A method for constructing a gas-liquid culture model of a multi-drug resistant pasteurella haemolytica invading an immortalized yak bronchial epithelial cell line

Abstract

The application discloses a kind of gas-liquid culture model construction methods of Pasteurella multocida infection immortal yak bronchial epithelial cell strain. It is related to the technical field of cell engineering. Including strain, model construction method and specific application. The application successfully constructs a yak bronchial epithelial cell monoclonal cell line using SV40T lentivirus, and the cell line has strong proliferation capacity and the ability to differentiate into mature bronchial epithelial cells. Then a bronchial epithelial cell gas-liquid culture model is established using a transwell polycarbonate membrane, and Pasteurella multocida is used for infection to establish an infection model, providing a good in vitro theoretical model for subsequent research on the pathogenesis of yak respiratory disease.

Description

Technical Field

[0001] This invention relates to the field of cell engineering technology, and more specifically to a method for constructing an air-liquid culture model of immortalized yak bronchial epithelial cell line infected with Pasteurella multocida. Background Technology

[0002] Yaks are one of the unique economic animals of the Qinghai-Tibet Plateau, living year-round in high-altitude, cold regions and are typical cold-resistant livestock. While the yak farming system is now quite sophisticated, the continued use of grazing methods makes it difficult to implement comprehensive vaccination programs, leading to the prevalence of respiratory diseases in yaks and significantly impacting the yak farming economy. Pasteurella multocida (Pm) causes many economically significant diseases in various livestock and wild animals, with high mortality and morbidity rates, and is closely related to the pathogenesis of respiratory diseases. Several studies have shown that the pathogenesis of Pasteurella multocida infection in its hosts is complex, but research on the mechanisms of Pasteurella multocida invasion and its interaction with the host remains limited.

[0003] Located at the interface between the animal body and the external environment, bronchial epithelial cells play a crucial role in maintaining respiratory homeostasis in yaks by secreting various mucus, regulating immunity, and engaging in ciliary movements to inhibit the invasion of pathogens entering the exhalation tract. Establishing a cell infection model based on the isolation and culture of yak bronchial epithelial cells can lay the foundation for studying the molecular mechanisms and interactions of pathogen infection in hosts. For research on the mechanisms of respiratory tract infection caused by pathogens entering the body through respiration, it is necessary to simulate the microenvironment of respiratory epithelial cells in vivo by exposing the cells to aerosols. Using exposure devices to expose the gas-liquid interface more closely resembles the state of respiratory epithelial cells exposed to compound aerosols in vivo; therefore, gas-liquid interface culture technology is an important method for culturing bronchial epithelial cells.

[0004] However, the limited tissue sources required for bronchial epithelial cell culture, low cell viability, slow proliferation rate, and lack of epithelial cell phenotypic differentiation ability severely restrict long-term and systematic experimental research. Researchers have used gene transfection and other technologies to introduce exogenous immortalization genes (hTERT, monkey kidney virus SV40, human herpesvirus, human papillomavirus, etc.) into cells such as porcine oral mucosal epithelial cells, canine bronchiolar epithelial cells, porcine ear, fetal and lung fibroblasts, and human corneal epithelial cells. These genes are integrated with target cell genes and promoted to express, enabling certain cells with poor passage performance, slow proliferation rate, and easy aging and death to proliferate indefinitely and achieve immortalization, providing valuable experimental materials for biological research.

[0005] Therefore, whether a method can be provided for constructing an air-liquid culture model of Pasteurella multocida infecting immortalized yak bronchial epithelial cell lines to overcome the above-mentioned technical deficiencies is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a method for constructing an air-liquid culture model of Pasteurella multocida infecting immortalized yak bronchial epithelial cell line.

[0007] Preservation Information: Yak bronchial epithelial cells SV40T-Yak BECs, deposited at China Center for Type Culture Collection, Wuhan University, Wuhan, China; deposit date: August 10, 2026; accession number: CCTCC NO: C2026159.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: An immortalized yak bronchial epithelial cell line, cell line: yak bronchial epithelial cells SV40T-YakBECs, preservation number: CCTCC NO: C 2026159.

