Construction method and application of immortalized chicken cecum epithelial cells
Through the transfection of simian vacuole virus 40 and puromycin screening, immortalized chicken cecum epithelial cell line was constructed, which solved the problem of poor passage of primary cells, achieved the characteristics of multiple passages and cell morphology stability, and was suitable for large-scale drug screening tests.
Patent Information
- Application Number
- CN202510363109.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-26
- Publication Date
- 2025-06-24
AI Technical Summary
Primary chicken cecum epithelial cells have poor passages and can only be passed on to the third generation, limiting the mechanism research of intestinal-related diseases and large-scale drug screening tests.
Immortalized and cultured primary chicken cecum epithelial cells were inoculated into culture plates, and transfected simian vacuole virus 40 was transfected, and transfected cells were screened with puromycin to construct an immortalized chicken cecum epithelial cell line.
The constructed immortalized chicken cecum epithelial cell line has multiple passage capabilities, and the cell morphology remains stable. It is suitable for large-scale coccidiosis drug screening tests, which reduces culture costs and ensures consistency of the test samples.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and more particularly to a method for constructing and applying immortalized chicken cecal epithelial cells. Background Art
[0002] The digestive system of poultry mainly consists of the beak, oral cavity, pharynx, esophagus, crop, glandular stomach, muscular stomach, duodenum, jejunum, ileum, cecum, colorectum, cloaca, liver, and glandular stomach. The intestine is not only an important organ for digestion and absorption of nutrients, but also an important immune organ of the body. Research shows that many intestinal diseases are related to the invasion of pathogens such as bacteria, viruses, parasites, and toxins. Chicken cecal epithelial cells cultured in vitro are often used for the research on the mechanism of various pathogens invading the body and causing related diseases and drug screening research. Among them, Eimeria tenella directly parasitizes the cecal part of chickens. Therefore, chicken cecal epithelial cells are often used as a drug treatment model for studying chicken coccidiosis in clinical practice. However, due to the characteristics of the cells themselves, the primary cultured chicken cecal epithelial cells have poor cell passage ability and can only be passed to the third generation, which is not conducive to the research on the mechanism of intestinal-related diseases and large-scale drug screening tests.
[0003] Therefore, there is an urgent need for a cell line that can be stably passaged and maintain a good cell state after multiple passages to meet the requirements. Summary of the Invention
[0004] In view of this, the present invention provides a method for constructing and applying immortalized chicken cecal epithelial cells.
[0005] To achieve the above object, the present invention adopts the following technical solutions:
[0006] A method for constructing immortalized chicken cecal epithelial cells, inoculating the isolated and cultured primary chicken cecal epithelial cells into a culture plate, culturing until the cells adhere to the wall, then inoculating simian vacuolating virus 40 for transfection, and then screening and identifying the transfected cells with puromycin to obtain immortalized chicken cecal epithelial cells.
[0007] Cell immortalization refers to the situation where, when normal cells cultured in vitro spontaneously or are affected by external factors, some cells escape from the crisis of proliferation and senescence and thus acquire the ability to proliferate indefinitely. In this invention, Simian-virus 40 (SV40) is transfected into chicken cecal epithelial cells to establish an immortalized cell line. The LT antigen fragment of SV40 integrates into the host cell, which can bind to and inhibit the expression of several cellular tumor suppressor genes including pRB and p53. At the same time, it can mediate the transformation of normal cells by blocking the transition of the cell cycle at the G1 / S phase and G2 / M phase, enabling the cells to enter a state of indefinite proliferation. The immortalized chicken cecal epithelial cell line constructed in this invention has the characteristics of being passaged multiple times and maintaining a stable cell morphology. Compared with primary cell culture, it can carry out large-scale coccidia drug screening tests, which can not only reduce the culture cost in the tests but also ensure the consistency of test samples, providing an adequate sample basis for the research and screening of later chicken coccidia drugs.
[0008] Preferably, the process of separation and culture is specifically as follows: Take 15-day-old SPF chicken embryos, isolate the ceca under sterile conditions, and wash them.
[0009] Cut the tissue into pieces and digest with thermolysin.
[0010] Resuspend the cell pellet with complete epithelial cell medium containing 10% FBS, inoculate it into a T25 cell culture flask coated with Matrigel, culture for 1 h, and remove fibroblasts.
