A goat endemic intranasal adenocarcinoma cell ena-1

Abstract

The application provides a new goat endemic intranasal adenocarcinoma cell ENA-1. The cell strain is preserved in China Center for Type Culture Collection (Wuhan), and the preservation number is CCTCC NO: C2022232. It is identified that the ENA-1 cell strain can grow rapidly in vitro, can be stably and continuously passed, retains the immunohistochemical characteristics of adenocarcinoma, can form a tumor after being inoculated subcutaneously in an immunodeficient nude mouse, and can form a tumor after being inoculated in a healthy goat for 8 months. The ENA-1 can be used as a cell material for the in-vitro treatment research of the goat endemic intranasal adenocarcinoma, can be used for constructing an animal model of the goat endemic intranasal adenocarcinoma, and has important significance for the research of the goat endemic intranasal adenocarcinoma.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, and specifically relates to a goat endemic nasal adenocarcinoma cell line. Background Technology

[0002] Enzootic nasal adenocarcinoma (ENA) is a contagious, chronic neoplastic disease in goats caused by the enzootic nasal tumor virus of goats (ENTV-2). ENA is prevalent globally, and has been reported in several provinces in China, with a recent trend of expansion, causing significant economic losses to the goat farming industry. Affected goats develop tumors in their nasal cavities; as the tumors enlarge, clinical symptoms such as serous nasal discharge and heavy breathing sounds appear. The disease has a long course, a low morbidity rate but a 100% mortality rate, and there are no vaccines or drugs for its control.

[0003] Tumor cell lines are crucial tools in tumor research, aiding in the study of tumorigenesis mechanisms and the screening of therapeutic drugs. Currently, research on endemic goat nasal adenocarcinoma cells is limited, and no identified endemic goat nasal adenocarcinoma cell lines are recorded in any database. Therefore, establishing a stable, morphologically homogeneous cell line for endemic goat nasal adenocarcinoma is of paramount importance for research on this disease. Summary of the Invention

[0004] The purpose of this invention is to provide a goat endemic nasal adenocarcinoma cell line. This cell line can form tumors after subcutaneous inoculation into immunodeficient nude mice. After nasal inoculation into healthy goats for 8 months, a tumor was observed to form in the ethmoid mucosa of the nasal cavity by computed tomography. This is helpful for studying the in vitro treatment and model establishment of goat endemic nasal adenocarcinoma.

[0005] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0006] A goat endemic intranasal adenocarcinoma cell line, named goat endemic intranasal adenocarcinoma ENA-1, is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: C2022232 and deposit date July 12, 2022.

[0007] The goat endemic intranasal adenocarcinoma cell line ENA-1 was observed to be polygonal in shape under an inverted optical microscope, and had numerous finger-like protrusions on its cell surface and a large number of intermediate filaments in its cytoplasm under a transmission electron microscope.

[0008] The doubling time of the goat endemic nasal adenocarcinoma cell line ENA-1 was approximately 36 hours.

[0009] Chromosomal karyotype analysis of the goat endemic intranasal adenocarcinoma cell line ENA-1 showed that it had an abnormal number of chromosomes.

[0010] The goat endemic intranasal adenocarcinoma cell line ENA-1 showed positive immunohistochemical expression of the glandular epithelial markers keratin 7 and keratin 18.

[0011] The histopathological features of the subcutaneous xenograft tumor of ENA-1 endemic nasal adenocarcinoma cells in goats in immunodeficient nude mice were encapsulated, well-differentiated glandular cells.

[0012] Eight months after the goat endemic nasal adenocarcinoma cell line ENA-1 was inoculated into healthy goats, a CT scan revealed tumor growth in the labyrinth of the ethmoid bone in the goat's nasal cavity.

[0013] The goat endemic nasal adenocarcinoma cell line ENA-1 of the present invention was obtained by the following method: tumor tissue of goats with endemic nasal adenocarcinoma was collected, primary tumor cells were isolated and cultured by tissue patch method, single-cell cloning was completed by serial dilution microplate method, and a purified tumor cell line was established.