[0009] This invention also provides a method for constructing an air-liquid culture model of Pasteurella multocida infecting immortalized yak bronchial epithelial cell lines, comprising the following steps: (1) Using lentiviruses containing the SV40T gene to infect yak bronchial epithelial cells, immortalized yak bronchial epithelial cell lines were obtained by screening with puromycin. (2) Immortalized yak bronchial epithelial cell lines were inoculated onto Transwell polycarbonate membranes and cultured. (3) Pasteurella multocida bacterial solution was inoculated into ALI culture medium B, mixed and precipitated, and then the bacterial solution was aspirated into the upper chamber of the Transwell polycarbonate membrane to establish an infection model.

[0010] Preferred: The optimal MOI for the lentivirus in step (1) is 100.

[0011] Preferred concentration: The concentration of puromycin used for screening in step (1) is 5 μg / mL.

[0012] Preferred: The culture in step (2) is as follows: after the cells have grown to 100% confluence, the lower chamber is replaced with ALI culture medium B, the upper chamber culture medium is removed, and the cells are cultured for more than 14 days until the transmembrane epithelial resistance value reaches its peak and remains stable.

[0013] Preferred concentration of Pasteurella multocida bacterial solution in step (3) is 1.0 × 10⁻⁶. 7 CFU / mL.

[0014] The present invention also provides the application of the above-described cell lines or models constructed by any of the above methods in the study of the pathogenic mechanisms of respiratory diseases in yaks.

[0015] The present invention also provides the application of the above-described cell lines or models constructed by any of the above methods in screening drugs against Pasteurella multocida or evaluating drug safety.

[0016] As can be seen from the above technical solution, compared with the prior art, this invention discloses a method for constructing an air-fluid culture model of immortalized yak bronchial epithelial cell line infected with Pasteurella multocida. The technical effects achieved are as follows: This invention successfully constructed a monoclonal cell line of yak bronchial epithelial cells using SV40T lentivirus, and this cell line has strong proliferative capacity and the ability to differentiate into mature bronchial epithelial cells. Subsequently, an air-fluid culture model of bronchial epithelial cells was established using Transwell polycarbonate membrane, and an infection model was established using Pasteurella multocida, providing a good in vitro theoretical model for subsequent research on the pathogenic mechanism of yak respiratory diseases. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 The attached figure shows images of lentivirus-transfected cells observed under optical and fluorescence microscopes provided by this invention.

[0019] Figure 2 The attached figure shows the bronchial epithelial cells of yaks provided by this invention. SV40T Gene expression level map.

[0020] Figure 3 The attached figure shows the SV40T-Yak BECs provided by this invention passed down to generation 40.

[0021] Figure 4 The attached figure is a diagram of keratin identification in yak bronchial epithelial cells provided by the present invention.

[0022] Figure 5 The attached figure is a growth curve of yak bronchial epithelial cells provided by the present invention.

[0023] Figure 6 The attached figure is a chromosome distribution diagram of yak bronchial epithelial cells provided by the present invention.

[0024] Figure 7The attached figure is a cell cycle diagram of yak bronchial epithelial cells provided by the present invention.

[0025] Figure 8 The attached figure shows apoptosis of yak bronchial epithelial cells provided by the present invention.

[0026] Figure 9 The attached figure is a verification diagram of the barrier function and membrane integrity of yak bronchial epithelial cells provided by the present invention.

[0027] Figure 10 The attached figure shows a 1×10 ohmmeter provided by this invention. 7 Figure showing the morphological changes of bronchial epithelial cells at different time points after infection with CFU / mL concentration Pm. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] This invention discloses a method for constructing an air-liquid culture model of immortalized yak bronchial epithelial cells infected with Pasteurella multocida. In this embodiment, raw materials, reagents, and instruments not explicitly mentioned are all commonly used in the art and commercially available; no brand restrictions are imposed. Operational steps not explicitly mentioned are routine processing steps and will not be described in detail here. Example 1

