section, do not dry the slice, heat in the microwave oven to 100 degrees to make it boil for 10 min, and then cool naturally. Wash with PBS for 5 min for 3 times. Prepare 5% BSA, dropwise infiltration to cover the entire tissue section, block at room temperature for 30 min. Use a pipette to suck the blocking solution, place it flat in a wet box, and drop 100-200 μl of prepared primary antibody (LaminB1 (CST, USA); p53 antibody (abcam, USA); SFTPC antibody (abclonal, China)) (antibody: 5% BSA = 1:100) on the section, and incubate overnight in a 4 ℃ refrigerator. Take the wet box out and warm it up for 30 min the next day, wash it with PBS for 5 min for 3 times. Dry the section with a clean absorbent paper, drop 100-200 μl of prepared secondary antibody (FITC-labeled goat anti-rabbit IgG (H+L) (Biogen, China); Cy3-labeled goat anti-mouse IgG (H+L) (Biogen, China)) (antibody: 5% BSA = 1:500) on the section, and incubate at room temperature for 1 h. Wash with PBS for 5 min for 3 times, dry the section with a clean filter paper, drop 10 μl of DAPI-containing anti-fluorescence quencher, and then use a cover glass to mount the section, and then use an inverted fluorescence microscope to collect images.

[0043] 6, Mouse BALF treatment

[0044] After lung function test, the mice were fixed on the sampling plate, the chest was opened, the lung tissue was fully exposed, the left lung was ligated with surgical thread, then 0.6 ml of normal saline was perfused into the right lung through the tracheal cannula, and after staying for a while, it was back-flushed, and the process was repeated for 6 times. The BALF collected in the first three and last three times was respectively placed in two different EP tubes. After collecting the BALF, the right lung hilum was ligated with surgical thread, and the upper, middle, lower and round leaf lung tissues were cut off, washed with normal saline to remove blood stains, and placed in a 1.5 ml EP tube and placed in a liquid nitrogen bottle. The left lung ligation thread was loosened, 10% neutral formalin was slowly dripped into the left lung tissue, and the left lung hilum was ligated again with the loosened surgical thread. The left lung was separated and placed in a centrifuge tube containing 10% neutral formalin, and fixed on a 4°C shaking bed for 24 hours.

[0045] 7, Protein extraction of mouse lung tissue

[0046] After the right lung of the mouse was taken out, it was divided into dry ice for temporary storage according to the upper, middle, lower and mesenchymal lobes. The lower lobe was weighed and placed in a tissue homogenizer tube. Freshly prepared protein lysis buffer was added according to the ratio of tissue weight: protein lysis buffer = 30 mg: 1 ml, and the tissue homogenizer was pre-cooled to 4°C. After homogenization, centrifugation was performed at 12000 rpm, 4°C for 30 min, and the supernatant was transferred to a new EP tube. The protein concentration was determined by BCA method according to the kit instructions, and each sample was diluted to the same concentration with protein lysis buffer, and 5xLoading Buffer was added. Boil in boiling water for 10 min to denature the protein, and store at -20°C after cooling.

[0047] 8, Western blotting

[0048] The denatured protein sample in step 5 was added to the pre-configured polyacrylamide gel hole (10 ul of protein sample was added to each hole), the voltage was adjusted to 80 V constant voltage electrophoresis, and the electrophoresis was stopped when the sample line was 1 cm away from the bottom of the glass plate. The electrophoresed gel was transferred to the PVDF membrane according to the conditions of 300 mA, 100 min, and after incubation with 5 % bovine serum albumin at room temperature for 1 hour, the film was cut according to the molecular weight of the target protein. Incubate overnight at 4°C in the prepared primary antibody diluent containing the corresponding antibody (MUC1 (MUC1-C): 1:1000 (USA abeam); P53: 1:1000 (USA abeam); P21: 1:1000 (USA abeam); P16: 1:1000 (USA abeam); β-actin: 1:10000 (China abclonal); GAPDH: 1:5000 (USA sigma)). After incubation of the primary antibody, the primary antibody was recovered and washed with the prepared TBST buffer for 10 min x 3 times, and the secondary antibody containing horseradish peroxidase was diluted in the primary antibody diluent at a ratio of 1:5000 (China Biyun Tian Biological Technology Institute) and incubated on a shaker at room temperature for 1 hour. After incubation of the secondary antibody, the TBST buffer was used again for 10 min x 4 times. Then the chemiluminescence developing solution was prepared, and the developing solution was dropped on the strip, and the Tanon5200 immunodetection system was used for development. After saving the developed strip picture, the Image J software was used for gray value analysis of the developed picture.

