A method for constructing immortal yak bronchial epithelial cell line hTERT-Yak BECs

Abstract

The application discloses a method for constructing an immortal yak bronchial epithelial cell strain hTERT-Yak BECs, and relates to the technical field of microorganisms. The application provides specific cell strains, a preparation method and application. The application successfully constructs a yak bronchial epithelial cell monoclonal cell line by using a pCI-neo-hTERT plasmid, and the cell line has strong proliferation ability and the ability of differentiating into mature bronchial epithelial cells.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to a method for constructing an immortalized yak bronchial epithelial cell line hTERT-Yak BECs. 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. The upper respiratory tract of yaks, composed of the trachea and bronchi, is the primary site for pathogens and harmful substances to reside. Under normal circumstances, the tracheal mucosa is the body's first line of defense against harmful external substances. Bronchial epithelial cells, located at the interface between the animal's body and the external environment, play a crucial role in maintaining respiratory homeostasis in yaks by secreting various mucus, regulating immunity, and using ciliary movements to inhibit the invasion of pathogens entering the exhalation tract. Establishing a cell model based on the isolation and culture of yak bronchial epithelial cells can lay the foundation for studying the molecular mechanisms and interactions of pathogenic microorganisms infecting the host.

[0003] Bronchial epithelial cell culture suffers from limited tissue sources, low cell viability, slow proliferation, lack of epithelial cell phenotypic differentiation, and limited passage times, making it difficult to meet the needs of most in vitro experimental studies. Therefore, constructing stable passaged yak bronchial epithelial cell lines using immortalization technology is of great significance for in vitro studies of the adhesion and infection mechanisms between bronchial epithelial cells of Pasteurella multocida. Cell immortalization refers to the process by which cultured cells escape proliferative senescence crises spontaneously or under the influence of external factors, thereby acquiring unlimited proliferative capacity. Under normal circumstances, cells undergo aging and death after a limited number of passages in vitro, which limits cell culture and subsequent experiments. Researchers use techniques such as gene transfection to introduce exogenous immortalization genes into target cells, integrating them with target cell genes and promoting their expression, thus constructing immortalized cell lines.

[0004] Currently, methods for establishing cell immortalization mainly include human telomerase catalytic subunit (hTERT)-mediated immortalization, viral gene transfection (such as simian kidney virus SV40, human herpesvirus, and human papillomavirus), hybridoma technology, and genetic modification. These methods enable the unlimited proliferation and immortalization of certain cells with poor passage performance, slow proliferation rates, and high senescence rates, providing valuable experimental materials for biological research. Compared with other methods, immortalized cells cultured through hTERT-mediated immortalization have significant advantages, not only preserving the original morphological characteristics and biological activity of the cells and improving the efficiency of cell immortalization, but also reducing the risk of malignant transformation. Studies have shown that the ribonuclease telomerase compensates for telomere lengthening by synthesizing new telomere repeat sequences at the 3' end of chromosomes. It consists of the human telomerase reverse transcriptase catalytic subunit (hTERT), telomerase RNA (hTR) components, two groups of H / ACA proteins (dyskerin, GAR1, NHP2, and NOP10), and telomerase Cajal body protein 1 (TCAB1). Normal cells can only continuously express the telomerase RNA components, but by introducing a vector containing the exogenous hTERT gene into cells via liposome transfection, telomerase activity is activated, allowing cells to proliferate in a relatively unlimited manner without entering the senescence stage and without altering their biological characteristics, thus achieving cell immortalization. Currently, the hTERT gene has been successfully introduced into the genomes of various cells to establish immortalized cell lines; however, there are no reports of successfully constructing immortalized bronchial epithelial cells in yaks. Therefore, this invention utilizes the pCI-neo-hTERT plasmid to construct immortalized bronchial epithelial cells of yaks, providing valuable experimental materials for the study of respiratory diseases in yaks. Summary of the Invention

[0005] In view of this, the present invention provides a method for constructing an immortalized yak bronchial epithelial cell line, hTERT-Yak BECs, focusing on establishing an immortalized cell line from respiratory cells of the yak, a unique economic animal of the Qinghai-Tibet Plateau, thus filling the challenge of primary cell isolation and culture in high-altitude areas. It provides a good cell model for in vitro research on yak respiratory diseases.