[0030] (1) Screening of primary bronchial epithelial cells from yaks Tracheal tissue from healthy, normally breathing yaks was collected up to the hilum. The tissue was rinsed with 75% alcohol, then rinsed twice with DPBS buffer (Shanghai Xiaopeng, China). In a laminar flow hood, the lung tissue was dissected from the tracheal fascia using a sterile scalpel. After rinsing thoroughly with DPBS containing antibiotics (Gbico, USA), the ear tissue was cut into pieces approximately 1 mm in size using sterile ophthalmic scissors. 3The tissue fragments were carefully cut and evenly transferred to the mouth of a T25 cell culture flask using sterile ophthalmic forceps. The tissue fragments were then spread evenly across the bottom of the flask using a pipette. 2 mL of fetal bovine serum (Shanghai Xiaopeng, China) was added, and the flask was incubated upside down in a 37 ℃, 5% CO2 incubator. After 6 h, the flask was gently inverted to allow the fetal bovine serum to infiltrate the tissue culture. After 24 hours, the medium was replaced with 2 mL of DMEM-F12 complete culture medium containing 15% fetal bovine serum (Shanghai Xiaopeng, China) (50 mL system: 7.5 mL fetal bovine serum + 500 μL penicillin antibody + 0.06 g NaHCO3 (Sigma, USA) + 125 μg insulin-transferrin-selenium supplement (Wuhan Pronosai, China) + 500 ngL-thyroxine (Dalian Meilun, China) + DMEM-F12 to make up to 50 mL). The culture medium was replaced every 2 days. The cells were cultured until passaged and cryopreserved to obtain yak bronchial epithelial cells, referred to as primary yak bronchial epithelial cells (Primary BECs).

[0031] (2) Optimal screening concentration of puromycin Puromycin (Solepro, China) was serially diluted with complete culture medium in the range of 1–10 μg / mL to prepare screening culture medium. Primary BECs were digested with 0.25% trypsin digestion solution (Gbico, USA) to prepare cell suspension, and then diluted at a concentration of 1 × 10⁻⁶. 4 Cells were seeded per well into 24-well plates. When approximately 40% of the cells adhered, they were screened using different gradients of puromycin in culture medium and incubated at 37°C in a 5% CO2 incubator. Cell growth was observed, and the screening medium was changed every 3 days. Later, based on the number of dead cells, the screening medium was changed every 1 day. The minimum puromycin concentration that killed all cells on day 7 was the optimal screening concentration.

[0032] The results showed that after screening with different concentrations of puromycin for 7 days, the lowest concentration that resulted in the complete death of yak bronchial epithelial cells was 5 μg / mL, and this concentration was selected as the optimal screening concentration of puromycin.

[0033] (3) Determination of lentivirus titer and optimal MOI value HEK 293T cells (Fenghui, China) were prepared into a cell suspension and diluted at a concentration of 1×10⁻⁶. 4 Seeds were inoculated per well into 96-well plates and incubated in a cell culture incubator. The packaged SV40T lentivirus solution (Hunan Fenghui, China) was set up with 5 titer gradients: 1×10⁻⁶. 8 TU / mL, 2×10 8 TU / mL, 5×10 8 TU / mL, 8×10 8 TU / mL, 1×109 TU / mL was added dropwise to HEK293T cells. After 48 h, for wells requiring puromycin for selection, 100 μL of virus-containing culture medium was aspirated, and 100 μL of DMEM-F12 complete culture medium containing 1.5 μg / mL puromycin and 15% fetal bovine serum was added. The culture medium was changed every 2 days, and the results were observed under a fluorescence microscope on day 5 to calculate the lentivirus titer.