[0014] The beneficial effects of this invention are:

[0015] This invention constructs a goat endemic nasal adenocarcinoma cell line, ENA-1, which is a novel goat endemic nasal adenocarcinoma cell line. It can be cultured in vitro in a culture medium, can be stably passaged, grows rapidly, retains the immunohistochemical characteristics of glandular epithelial cells, and can form tumors after subcutaneous inoculation in immunodeficient nude mice. After 8 months of nasal inoculation in healthy goats, it can form tumors. It can be used as cell material for in vitro treatment research of goat endemic nasal adenocarcinoma and for constructing animal models of goat endemic nasal adenocarcinoma. Attached Figure Description

[0016] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments:

[0017] Figure 1 Photograph of ENA-1 endemic nasal adenocarcinoma cells from goats under an inverted phase-contrast microscope, magnified 400 times.

[0018] Figure 2 Transmission electron microscope image of ENA-1 endemic nasal adenocarcinoma cells in goats.

[0019] Figure 3 Growth curve of ENA-1, a local goat nasal adenocarcinoma cell line.

[0020] Figure 4 Chromosome analysis results of ENA-1, a local goat nasal adenocarcinoma cell line, magnified 1000 times.

[0021] Figure 5 Immunofluorescence results of CK7 and CK18, glandular epithelial markers, in ENA-1 endemic nasal adenocarcinoma cells of goats.

[0022] Figure 6 Pathological observation results of ENA-1 endemic nasal adenocarcinoma cells from goats in nude mice, magnified 400 times.

[0023] Figure 7 Image of a nasal cavity CT scan 18 months after inoculation of a healthy goat with endemic goat nasal adenocarcinoma cells (ENA-1). Detailed Implementation

[0024] Example 1: Isolation, purification, and cryopreservation of ENA-1, a type of endemic goat nasal adenocarcinoma.

[0025] 1.1 Sample Collection and Processing

[0026] A 1cm mass was taken from the nasal cavity of a goat that died from endemic nasal adenocarcinoma. 3 The samples were washed three times in PBS containing antibiotics (200 U / mL penicillin + 0.2 mg / mL streptomycin + 0.1 mg / mL ceftiofur sodium) to remove as much surface microorganisms, mucus, and blood as possible, and then brought back to the laboratory within 30 minutes for further processing.

[0027] Take a 6cm diameter cell culture dish, transfer the tumor into the dish, and cut it into 1mm pieces with a sterile scalpel blade. 3 Small pieces of tissue. Take several 25cm² cell culture flasks, place the chopped tissue pieces at the bottom of each flask, about 20-30 pieces per flask, and slowly add about 1mL of RPMI-1640 medium (containing 10% fetal bovine serum, 100U / mL penicillin, and 0.2mg / mL streptomycin). Incubate in a 37℃, 5% CO2 cell culture incubator.

[0028] 1.2 Cell passage

[0029] On day 3 after tissue block inoculation, add culture medium to 5 mL. Observe cell growth and morphology daily, changing the culture medium every 3 days. After 7 days, the cells will cover approximately 80% of the bottom of the flask and are ready for passage. Discard all old culture medium and pre-wash the cells twice with 3 mL of D-PBS containing 1 mmol / L EDTA. Add 1 mL of 0.25% trypsin, gently agitate the culture flask to spread the trypsin to the cell surface, and digest for 5 min. Add 5 mL of culture medium to stop digestion, thoroughly disperse the cells by pipetting, and transfer the entire cell suspension to a sterile 10 mL centrifuge tube. Centrifuge at 1200 rpm for 5 min, carefully aspirate the supernatant, and repeatedly pipette the dispersed cell clumps with 1 mL of fresh culture medium. Count the cells using a hemocytometer at a rate of 2 × 10⁻⁶ cells / mL. 5 The cells / mL were passaged and cultured further.