[0006] Preservation Information: hTERT-Yak BECs, deposited at China Center for Type Culture Collection, Wuhan University, Wuhan, China; deposit date: August 10, 2026; accession number: CCTCC NO: C2026145.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: An immortalized yak bronchial epithelial cell line, the cell line being hTERT-YakBECs, with accession number CCTCC NO: C 2026145.

[0008] The present invention also provides a method for preparing the cell line as described above, comprising the following steps: (1) Screening of primary bronchial epithelial cells from yaks; (2) Transfect plasmids containing the human telomerase reverse transcriptase hTERT gene into primary bronchial epithelial cells; (3) Use screening medium to screen the transfected cells to obtain positive clones, which are the cell lines.

[0009] Preferred: The screening medium in step (3) is a medium containing genimycin sulfate; the screening concentration is 800 μg / mL.

[0010] The present invention also provides the application of the above-mentioned cell lines in constructing an in vitro research model of respiratory diseases in yaks.

[0011] Preferred: Yak respiratory diseases are caused by Pasteurella multocida infection.

[0012] The present invention also provides the application of the above-mentioned cell lines in screening or evaluating drugs for treating respiratory diseases in yaks.

[0013] The present invention also provides the application of the above-mentioned cell lines in yak farming.

[0014] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method for constructing an immortalized yak bronchial epithelial cell line hTERT-Yak BECs. The technical effect achieved is that the present invention successfully constructed a yak bronchial epithelial cell monoclonal cell line using the pCI-neo-hTERT plasmid, and the cell line has strong proliferation ability and the ability to differentiate into mature bronchial epithelial cells. Attached Figure Description

[0015] 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.

[0016] Figure 1 The attached figure shows the bronchial epithelial cells of yaks provided by this invention. hTERT (i.e., HTERT) gene expression level map.

[0017] Figure 2 The attached figure shows the hTERT-Yak BECs provided by this invention up to generation 40.

[0018] Figure 3 The attached figure is a diagram of keratin identification in yak bronchial epithelial cells provided by the present invention.

[0019] Figure 4 The attached figure is a growth curve of yak bronchial epithelial cells provided by the present invention.

[0020] Figure 5 The attached figure is a chromosome distribution diagram of yak bronchial epithelial cells provided by the present invention. Figure 6 The attached figure is a cell cycle diagram of yak bronchial epithelial cells provided by the present invention.

[0021] Figure 7 The attached figure shows apoptosis of yak bronchial epithelial cells provided by the present invention. Detailed Implementation

[0022] 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.

[0023] This invention discloses a method for constructing the immortalized yak bronchial epithelial cell line hTERT-Yak BECs. In this embodiment, raw materials, reagents, and instruments not explicitly mentioned are all commonly used in the art and commercially available; no brand is specified. Operational steps not explicitly mentioned are routine processing steps and will not be described in detail here. Example 1

[0024] (1) Screening of primary bronchial epithelial cells from yaks Tracheal tissue from healthy, normally breathing yaks, up to the hilum, was collected. 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 blade. After rinsing thoroughly with DPBS containing antibiotics (Gbico, USA), the tissue was cut into pieces approximately 1 mm in size using sterile ophthalmic scissors. 3Tissue blocks were cut and evenly transferred to the mouth of a T25 cell culture flask using sterile ophthalmic forceps. The tissue blocks 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 inverted for culture. After 6 h, the flask was gently inverted to allow the fetal bovine serum to infiltrate the tissue culture. After 24 h, the culture medium was replaced with 2 mL of DMEM-F12 complete medium (Shanghai Xiaopeng, China) containing 10% fetal bovine serum (50 mL system: 7.5 mL fetal bovine serum + 500 μL double antibiotic + 0.06 g NaHCO3 (Sigma, USA) + 125 μg insulin-transferrin-selenium supplement (Wuhan Pronosai, China) + 500 ng L-thyroxine (Dalian Meilun, China) + DMEM-F12 to bring the total to 50 mL). The culture medium was changed 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).