[0034] The original BECs were divided into 1×10 4 Cells were seeded per well into 96-well plates and cultured overnight. Six MOI values ​​were set: MOI=0, 12.5, 25, 50, 100, and 200. The required virus volume for each MOI value was calculated based on the formula: (MOI × number of infected cells) / virus titer × 1000. Each group was replicated in triplicate. After 24 h of culture, the medium was replaced with DMEM-F12 complete medium containing 15% fetal bovine serum (FBS) and 8 μg of polybrene, corresponding to the required virus volume for each MOI value. For the MOI=0 group, 100 μL of DMEM-F12 complete medium containing 15% FBS was added. Twelve hours after infection, the medium was replaced with 100 μL of DMEM-F12 complete culture medium containing 15% fetal bovine serum, and cell morphology was observed under a fluorescence inverted microscope. The culture medium was changed every two days. Seventy-two hours after infection, the abundance of fluorescence expression was observed. The infection efficiency was about 50%. The infection conditions and MOI value corresponding to the group with good cell growth were the optimal MOI value for SV40T lentivirus.

[0035] like Figure 1 (A: HEK 293T cells under an optical microscope; B: HEK 293T cells under a fluorescence microscope; C: Fluorescence image of yak bronchial epithelial cells at MOI=100) As shown, cell counting revealed an average copy number of 2.0 × 10⁻⁶ cells after lentivirus infection. 10 Based on the lentivirus titer formula, the viral titer is calculated to be 2 × 10⁻⁶ copies / μL. 8 The TU / mL indicates that a large amount of SV40T lentivirus has been transfected into HEK 293T cells. Infection of primary BECs with SV40T lentivirus revealed that cell fluorescence efficiency gradually increased with increasing MOI value, with the highest fluorescence abundance observed at an MOI of 100 at 72 h. Therefore, MOI=100 was selected as the optimal MOI value for lentivirus.

[0036] (4) Transfect SV40T lentivirus into yak bronchial epithelial cells and screen for positive monoclonal cell lines. When the obtained primary BECs with good growth status reached 80%, the optimal MOI value (100) SV40T lentivirus solution was diluted 1:1 using DMEM-F12 and added dropwise to the cells to be transfected. After gentle mixing, DMEM-F12 culture medium containing 8 μg polybrene was added, and the cells were placed in a 37 ℃, 5% CO2 incubator for 6 h for transfection. The culture medium was then replaced with DMEM-F12 complete culture medium containing 15% fetal bovine serum. When transfected cells reached 80% confluency, they were passaged and seeded into new 6-well plates, with transfection wells and negative control wells. When the cell adhesion rate reached 20%, the optimal purine (5 μg / mL) selection medium was added to each well. The cell status was observed daily, and the selection medium was replaced with the same concentration every 2 days. When all cells in the negative control wells died, the concentration of purine was halved (2.5 μg / mL), and the cells were cultured in a 37 ℃, 5% CO2 incubator. The selection medium was replaced every 2 days until positive clones appeared (named SV40T-Yak BECs), which were then preserved using the patented procedure.

[0037] Positive clones were digested with 0.25% trypsin digestion solution, diluted using the limiting dilution method, and plated into 96-well plates for single-cell passage and expansion culture. The cells were then cryopreserved and continuously passaged.

[0038] (5) Identification of bronchial epithelial cells of immortalized yak Total RNA was extracted from primary BECs and SV40T-Yak BECs using the Trizol method. Using the extracted total RNA as a template, a reverse transcription system was prepared according to the HiScript II Q RT SuperMix reverse transcription kit (Shanghai Sangon Biotech, China) on an ice-water bath. The reaction was carried out at 50 °C for 15 min, followed by a reaction at 85 °C for 5 s. The reverse transcription product was stored at -20 °C for later use. Using the reverse transcription product as a template, bronchial epithelial cells were designed. SV40T Gene primer sequences (Table 1) and β-actin Internal reference gene sequence SV40T Gene RT-PCR reaction. RT-PCR reaction system: 1 μL each of upstream and downstream primers, 2 μL cDNA template, ArtiCan... ATM SYBR qPCRMix 12.5 μL, ddH2O 3.5 μL. RT-PCR reaction conditions: 95 ℃ pre-denaturation for 20 s, 95 ℃ denaturation for 10 s, 60 ℃ annealing for 20 s, for a total of 40 cycles; melting curve temperature 65-95 ℃; using 2 -∆∆CT The expression differences of the target gene between the control group and the assay group were analyzed.