[0030] 1.3 Monoclonal cell purification and culture

[0031] When the cells reached passage 5, the number of fibroblast-like cells was relatively small, and the cell growth rate stabilized. Cells were routinely digested according to the method described in section 1.2, centrifuged, and resuspended. The cell count was performed three times using a hemocytometer, and the average value was taken. A serial dilution microplate method was used to gradually dilute the cells to a final concentration of 10 cells / mL. Five 96-well plates were prepared, and 100 μL of the thoroughly pipetted cell suspension was added to each well. Each well was observed under an inverted microscope, and wells with no observed cells or more than one cell were removed. The plates were incubated for 3 days, and culture medium was added to a final volume of 200 μL. When the cells covered 50% of the well bottom, they were digested with a small amount of trypsin and transferred to 24-well plates for further culture to establish single clones.

[0032] 1.4 Cell cryopreservation

[0033] When the cells reach the logarithmic growth phase, digest and centrifuge them as described in section 1.2. After removing the supernatant, add 1-2 mL of freshly prepared cell cryopreservation medium (DMSO: fetal bovine serum: 1640 volume ratio = 1:3:6). After thorough pipetting, count the cells under a microscope using a hemocytometer. Add more cell cryopreservation medium to adjust the cell count to 1 × 10⁻⁶ cells / year. 6 The samples were dispensed at a rate of 1 / mL into cryovials. The cryovials were then placed in a programmed cooling cryovial box and stored at -80°C overnight. After storage, the cryovials were transferred to a liquid nitrogen tank for preservation.

[0034] 1.5 Cell resuscitation

[0035] Remove the cryovials from the liquid nitrogen container and immediately place them in a 37°C water bath. Gently shake the cryovials to thaw the cells rapidly within 1 minute. Remove the cryovials, disinfect the tube walls with alcohol swabs, and place them in a clean bench. Transfer the cell suspension to a 25cm... 2Add culture medium to the culture flask to a final volume of 5 mL, and gently shake the flask to distribute the cells evenly. After 24 hours, most cells will adhere to the culture flask; replace the culture medium with fresh medium.

[0036] The 9th generation cell line, after purification of monoclonal cells, was deposited at the China Center for Type Culture Collection (CCTCC) and named ENA-1, a goat endemic intranasal adenocarcinoma cell line. The deposit address is Wuhan University, Wuhan, China, the accession number is CCTCC NO: C2022232, and the deposit date is July 12, 2022.

[0037] like Figure 1 As shown, endemic nasal adenocarcinoma cells in goats adhere to the wall and grow. The cells are similar in size, mostly elongated or triangular in shape, with obvious nuclei and abundant cytoplasm, and are similar in morphology to epithelial-like cells.

[0038] Example 2: Ultrastructural characteristics of ENA-1 endemic nasal adenocarcinoma cells in goats

[0039] ENA-1, a type of endemic goat nasal adenocarcinoma, was seeded into a 10cm diameter cell line. 2 On a cell culture dish, when the cells cover 80% of the bottom, remove the old culture medium, wash three times with PBS, remove the PBS, and gently scrape the cells in one direction along the bottom of the culture dish with a cell scraper. Collect the white, strip-shaped cell clumps with a pipette and place them in a 1.5 mL centrifuge tube. Centrifuge at 1800 r / min for 10 min, and slowly add 1 mL of 2.5% glutaraldehyde along the tube wall for fixation. Dehydrate the cells using a gradient of ethanol, embed them in epoxy resin to prepare ultrathin sections, and observe them using a Hitachi H-7650 transmission electron microscope after staining with uranium acetate and lead citrate.

[0040] like Figure 2 As shown, the ultrastructure of the endemic nasal adenocarcinoma cell line from goats reveals numerous finger-like projections on the cell surface and tight junctions between cells. The cells are similar in shape, mostly round or oval. The nucleus-to-cytoplasm ratio is not high; the nucleus is regularly oval with no atypia observed in several fields of view, and is relatively bright, predominantly composed of euchromatin. Numerous intermediate filaments are distributed in the cytoplasm, forming a fine network that runs between organelles. The rough endoplasmic reticulum is dilated to varying degrees, and the mitochondrial cristae are also dilated to varying degrees. Retrovirus-like particles ranging in size from 80-120 nm are also visible in some parts of the cytoplasm.