[0049] The antibodies used are as follows: anti-MUC1-C antibody (abcam, ab109185); anti-β-actin antibody (Santa, #4695).

[0050] 9. Statistical analysis

[0051] The measurement data conforming to the normal distribution is (Mean ± SD), and if the measurement data cannot be corrected for skewness distribution, Median (IQR) is used. The mean comparison between two groups of samples applies t test, and for multiple groups of samples conforming to normal distribution and variance, one-way ANOVA is used for overall mean comparison, and then LSD method is used for pairwise comparison; if the sample does not conform to the normal distribution or the variance is not uniform, the data is transformed (sqrt, lg, exp, etc.) and then the normality test and variance homogeneity test are performed. If it still does not conform to the variance homogeneity or the normal distribution, non-parametric test is used. GraphPad Prism 9.4.1 software is used for statistical analysis, and P < 0.05 is considered to have statistically significant difference.

[0052] Example 2

[0053] 1. Animal grouping

[0054] 6-8 weeks wild type and Muc1 - / - Rats (all purchased from Guangzhou Sailabimo Biotechnology Co., Ltd.) were divided into air control and smoke exposure groups (n=10 in each group) according to experimental grouping. After the modeling was completed, the lung tissues of the rats were taken for pathological section and protein extraction.

[0055] 2、Muc1 - / - Rat gene identification

[0056] A small amount of mouse toes was cut into a pre-labeled 1.5 ml EP tube, 200 μl of mouse tail PCR lysis solution and 10 μl of proteinase K were mixed for each mouse, 200 μl of mouse tail lysis solution + proteinase K mixture was added to each EP tube, and it was placed in a pre-heated 55℃ water bath overnight (about 10-12 h). The water bath was warmed to 85℃, and the proteinase K was inactivated by water bath for 1 h. The primer sequences used for identification are shown in Table 4,

[0057] Table 4

[0058]

[0059] According to the system as shown in Table 2, the corresponding primers were replaced, and PCR amplification was carried out according to the conditions as shown in Table 3. During the PCR process, 100 μl of newly prepared 1x TAE was measured with a graduated cylinder into a conical flask, 2 g of agarose powder was weighed and added, and it was shaken to mix well. It was placed in a microwave oven, and the middle fire was boiled for 3-5 min until the solution was clear and transparent. When the temperature decreased to a temperature that could be touched by hand (it could also be cooled by washing with cold water), 5 μl of nucleic acid dye was added, and the conical flask was shaken to mix well. Pour into the gel plate with clean combs, cool for about 30 min. After the PCR reaction was completed, 5 μl of each PCR product and 1 μl of 6x loading buffer were mixed in the plastic wrap and added to the loading well. After loading, electrophoresis was carried out at 120v for 30 min. After electrophoresis, the agarose gel was immediately taken out and placed in the gel imaging system for imaging. According to the results, the mouse genotype was determined.

[0060] 3、The experimental process of the tobacco smoke exposure rat model was the same as that of Example 1 step 3. The experimental process of the rat lung tissue pathology was the same as that of Example 1 step 4. The experimental process of the rat BALF treatment was the same as that of Example 1 step 6. The experimental process of the rat lung tissue protein extraction was the same as that of Example 1 step 7. The experimental process of Western blotting was the same as that of Example 1 step 8. The statistical analysis was the same as that of Example 1 step 9.

[0061] Example 3

[0062] 1、Animal grouping

[0063] 6-8 weeks old wild type and Muc1 AT2- / - Mice (all purchased from Guangzhou Sailabimo Biotechnology Co., Ltd.) were obtained from ENSEMBL, UCSC and Refseq databases. CRISPR / Cas9 gene knockout technology was used to construct the mice. The Muc1 segment was cut in AT2 to construct the Muc1 segment selectively knockout mice in mAT2, and the success of gene conditional knockout was verified by qRT-PCR and WB. According to the experimental grouping (n=3 in each group), after intraperitoneal injection of tamoxifen (tamoxifen injection dose was 75 mg / kg, injection scheme was 1 day interval after injection for 2 days, a total of 4 days of injection), the positive control mice were exposed to air for 6 months, and the positive control mice were exposed to tobacco smoke (CS) generated by lighting red plum brand filter cigarettes (China Guangdong Tobacco Industry Company) (9 / h, 2 hours / time, 2 times / day, 6 days / week) for 6 months (exposure box size was 60 50 50cm), the lung function of the mice was detected, and the lung tissue was taken for pathological section and protein extraction.