[0039] Table 1

[0040] like Figure 2 , Figure 3 As shown, in immortalized SV40T-Yak BECs SV40T Gene expression levels were significantly higher than those of primary BECs ( P <0.01), indicating SV40T The gene can be stably expressed in immortalized bronchial epithelial cells, and after passage, SV40T-Yak BECs can be cultured for more than 40 generations.

[0041] (6) Analysis of the growth characteristics of bronchial epithelial cells in yaks ① Identification of keratin in yak bronchial epithelial cells When the primary BECs and SV40T-Yak BECs reached a density of 80-90%, the cell suspension was diluted with 1×10⁻⁶ cells / mL. 6 Cells / mL were slowly added dropwise to a 6-well culture plate containing a coverslip and incubated in a CO2 incubator. Once the cells on the coverslip were confluent, 4% paraformaldehyde solution (Biosharp, China) was added for fixation at room temperature for 15 min. Cell smears were then picked up with sterile scissors and placed in a new culture dish. A 1:400 diluted mouse anti-human keratin 18 monoclonal antibody (Bio-Sen, China) was added and incubated at 4 ℃ for 24 h. Then, a 1:500 diluted goat anti-mouse FITC IgG (Bio-Sen, China) was added and incubated at 37 ℃ in the dark for 30 min. The bronchial epithelial cell status was observed under a fluorescence inverted microscope.

[0042] like Figure 4 (A: Keratin identification of primary BECs; B: Keratin identification of SV40T-Yak BECs) As shown, both primary BECs and SV40T-Yak BECs showed green fluorescence on their cell membranes, but no fluorescence was observed in the cell nuclei, proving that the immortalized positive monoclonal cells obtained were yak bronchial epithelial cells.

[0043] ② Growth curve of yak bronchial epithelial cells When the primary generation BECs and SV40T-Yak BECs reach a density of 80-90%, they are grown at a rate of 1×10⁻⁶. 3 3 cells / mL were passaged and seeded into 96-well plates. Every 1 hour, 10 μL of CCK8 reagent (Solepro, China) was added to 3 wells. The cells were gently mixed and incubated at 37°C with 5% CO2 for 2 hours. The absorbance was read at 450 nm using a multi-mode microplate reader. The growth curve of bronchial epithelial cells was plotted using Graphpad Prism 8 software.

[0044] like Figure 5As shown, the growth curves of both primary BECs and SV40T-Yak BECs are typical "S"-shaped, with a rapid increase starting from day 2 and continuing until day 5, indicating that the cells are in good growth condition and proliferate rapidly within 2-5 days. From day 6 onwards, the cells enter a plateau phase with slow proliferation, indicating that SV40T-Yak BECs have similar growth capabilities to newly isolated primary BECs.

[0045] ③ Chromosomal karyotype analysis of yak bronchial epithelial cells When the primary BECs and P30 generation SV40T-Yak BECs reached a growth density of 70-80% (logarithmic growth phase), they were incubated in culture medium containing 0.1 µg / mL colchicine (source leaf, China) at 37°C for 2 h. Then, they were treated with 0.075 M KCl solution (Sigma, Shanghai, China) in a 37°C water bath for 30 min to achieve hypotonic treatment. After the hypotonic treatment, the cells were fixed with methanol (Thermo Fisher Scientific, USA)-glacial acetic acid (Thermo Fisher Scientific, USA) solution (3:1). This was repeated 3 times. Fresh fixative was added, and 100 µL of cell suspension was dropped onto a pre-cooled glass slide at -20°C. The slides were stained with Giemsa staining solution (Solepro, China) for 15 min. After rinsing with running water, the cells were observed.

[0046] like Figure 6 (A: Chromosomes of primary BECs; B: Chromosomes of P30 generation SV40T-Yak BECs) As shown, the primary cells and P30 generation SV40T-Yak BECs mostly have 60 chromosomes, and their karyotypes are consistent with the yak chromosome atlas, indicating that the chromosomes of immortalized cells have not undergone significant changes and can maintain genetic integrity during long-term passage.