[0041] Example 3: Growth curve of ENA-1 endemic nasal adenocarcinoma cells in goats

[0042] Goat endemic intranasal adenocarcinoma cells ENA-1 were seeded into 24-well plates, with 1 mL of 1.5 × 10⁻⁶ cells per well. 4Three parallel control groups were set up with a well-drained cell suspension of cells / mL. Cells in the 24-well plates were over-digested at the same time each day to ensure complete cell detachment. The number of cells in each well was counted using a hemocytometer for 10 consecutive days. A cell growth curve was plotted with the number of culture days on the x-axis and the number of cells per well on the y-axis.

[0043] like Figure 3 As shown, the cell growth curve exhibits an "S" shape, encompassing three typical phases: adaptation, logarithmic growth, and plateau. The doubling time of ENA-1 cells, calculated using the formula, is approximately 36 hours.

[0044] Example 4: Chromosomal karyotype analysis of ENA-1 endemic nasal adenocarcinoma cells in goats

[0045] When cells reached the late logarithmic growth phase, colchicine was added to the culture medium to a final concentration of 0.2 μg / mL, and the incubation time was 2 h. The culture medium was then removed, the cells were washed once with PBS, and the cells were routinely digested and collected into 10 mL centrifuge tubes. 8 mL of 0.075 mol / L KCl hypotonic solution (pre-warmed to 37°C) was added, and the cell clumps were gently agitated. The tubes were then incubated in a 37°C water bath for 20 min. After centrifugation at 1000 rpm for 5 min, the supernatant was removed, and 1 mL of freshly prepared fixative (glacial acetic acid:anhydrous ethanol, volume ratio = 1:3, stored on ice) was added for pre-fixation at room temperature for 5 min. After centrifugation at 1000 rpm for 5 min, the supernatant was removed, and 10 mL of fixative was added for fixation at room temperature for 20 min. After centrifugation at 1000 rpm for 5 min, the supernatant was removed, and 10 mL of fixative was added for further fixation at room temperature for 30 min. After centrifugation at 1000 rpm for 5 min, the supernatant was removed, and approximately 0.5 mL of liquid remained for slide preparation. Take a clean glass slide frozen at -4℃, and use a pipette to drop one drop of cell suspension onto the slide from a height of 15cm. Immediately fix the slide by quickly passing it over an alcohol lamp flame several times. After the fixative evaporates, cell particles can be seen on the slide. Stain with new Giemsa staining solution for 20 minutes, rinse the slide with running water, and examine it under a microscope after air drying.

[0046] Select well-dispersed mitotic figures with clear chromosome outlines under a low-power microscope, then switch to high-power and oil immersion microscopes. Count the chromosomes in 50 mitotic figures under oil immersion. The normal chromosome number of a goat is 2n = 60. Figure 4 As shown, the chromosome number of ENA-1 cells ranged from 51 to 116, with the majority being 80 to 112. Aneuploid karyotypes were predominant, with subdiploid and subtetraploid cells being more common, while diploid cells were rare, indicating that the chromosomes of this cell type are unstable.

[0047] Example 5: Detection of ENA-1-specific molecular expression in endemic goat nasal adenocarcinoma cells

[0048] Cytokeratin is an important marker for identifying epithelial cells, with CK7 and CK18 being commonly used indicators in the study of glandular epithelial cells. Cell slides were prepared as follows, and the expression of glandular epithelial markers CK7 and CK18 in sheep endemic nasal adenocarcinoma cell line ENA-1 was detected by immunofluorescence: A 12-well plate was prepared, one drop of culture medium was added to the bottom of each well, a cleaned and sterilized round coverslip was placed, and 1 mL of culture medium containing 4 × 10⁻⁶ cells was seeded into each well. 4 Cell suspensions were cultured using standard methods until cells covered approximately 60% of the slide. The cells were washed twice with PBS, and each well was fixed with 1 mL of 4% paraformaldehyde for 20 min. After washing once with PBS, the cells were permeated with 0.5% Triton X-100 for 20 min. After washing three times with PBS, the cells were blocked with 3% fetal bovine serum at room temperature for 30 min. The blocking solution was removed, and CK7 and CK18 primary antibody dilutions (1:100 dilution) were added, and the cells were incubated overnight at 4°C. After washing three times with PBS, horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody dilutions (1:200 dilution) were added, and the cells were incubated at room temperature for 50 min. After washing three times with PBS, the cell nuclei were counterstained with DAPI staining solution and incubated at room temperature in the dark for 10 min. After washing three times with PBS, the cells were mounted with anti-fluorescence quenching mounting medium and observed under a fluorescence microscope.