[0025] (2) Optimal screening concentration of genimycin sulfate (G-418) G-418 was serially diluted 12-fold using DMEM-F12 to prepare a selection culture medium. Primary BECs were digested with 0.25% trypsin digestion solution (Gbico, USA) to prepare a cell suspension, and then diluted at a concentration of 1×10⁻⁶ cells / mL. 4 Cells were seeded per well in 24-well plates. When 40% of the cells adhered, the medium was replaced with G-418 selection medium at different gradients, with each gradient repeated three times. The plates were then incubated at 37 ℃ in a 5% CO2 incubator. Cell growth was observed, and the selection medium was replaced every 3 days. Later, based on the number of dead cells, the medium was replaced every 1 day. The minimum G-418 concentration that killed all cells on day 14 was the optimal selection concentration.

[0026] The results showed that after screening with different concentrations of G418 for 14 days, the lowest concentration that resulted in the complete death of yak bronchial epithelial cells was 800 μg / mL. This concentration was selected as the optimal screening concentration of G418, i.e., the concentration of genimycin sulfate screening culture medium was 800 μg / mL.

[0027] (3) pCI-neo-hTERT plasmid transfection and positive cell screening Following the instructions of the Lipofectamine 2000 transfection kit (Thermo Fisher, USA), pCI-neo-hTERT plasmid (Hunan Fenghui, China) was transfected into primary BECs. After 6 h of transfection in a 37 ℃, 5% CO2 incubator, the culture medium was replaced with DMEM-F12 containing 10% fetal bovine serum. When the transfected cells reached 80% confluency, they were passaged and seeded into new 6-well plates. Once the cell adhesion rate reached 20%, the medium was replaced with G-418 (Solepro, China) selection medium. Cell growth was observed daily, and the medium was replaced with the same concentration every 2 days. After all cells in the negative control wells died, the G418 concentration was halved (400 μg / mL), and the cells were incubated at 37 ℃, 5% CO2, with the selection medium replaced every 2 days until positive clones appeared. Immortalized hTERT-YakBECs were obtained and preserved using patent procedures.

[0028] 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.

[0029] (4) Identification of bronchial epithelial cells of immortalized yak Total RNA was extracted from primary BECs and hTERT-Yak BECs using the Trizol method. Using the extracted total RNA as a template, a reverse transcription system was prepared according to the instructions of 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 an incubation at 85 °C for 5 s. The reverse transcription products were stored at -20 °C for later use. Using the reverse transcription products as a template, bronchial epithelial cells were designed. hTERT Gene primer sequences and β-actin Internal reference gene sequence hTERT Gene RT-PCR reaction (Table 1). RT-PCR reaction system: 1 μL each of upstream and downstream primers, 2 μL cDNA template, ArtiCan... ATM SYBR qPCR Mix 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.

[0030] Table 1

[0031] like Figure 1 , Figure 2As shown, in immortalized hTERT-Yak BECs hTERT Gene expression levels were significantly higher than those of primary BECs ( P <0.01), indicating hTERT The gene can be stably expressed in immortalized bronchial epithelial cells, and after passage, hTERT-Yak BECs can be cultured for more than 40 generations.

[0032] (5) 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 hTERT-Yak BECs reach a density of 80-90%, the cell suspension is 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°C for 24 h. Then, a 1:500 diluted goat anti-mouse FITC IgG (Bio-Sen, China) was added and incubated at 37°C in the dark for 30 min. The bronchial epithelial cell status was observed under a fluorescence inverted microscope.

[0033] like Figure 3 As shown in the figure (A: keratin identification of primary BECs; B: keratin identification of hTERT-Yak BECs), both primary BECs and hTERT-Yak BECs showed green fluorescence on their cell membranes, while no fluorescence was observed in the cell nuclei, proving that the immortalized positive monoclonal cells obtained were yak bronchial epithelial cells.