[0047] ④ Detection of bronchial epithelial cell cycle in yaks Once the primary BECs and SV40T-Yak BECs reached a density of 80-90%, they were digested and then diluted to 1×10⁶ cells using DMEM-F12 complete culture medium containing 15% fetal bovine serum. 6Cells per mL were collected. 1 mL of cell suspension was transferred to a 2 mL sterile centrifuge tube and centrifuged at 1500 r / min for 5 min. The supernatant was discarded, and the cells were washed with pre-chilled DPBS. The above steps were repeated once. The precipitate was mixed with a small amount of residual DPBS, and pre-chilled 70% ethanol at -20 °C was slowly added while shaking. The mixture was then fixed at -20 °C. After fixation, the cells were centrifuged at 1500 r / min for 5 min. The supernatant was discarded, and the cells were washed twice with DPBS. 500 μL of PBS containing 50 μg / mL PI and 100 μg / mL RNase A was added. The cells were incubated at 4 °C in the dark for 20 min. The cell cycle was then detected by flow cytometry, and a cell cycle diagram was plotted using MFLT32 software.

[0048] like Figure 7 (A: Primary cell cycle; B: SV40T-Yak BECs cell cycle; C: Percentage of different cell cycle stages in primary and immortalized cells; ** indicates highly significant differences) P <0.01), * indicates a significant difference ( P As shown in the figure (<0.05), the proportions of primary bronchial epithelial cells in G1, S, and G2 phases of primary BECs were 53.09±0.16%, 15.16±0.14%, and 31.75±0.16%, respectively, while those of SV40T-Yak BECs were 23.32±0.11%, 50.31±0.14%, and 26.37±0.15%, respectively. There were highly significant differences between primary BECs and SV40T-Yak BECs in both the G1 and S cell cycle stages. P <0.01, with significant differences in the G2 cell cycle stage ( P <0.05).

[0049] ⑤ Detection of apoptosis in yak bronchial epithelial cells After digesting the cells according to the above steps, resuspend the cells in 1× Binding Buffer; add 5 μL Annexin V-FITC (Elec, China) and 10 μL PI (Elec, China) to each tube, gently vortex to mix, and incubate at room temperature in the dark for 20 min. Within 1 h, use flow cytometry to detect apoptosis in Annexin V-FITC (Ex=488 nm; Em=530 nm) and PI (Ex=535 nm; Em=615 nm), and use FlowJo v10.8.1 software to analyze and draw apoptosis detection maps and cell percentage tables.

[0050] like Figure 8(A: Primary cell apoptosis; B: SV40T-Yak BECs cell apoptosis; C: Apoptosis rate of primary and immortalized cells) As shown, the apoptosis rate of primary BECs (29.93±0.13%) was significantly higher than that of SV40T-Yak BECs (15.51±0.12%). P <0.01), combined with the above cell growth viability test results, indicates that immortalized cells SV40T-Yak BECs are more conducive to cell proliferation.

[0051] (7) Establishment of a gas-fluid model of Pasteurella multocida infecting yak bronchial epithelial cells ①Culturing yak bronchial epithelial cells at the air-liquid interface Add 500 μL of extension-phase culture medium to the lower chamber of a 24-well culture plate. Using sterile forceps, place the Transwell polycarbonate membrane (embedded sleeve) (Corning, USA) into the culture plate. Subculture the aforementioned primary BECs and SV40T-Yak BECs onto the Transwell polycarbonate membrane using extension-phase culture medium (50 mL system: 1 mL PneumaCult-Ex 50×Supplement (Stemcell, Canada) + 48.45 mL PneumaCult-Ex Basal Medium (Stemcell, Canada) + 50 μL hydrocortisone (Shanghai Jizhi, China) + 500 μL double antibiotics). Incubate at 37 ℃ in a 5% CO2 incubator. Every 24 h, the upper and lower chambers of the Transwell polycarbonate membrane are simultaneously replaced with the expansion phase culture medium. When the cells grow to 100%, the lower chamber culture medium is replaced with 500 μL of ALI culture phase B culture medium (50 mL of solution A: 5 mL PneumaCult-ALI 10×Supplement (Stemcell, Canada) + 44.5 mL PneumaCult-ALI Basal Medium (Stemcell, Canada) + 500 μL of penicillin-dextrose antibody; 40 mL of solution B: 39.32 mL of solution A + 400 μL PneumaCult-ALI MaintenanceSupplement (Stemcell, Canada) + 80 μL of 0.2% heparin solution (Solepro, China) + 200 μL of hydrocortisone). The upper chamber culture medium is then removed to establish an ALI interface culture model. The cells are cultured in a 37 ℃, 5% CO2 incubator, with the lower chamber culture medium replaced every 24 h.