[0064] 2、Muc1 AT2- / - Mouse gene identification

[0065] A small amount of mouse toe was cut into a pre-labeled 1.5 ml EP tube, 200 μl of mouse tail lysis buffer and 10 μl of proteinase K were mixed, 200 μl of mouse tail lysis buffer + proteinase K mixture was added to each EP tube, and it was placed in a pre-heated 55°C water bath overnight (about 10-12h), the water bath was heated to 85°C, and the water bath was heated for 1h to inactivate proteinase K. The following primer sequences were used for gene identification:

[0066] Muc1-flox identification primer is as follows:

[0067] F1, SEQ ID NO. 6: 5'-GGCAAATAATACTCTTTGTGGGGA-3'

[0068] R1, SEQ ID NO. 7: 5'-TGCTTCTGGCTCATTTATTCCTCTA-3'

[0069] MT: 266bp; WT: 204bp;

[0070] F2, SEQ ID NO. 8: 5'-TACTCATTAGGATGCCCAGAGGT-3'

[0071] R2, SEQ ID NO. 9: 5'-TAAAACTCCCTCAGCACACTCATC-3'

[0072] MT: 255 bp; WT: 182 bp;

[0073] According to the system as shown in Table 2, replace the corresponding primers, and perform PCR amplification according to the conditions as shown in Table 3. During the PCR process, 100 μl of newly prepared 1x TAE is measured by a measuring cylinder into a conical flask, 2 g of agarose powder is weighed and added, and shaken to mix well. Place in a microwave oven, medium heat for 3-5 min, boil until the solution is clear and transparent. When the temperature drops to a temperature that can be touched by hand (it can also be accelerated by washing with cold water), add 5 μl of nucleic acid dye, shake the conical flask to mix well. Pour into a clean gel plate with a comb inserted, cool for about 30 min. After the PCR reaction is completed, 5 μl of each PCR product and 1 μl of 6x loading buffer are mixed in the plastic wrap and added to the loading well. After loading is completed, electrophoresis is performed at 120 v for 30 min. After electrophoresis, immediately remove the agarose gel and place it in the gel imaging system for imaging. According to the results, the mouse genotype is determined.

[0074] 3. The experimental process of the tobacco smoke exposure mouse model is the same as that of Example 1 Step 3. The experimental process of the mouse lung tissue pathology is the same as that of Example 1 Step 4. The experimental process of the mouse BALF treatment is the same as that of Example 1 Step 6. The experimental process of the mouse lung tissue protein extraction is the same as that of Example 1 Step 7. The experimental process of Western blotting is the same as that of Example 1 Step 8. The statistical analysis is the same as that of Example 1 Step 9.

[0075] Example 4

[0076] 1. 16 HBE cell culture

[0077] Human airway epithelial cells 16 HBE were cultured in 10% FBS-DMEM (GIBCO) high-sugar medium at 37°C, 5% CO2. After 50 generations of culture, the cells were harvested, and 2x10 5 cells were inoculated in a six-well cell culture plate. After 24 hours, the cell morphology was observed under a microscope, and the cells adhered and grew well, reaching about 70% contact. The old culture medium was discarded, and 1% FBS-DMEM high-sugar medium was replaced. After 24 hours of starvation culture, transfection or 2% CSE treatment was started.

[0078] 2. A549 cell culture

[0079] A549 cells were cultured in 1640 (GIBCO) medium containing 10% FBS, and were cultured for 50 generations at 37℃, 5% CO2. When the cells grew to the logarithmic growth phase, the cells were harvested, and 2x10 5

[0080] 3、hAT2 cell culture

[0081] Human primary alveolar type II epithelial cells hAT2 cells (purchased from ABM Company in Canada [Applied Biological Materials Inc.], China agent is Zhenjiang Aibimeng Biological Technology Co., Ltd., and the item number is TO482) were cultured in a finished medium with the same item number TO482 (purchased from ABM Company in Canada [Applied Biological Materials Inc.], China agent is Zhenjiang Aibimeng Biological Technology Co., Ltd.), and were cultured for 10 generations at 7℃, 5% CO2. When the cells grew to the logarithmic growth phase, the cells were harvested, and 2x10 5