[0034] ② Growth curve of yak bronchial epithelial cells When the primary generation of BECs and hTERT-Yak BECs reach a density of 80-90%, they are grown at a rate of 1×10⁻⁶. 3 Cells / mL were passaged and seeded into 96-well plates. Every 1 hour, 3 wells were selected and 10 μL of CCK8 reagent (Solepro, China) was added. 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-functional microplate reader (FLX800, BioTek). The growth curve of bronchial epithelial cells was plotted using Graphpad Prism 8 software.

[0035] like Figure 4As shown, the growth curves of both primary BECs and hTERT-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 immortalized cell lines have similar growth capabilities to newly isolated primary cell lines.

[0036] ③ Chromosomal karyotype analysis of yak bronchial epithelial cells When the primary BECs and P30 generation hTERT-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.

[0037] like Figure 5 As shown (A: chromosomes of primary BECs; B: chromosomes of P30 generation hTERT-Yak BECs), both primary cells and P30 generation hTERT-Yak BECs have 60 chromosomes, and their karyotypes are consistent with the yak chromosome atlas, indicating that these immortalized cells can maintain genetic integrity during long-term passage.

[0038] ④ Detection of bronchial epithelial cell cycle in yaks Once the primary BECs and hTERT-Yak BECs reached a density of 80-90%, they were digested and then diluted to 1×10⁻⁶ cells using DMEM-F12. 6 Cells were counted at 1 / mL. 1 mL of cell suspension was placed in a 2 mL sterile centrifuge tube and centrifuged at 1500 rpm for 5 min. The supernatant was discarded, and the cells were washed with pre-cooled DPBS. The above steps were repeated once. After fixation, the cells were centrifuged at 1500 rpm 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 drawn using MFLT32 software.

[0039] like Figure 6As shown (A: cell cycle of primary BECs; B: cell cycle of hTERT-Yak BECs; C: percentage of different cell cycle stages in primary and immortalized cells; ** indicates highly significant differences). P <0.01), and found that the proportions of primary BECs in G1, S, and G2 phases were 53.09±0.16%, 15.16±0.14%, and 31.75±0.16%, respectively, while the proportions of hTERT-Yak BECs in G1, S, and G2 phases were 41.48±0.20%, 29.83±0.17%, and 28.69±0.12%, respectively. Primary BECs were significantly higher than hTERT-Yak BECs in G1 phase ( P <0.01, significantly lower than hTERT-Yak BECs in the S phase ( P <0.05).

[0040] 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.

[0041] like Figure 7 As shown in the figures (A: apoptosis of primary BECs; B: apoptosis of hTERT-Yak BECs; C: apoptosis rate of primary and immortalized cells), the apoptosis rate of primary BECs (29.93±0.13%) was significantly higher than that of hTERT-Yak BECs (25.16±0.06%). P <0.05), combined with the above cell growth viability test results, indicates that immortalized cells hTERT-Yak BECs are more conducive to cell proliferation than primary cells.

[0042] 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.

[0043] 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 was hTERT-Yak BECs, with accession number CCTCC NO: C 2026145.

2. A method for preparing a cell line as described in claim 1, characterized in that, Includes the following steps: (1) Screening of primary bronchial epithelial cells from yaks; (2) Transfect the plasmid containing the human telomerase reverse transcriptase hTERT gene into the primary bronchial epithelial cells; (3) Use screening medium to screen the transfected cells to obtain positive clones, which are the cell lines described above.

3. The preparation method according to claim 2, characterized in that, The screening medium mentioned in step (3) is a medium containing genimycin sulfate; the screening concentration is 800 μg / mL.

4. The application of the cell line described in claim 1 in constructing an in vitro research model of respiratory diseases in yaks.

5. The application as described in claim 4, characterized in that, The respiratory disease in yaks is caused by infection with Pasteurella multocida.

6. The use of the cell line of claim 1 in screening or evaluating drugs for treating respiratory diseases in yaks.

7. The application of the cell line according to claim 1 in yak farming.

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