[0052] ② Measurement of transmembrane epithelial resistance When the cells on the Transwell polycarbonate membrane reached 80% confluence during the culture period, the original culture medium was discarded. Preheated DPBS (37 °C) was added to the bottom and top sides of the Transwell polycarbonate membrane, and the membrane was equilibrated in a 37 °C, 5% CO2 incubator for 20 min. At the same time, the fixed electrode of the ERS-2 cell resistance meter was placed in the preheated DPBS (37 °C) for equilibration for 20 min, and then preheated DPBS was added again. The fixed electrode was inserted into the Transwell polycarbonate membrane using sterile forceps, and the resistance measurement result was collected at the first point. When the value remained stable for 3 seconds, the transmembrane epithelial resistance value was recorded. The above steps were repeated with one blank carrier to obtain a blank control value. The transmembrane epithelial resistance of the ALI gas-liquid model was calculated according to TEER = (measured resistance value - blank value) × monolayer surface area (Q·cm²).

[0053] like Figure 9 (A. Transmembrane resistance of the bronchial epithelial cell barrier in yak; B. Comparison of fluorescence intensity between primary BECs and SV40T-Yak BECs at different concentrations of fluorescein hydrazine; C. Lactate dehydrogenase release from primary BECs and SV40T-Yak BECs at the same culture time; ** indicates extremely significant difference) P <0.01), * indicates a significant difference ( P As shown in A in <0.05), both primary BECs and SV40T-Yak BECs had low transmembrane epithelial resistance immediately after inoculation, close to the resistance of the blank membrane. From day 3 to 7, the cells gradually proliferated and formed a monolayer, and the TEER value began to rise, indicating that the barrier function was gradually established. Differentiation began after day 7. The TEER value of SV40T-Yak BECs gradually increased during the differentiation culture stage, while the TEER value of primary BECs showed a decreasing trend after day 8, and rose to form a stable monolayer after day 10. The TEER value reached its peak after 14 days of ALI culture. At this stage, cell differentiation and growth were vigorous, and the monolayer cells fused. After that, the TEER value was basically stable, indicating that both types of cells can form a complete barrier function under ALI cell differentiation culture.

[0054] ③ Diffusivity detection LY working solution (100 μg / mL) of fluorescein carbazide (Thermo Fisher, USA) was prepared using DPBS containing calcium and magnesium ions. When the cells reached 80% confluence, the original culture medium was discarded, and 0.5 mL of LY working solution was added to the top side of the Transwell polycarbonate membrane, and 1 mL of DPBS was added to the bottom side. The membrane was incubated at 37 °C in a 5% CO2 incubator for 1 h to allow cell diffusion. After incubation, the culture medium on the bottom side of the Transwell polycarbonate membrane, along with the standard working solution and blank control, was read at 485 nm and 535 nm absorbance using a multi-mode microplate reader to obtain a moderate PMT gain.

[0055] like Figure 9 As shown in Figure B, the fluorescence intensity of both primary BECs and SV40T-Yak BECs generally decreased with decreasing fluorescein hydrazine concentration, indicating that fluorescence intensity is correlated with fluorescein hydrazine concentration. Significant differences in fluorescence intensity were observed between the two cell types at concentrations of 3.125 and 25 μg / mL. P <0.05); at a concentration of 50 μg / mL, there was a highly significant difference between the two cell types ( P The difference was <0.01, indicating that the two cell types had different diffusion abilities to fluorescein hydrazine, reflecting differences in the integrity and permeability of their cell membranes.