[0082] 4、Extraction of cell protein

[0083] The cells in the six-well plate were washed with pre-cooled PBS for 3 times, 80 μl of cell lysis solution was added to each well, and the plate was placed on a shaker and incubated on ice for 30 min. Then the cells were collected with a cell scraper and transferred to a pre-cooled 1.5 ml centrifuge tube and incubated on ice for 10 min. After centrifugation at 4℃, 14000 rpm for 30 min, the supernatant was carefully transferred to a new centrifuge tube, and the protein concentration was determined using the BCA method according to the kit instructions. After the same concentration of protein lysis solution was added to each sample, 5xLoading Buffer was added, and the protein was boiled for 10 min to denature the protein completely. After cooling, it was stored at -20℃.

[0084] 5、Extraction of cell RNA

[0085] ​​The cells in the six-hole plate were homogenized with 1 ml Trizol, and the total RNA in the cells was extracted. The tissue homogenate was mixed with 200 μl of chloroform and shaken, and then left at room temperature for 3 min, centrifuged for 15 min (12000 g, 4 ℃), and the upper aqueous phase was slowly aspirated into another 1.5 ml centrifuge tube. An equal volume of isopropanol was added, mixed, left at room temperature for 10 min, and centrifuged for 10 min (12000 g, 4 ℃). The supernatant was discarded, 1 ml of 75% ethanol was added to wash the RNA precipitate at the bottom of the tube, centrifuged for 5 min (7500 g, 4 ℃), the supernatant was discarded, and the RNA precipitate was dissolved in an appropriate amount of DEPC water after being dried until there was no alcohol smell. The concentration was determined, and then the reverse transcription system was prepared for cDNA synthesis, and the transcription level changes of the target molecules were detected by real-time fluorescent quantitative PCR.

[0086] 6、Cell galactosidase activity detection

[0087] The operation process is as follows: the cells are inoculated on 12-well plate cell slides, washed with pre-cooled PBS after intervention, and fixed with 2% formaldehyde / 0.2% glutaraldehyde for 15 minutes. Prepare X-gal staining solution, add 500 μL per well, incubate at 37°C in the dark for 12-16 hours. After staining, the liquid is aspirated, 0.1% nuclear fast red is used for re-staining for 5 minutes, and PBS is used for washing. Observe the blue staining under a microscope. Transfer the slide to a glass slide and use a pathological section scanning machine to obtain an image.

[0088] The experimental results of the above examples 1-4 are as follows:

[0089] 1、Muc1 - / - Mice develop more severe lung epithelial cell aging with age

[0090] The results of paraffin sections of lung tissues of 3-month-old, 6-month-old and 18-month-old mice showed that with the increase of age, the inflammatory cells around the airway of mice gathered, the number of cells increased, leading to thickening of the tube wall, and the surrounding inflammatory cell infiltration was more obvious after knocking out Muc1 Figure 1 A); The expression of inflammatory factors IL-6, KC, IL-4 and TNF-α in the bronchoalveolar lavage fluid of mice increased from 6 months to 3 months, and from 18 months to 6 months, and the increase of the Muc1 - / - group was more obvious. However, there was no significant difference between the 3-month-old, 6-month-old Muc1 + / + group and the Muc1 - / - group, and the increase after knocking out Muc1 in 18-month-old mice had statistical difference Figure 1 B-E); The changes of lamin B1, a cell aging marker, were observed by selecting lung tissue sections of 18-month-old mice. Immunofluorescence of tissue sections found that the expression of lamin B1 in the Muc1 + / +Compared with the control group, Muc1 - / - The fluorescence intensity of mouse lamin B1 (green light) was significantly reduced, especially in bronchial cells. Figure 1 F); in addition to Muc1 + / + Compared with the control group, 18-month-old Muc1 - / - Mouse Sftpc + p53 + The proportion of cells increased significantly ( Figure 1 These results collectively indicate that the airway and alveolar epithelial cells in the lung tissue of Muc1 knockout mice are aged more rapidly.

[0091] 2. Muc1 knockout exacerbates lung inflammation, airway remodeling, mucus hypersecretion, and emphysema in CS-induced COPD model mice.