[0049] like Figure 5 As shown, under a fluorescence microscope, the cytoplasm of ENA-1 cells expressed CK7 and CK18, indicating that the cells originated from glandular tissue epithelium.

[0050] Example 6: Histopathological examination of subcutaneous xenograft tumors of the goat endemic nasal adenocarcinoma cell line ENA-1 in immunodeficient nude mice.

[0051] When the cells reached the late logarithmic growth phase, they were routinely digested according to the method in section 1.2, and the cells were resuspended in RPMI-1640 medium without serum and antibiotics to prepare a cell suspension. The cell count was calculated three times using a hemocytometer, and the average value was taken. The cell count was then adjusted to 1 × 10⁻⁶. 7 Tumor cells / mL. Four-week-old immunodeficient nude mice (half male and half female) were selected. The skin under the left armpit was disinfected with alcohol, and 0.2 mL of tumor cell suspension was injected subcutaneously. After four weeks of feeding, tumors grew in the armpits of the nude mice. The mice were anesthetized and sacrificed, and the transplanted tumors were removed, fixed in 10% neutral formalin solution, embedded in paraffin, and used to prepare pathological sections. After hematoxylin-eosin staining, the sections were observed under a regular optical microscope.

[0052] like Figure 6As shown, hematoxylin-eosin staining of ENA-1 nude mouse subcutaneous xenograft samples revealed that the tumor cells were surrounded by a capsule composed of multiple layers of connective tissue. The tumor cells were well-differentiated, with some forming glandular tubular structures. The cell boundaries were distinct, and the cytoplasm was eosinophilic or neutrophilic. The nuclei were round or oval, lightly stained and unevenly, located in the center or on one side of the cell, and mitotic figures were rare. This is consistent with the pathological characteristics of well-differentiated / low-grade adenocarcinoma.

[0053] Example 7: CT scan after nasal inoculation of the goat endemic nasal adenocarcinoma cell line ENA-1 into healthy goats.

[0054] When the cells reached the late logarithmic growth phase, they were digested as described in section 1.2, resuspended in PBS, and the cell count was calculated three times using a hemocytometer, with the average value taken. The cell count was then adjusted to 2 × 10⁶ cells / year using PBS. 6 Cell suspension at 0.01 mL / kg xylazine hydrochloride was administered intramuscularly to healthy goats under general anesthesia. Under head CT guidance, the location of the ethmoid labyrinth in the nasal cavity was confirmed, and 5 mL of cell suspension was injected into the unilateral ethmoid labyrinth mucosa through a 3 mm diameter rubber catheter. Goats underwent head CT scans every 4 weeks under general anesthesia to reconstruct bone and soft tissue windows and observe tumor growth.

[0055] like Figure 7 As shown, about 8 months later, a CT scan revealed a soft tissue density mass in the ethmoid labyrinth of the nasal cavity on the left side of the goat inoculated cell side. The mass had irregular edges and was approximately 11mm × 9mm × 2mm in size, with a CT value of approximately 60HU. The mass caused damage to the ethmoid labyrinth structure.

[0056] In summary, the ENA-1 cell line possesses the characteristics of endemic goat nasal adenocarcinoma tumor cells, meets the criteria for cell line establishment, and is a novel endemic goat nasal adenocarcinoma tumor cell line. It can be used as cell material for studying in vitro treatment of endemic goat nasal adenocarcinoma and can be used to construct animal models of endemic goat nasal adenocarcinoma.

Claims

1. A goat endemic nasal adenocarcinoma cell line, characterized in that, It was named ENA-1, a type of endemic goat nasal adenocarcinoma cell, and deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2022232.

2. The application of the goat endemic nasal adenocarcinoma cell line of claim 1 in the construction of a nude mouse tumor animal model.

3. The application of the goat endemic nasal adenocarcinoma cell line of claim 1 in the construction of a goat tumor animal model.

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