[0056] ④ Cytotoxicity test Lactate dehydrogenase (LDH) release in basal-side cell culture medium from Transwell polycarbonate membranes was measured according to the manufacturer's instructions (Solepro, China). Basal-side cells were collected after the addition of fluorescein hydrazine, digested with 0.25% trypsin, and then 1 mL of extraction buffer was added. Cells were sonicated, centrifuged at 8500 r / min for 10 min at 4 ℃, and 50 μL of supernatant was collected. The detection reagent was added sequentially, and the absorbance was measured at 450 nm. LDH (U / 10) was then measured. 4 cell) = [sample pyruvate content × V] 样本 ×10 3 ] / [(500 / V 提取液 ×V 样本 LDH activity units were calculated using the reaction time. All measurements were corrected for the background of the cell culture medium and normalized to the maximum lactate dehydrogenase release after control cell lysis.

[0057] like Figure 9 As shown in Figure C, the LDH release amounts of primary BECs and SV40T-Yak BECs are similar, indicating that under ALI gas-liquid interface culture conditions, the two types of bronchial epithelial cells exhibit similar LDH diffusion with little difference.

[0058] ⑤ A gas-fluid model of Pasteurella multocida infecting yak bronchial epithelial cells When the SV40T-Yak BECs cells reached 80-90% confluence, the Pasteurella multocida bacterial suspension was diluted to 1.0 × 10⁻⁶. 7 The bacterial culture was centrifuged at 3000 r / min for 5 min using a low-speed centrifuge at a concentration of CFU / mL. The supernatant was discarded, and 100 μL of LALI culture medium in culture period B was added and mixed to form a precipitate. The bacterial culture was then aspirated into the upper chamber of the Transwell polycarbonate membrane to establish an infection model.

[0059] like Figure 10As shown, SV40T-Yak BECs showed no significant changes after 2 h of infection. After 4 h of infection, the cells adhered well, had normal morphology, and the culture medium was clear. After 6 h of infection, a small number of cells died, but overall, the cells still adhered well. After 16 h of infection, the cells became rounded and detached, and a large number of cells aggregated and the mortality rate continued to increase.

[0060] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0061] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An immortalized yak bronchial epithelial cell line, characterized in that, The cell line in question is SV40T-Yak BECs, with accession number CCTCC NO: C 2026159.

2. A method for constructing an air-liquid culture model of *Pasteurella multocida* infecting immortalized yak bronchial epithelial cell lines, characterized in that, Includes the following steps: (1) Using lentiviruses containing the SV40T gene to infect yak bronchial epithelial cells, immortalized yak bronchial epithelial cell lines were obtained by screening with puromycin. (2) The immortalized yak bronchial epithelial cell line was inoculated onto a Transwell polycarbonate membrane and cultured. (3) Pasteurella multocida bacterial solution was inoculated into ALI culture medium B, mixed and precipitated, and then the bacterial solution was aspirated into the upper chamber of the Transwell polycarbonate membrane to establish an infection model.

3. The method as described in claim 2, characterized in that, In step (1), the optimal MOI for lentivirus is 100.

4. The method as described in claim 3, characterized in that, The concentration of puromycin used for screening in step (1) is 5 μg / mL.

5. The method as described in claim 4, characterized in that, The specific culture described in step (2) is as follows: after the cells have grown to 100% confluence, the lower chamber is replaced with ALI culture medium B, the upper chamber culture medium is removed, and the cells are cultured for more than 14 days until the transmembrane epithelial resistance value reaches its peak and remains stable.

6. The method as described in claim 5, characterized in that, The concentration of the Pasteurella multocida bacterial solution in step (3) is 1.0 × 10⁻⁶. 7 CFU / mL.

7. The application of the cell line of claim 1 or the model constructed by any of the methods described in claims 2-6 in the study of the pathogenic mechanism of respiratory diseases in yaks.

8. The use of the cell line of claim 1 or the model constructed by any of the methods described in claims 2-6 in screening drugs against Pasteurella multocida or evaluating drug safety.

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