[0092] Six months after CS exposure to establish the model, the wild type, namely Muc1, was obtained. + / + Group mice and Muc1 - / - The mice were significantly lighter than the air control group and lighter than Muc1. + / + Body weight of CS group mice, Muc1 - / - The CS group mice experienced a more significant reduction in body weight. Figure 2 A) qPCR and Western blotting results showed that Muc1 + / + The levels of Muc1 mRNA and protein in the lung tissue of CS group mice were significantly higher than those in their Muc1 group. + / + CTL group; Muc1 - / - In both the CS and CTL groups, Muc1 mRNA and protein were not expressed in the mice. Figure 2 (BD); HE staining was performed on mouse lung tissue sections. The results showed that in both groups of CS mice, the bronchial mucosal folds were thickened, bronchial epithelial cells were sloughed off, and a large amount of inflammatory infiltration was observed in the submucosa and bronchial wall, with luminal narrowing. The bronchial inflammation index score showed that compared with Muc1, + / + CS group mice, Muc1 - / - The bronchial inflammation index was more significantly increased in the CS group mice. Figure 2 EF); the alveolar structures in the lung tissues of the two groups of CS mice were of different sizes, some alveolar septa were broken, and alveoli fused to form bullae, and Muc1 - / - The pathological changes of emphysema in the lungs were more obvious in the CS group mice. Figure 2 GH); Masson staining of mouse lung tissue sections showed that Muc1 + / + CS group and Muc1 - / - Collagen deposition in the airway walls of CS group mice was significantly increased compared to that in CTL group mice, with Muc1 showing the most significant increase. - / - CS group mice compared with Muc1 + / + The increase was more pronounced in the CS group mice.Figure 2 I-J) ; Muc1 + / + CS group and Muc1 - / - The number of airway goblet cells in CS group mice was significantly increased compared with that in the corresponding CTL group mice; Muc1 - / - The number of airway goblet cells in CS group mice was significantly increased compared with that in the corresponding CTL group mice; Muc1 + / + The airway goblet cell proliferation in CS group was more obvious (Fig. K-L). These results collectively indicated that knocking out Muc1 aggravated the lung inflammation, airway remodeling, mucus hypersecretion and emphysema in CS-induced COPD model mice.

[0093] 3. Knocking out Muc1 aggravated the lung inflammation, airway remodeling, mucus hypersecretion and emphysema in CS-induced COPD model rats

[0094] After 6 months of CS exposure modeling, wild type, i.e. Muc1 + / + group rats and Muc1 - / - The body weight of CS group rats was significantly reduced compared with that of air control group, and was more obvious than that of Muc1 + / + The body weight of CS group rats, Muc1 - / - The body weight of CS group rats was more obviously reduced than that of Muc1 Figure 3 A) ; immunohistochemistry and Western blotting results showed that: Muc1 + / + The Muc1 protein level in lung tissue of CS group rats was significantly higher than that of Muc1 + / + CTL group; Muc1 - / - In both CS group and CTL group rats, Muc1 protein was not expressed (Fig. A). Figure 3 B-C) ; HE staining of rat lung tissue found that the lung tissue of two groups of CS rats had thickened airway (small airway <1000 μm; medium airway: 1000-2000 μm) mucosal folds, bronchial epithelial cell shedding, submucosal and wall could be seen a large number of inflammatory infiltrates, lumen stenosis, compared with Muc1 + / + CS group, Muc1 - / - The bronchial inflammatory cell infiltration in CS group rats was more obvious (Fig. B-C). Figure 3 D) ; the lung tissue of two groups of CS rats had disordered alveolar structure, rat alveolar cavity was enlarged, part of alveolar septum was broken, fused into pulmonary bulla, and presented typical emphysema pathological changes. Compared with Muc1 + / + CS group rats, Muc1 - / - The emphysema pathological changes in CS group rats were more obvious (Fig. D). Figure 3 E) ; Masson staining of rat lung tissue sections showed that: Muc1 - / - CS group and Muc1 + / + The airway wall collagen deposition in CS group rats was significantly increased compared with that in the corresponding CTL group rats; Muc1 - / - The airway wall collagen deposition in CS group rats was significantly increased compared with that in the corresponding CTL group rats; Muc1+ / + CS group airway collagen deposition was more obvious ( Figure 3 F); rat lung tissue sections and PAS staining showed that Muc1 - / - CS group and Muc1 + / + CS group rats airway goblet cell number was significantly increased than its CTL group rats; Muc1 - / - CS group rats than Muc1 + / + CS group airway goblet cell proliferation was more obvious ( Figure 3 G); At the same time, Muc1 - / - CS group and Muc1 + / + CS group rats BALF mucin Muc5ac and muc5b levels were higher than their respective CTL group; Muc1 + / + CS group rats, mucin Muc5ac and Muc5b in Muc1 - / - CS group increased more obviously ( Figure 3 H-I). These results collectively indicate that knockout Muc1 aggravates CS-induced COPD model rats lung inflammation, airway remodeling, mucus hypersecretion and emphysema.

[0095] 4, knockout Muc1 in AT2 aggravates CS-induced COPD model mice lung inflammation and emphysema

[0096] Extraction of wild type and Muc1 AT2- / - Western blot detection of Muc1 expression changes in mouse lung tissue, the results showed that wild type CS group mice lung tissue Muc1 protein levels were significantly higher than its air control group; Muc1 AT2- / - Muc1 protein levels were significantly reduced in both CS and CTL groups of mice ( Figure 4 A-B); HE staining of mouse lung tissue sections showed that the size of alveolar structure in the lung tissue of two groups of CS mice was different, part of the alveolar septum was broken, and alveolar fusion formed lung bullae, compared with wild type mice, Muc1 AT2- / - The average alveolar intercept of mice was significantly increased, and Muc1 AT2- / - Mice also had more obvious inflammatory infiltration and emphysema ( Figure 4 C-D); HE staining results of mouse BALF after shaking showed that wild type mice and Muc1 AT2- / - Mice compared with their respective air control group BALF total number was significantly increased, Muc1 AT2- / - Mice compared with the same modeling way wild type mice BALF total number further increased ( Figure 4 E-F). These results collectively indicate that knockout Muc1 in AT2 also aggravates CS-induced COPD model mice lung inflammation and emphysema.

[0097] 5. Knocking out MUC1 aggravates CSE-induced lung epithelial cell senescence

[0098] 16HBE cells were transfected with overexpression plasmid or siRNA for 48 h, and then subjected to senescence-associated galactosidase staining. Compared with the pcDNA3.1 vector-transfected cells, the number of cells stained dark blue was less in the MUC1 overexpression plasmid-transfected cells. However, compared with the siRNA control-transfected cells, the number of cells stained dark blue was more in the MUC1 siRNA-transfected cells. Figure 5 A); Western blot analysis of the cell proteins extracted from the siRNA NC- and MUC1-transfected 16HBE cells showed that the expression of senescence-associated proteins p53 and pl6 was increased in the si-MUC1-transfected cells compared with the si-NC-transfected cells. Figure 5 B-D); Western blot analysis showed that knocking out MUC1 significantly upregulated the level of senescence marker P21 protein in A549 cells stimulated by CSE compared with the control. Figure 5 E-F); qPCR results showed that the mRNA levels of IL-6 and IL-8 were significantly increased in A549 cells after knocking out MUC1, regardless of whether the cells were stimulated by CSE. Figure 5 G-H); In hAT2 cells, senescence-associated galactosidase staining showed that the number of cells stained dark blue was increased after CSE stimulation, and the number of cells stained dark blue was significantly increased in the control group after knocking out MUC1, and the number of dark blue cells was further increased after CSE stimulation. Figure 5 I). These cell experimental results showed that knocking out MUC1 could aggravate lung epithelial cell senescence, and the lung epithelial cell senescence was aggravated after CSE induction.

[0099] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A method of constructing a model of lung epithelial cell senescence in COPD, characterized by, The Muc1 gene knockout mice are exposed to tobacco smoke for 6-8 weeks, and / or the Muc1 knockout rats are exposed to tobacco smoke for 6-8 weeks, and / or the Muc1 type II alveolar epithelial cell knockout mice are exposed to tobacco smoke at the age of 6-8 weeks, and then the mice are injected with tamoxifen intraperitoneally, and the tobacco smoke exposure is performed one week later.

2. The method of claim 1, wherein, The injection dose of the tamoxifen is 75 mg / kg.

3. The method of claim 1, wherein, The injection time of the tamoxifen is 4 days.

4. The method of claim 1, wherein, The injection frequency of the tamoxifen is 2 days / time.

5. The method of claim 1, wherein, The modeling time of the large / small mouse COPD lung epithelial cell aging model is 3-8 months.

6. The use of the method of claim 1 in constructing a COPD lung epithelial cell aging model.

7. The use of the COPD lung epithelial cell aging model obtained by the construction method of claim 1 in COPD and its basic research.

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