[0011] Collect the cell culture medium, centrifuge, resuspend the pellet with complete epithelial cell medium, inoculate it into a new T25 cell culture flask coated with Matrigel, and continue to culture in an incubator.
[0012] Preferably, the process of inoculating Simian-virus 40 for transfection is specifically as follows: Inoculate primary chicken cecal epithelial cells into a 6-well plate, place it in an incubator at 37 °C with saturated humidity and containing 5% CO2 for culture.
[0013] After the cells grow for 24 h, change the medium with complete epithelial cell medium, add 1 mL of complete epithelial cell medium and 20 μL of Simian-virus 40, mix well and continue to culture.
[0014] Observe the cell state after 12 h, change to fresh medium and continue to culture.
[0015] When the cells cover the entire bottom of the plate, digest the cells with 1 mL of trypsin-EDTA digestion solution and passage them to a T25 cell culture flask for standby passage.
[0016] Preferably, the process of screening transfected cells with puromycin is as follows: inoculate the cells transfected with simian vacuolating virus 40 into a 24-well plate, place it at 37°C in an incubator with saturated humidity and 5% CO2 for culture;
[0017] After culturing for 24 h, discard the culture medium in the culture plate, add complete epithelial cell medium containing 1 μg / mL puromycin, and incubate;
[0018] Replace the fresh complete epithelial cell medium for culture at 48 h, and observe the survival ratio of the cells every day;
[0019] The cells that survive within 2 d are the successfully transfected cells.
[0020] Preferably, the process of the identification is as follows: detect whether there is PCK protein signal in the cells by indirect immunofluorescence method. If there is, it is considered successful. In the indirect immunofluorescence method, the primary antibody is rabbit PCK, and the secondary antibody is goat anti-rabbit secondary antibody.
[0021] Another object of the present invention is to provide an immortalized chicken cecal epithelial cell line constructed by the above method.
[0022] Another object of the present invention is to provide the application of the chicken cecal epithelial cell line constructed by the above construction method or the above chicken cecal epithelial cell line in the preparation of a cell model for Eimeria invasion.
[0023] Beneficial effects: The immortalized chicken cecal epithelial cell line constructed by the present invention has the characteristics of being passaged multiple times and maintaining a stable cell morphology. Compared with primary cell culture, large-scale screening tests for coccidiosis drugs can be carried out, which can not only reduce the culture cost in the test, but also ensure the consistency of test samples, providing a sufficient sample basis for the research and screening of coccidiosis drugs in the later stage. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.
[0025] Figure 1 It is the morphology of the primary chicken cecal epithelial cells of the present invention; among them, A is 10×; B is 20×;
[0026] Figure 2 It is the morphology of the primary chicken cecal epithelial cells transfected with EF1α-SV40-IRES-puromycin of the present invention; among them, A is 10×; B is 20×;
[0027] Figure 3 This is the cell morphology of the immortalized chicken cecal epithelial cells at the initial stage of subculture; among them, A is 10×; B is 20×;
[0028] Figure 4 This is the cell morphology of the immortalized chicken cecal epithelial cells subcultured to the 15th generation; A is 10×; B is 20×;
[0029] Figure 5 This is the immunofluorescence detection (20×) of the marker PCK of the immortalized chicken cecal epithelial cells of the present invention; among them, A is the DAPI staining map; B is the PCK staining map;
[0030] Figure 6 This is the morphology of the immortalized chicken cecal epithelial cells invaded by coccidia; among them, A is 10×; B is 20×. Detailed implementation manners
[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
[0032] The embodiments of the present invention disclose a method for constructing immortalized chicken cecal epithelial cells. All raw materials used in the present invention are commercially available, and no specific limitation is made on their sources. For example, the penicillin-streptomycin mixture, the cell climbing slides dedicated to 24-well plates, paraformaldehyde, Triton X-100, goat serum, trypsin-EDTA digestive solution (0.25%) can be purchased from Beijing Solarbio Science & Technology Co., Ltd.; the epithelial cell medium and the complete epithelial cell medium can be purchased from Hefei Wanwu Biotechnology Co., Ltd.; the DMEM high-glucose medium can be purchased from Thermo Fisher Scientific of the United States; PCK and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) can be purchased from Wuhan proteintech Co., Ltd.; fetal bovine serum can be purchased from Gibico of the United States; the thermolysin digestive solution can be purchased from MedChemExpress; EF1α-SV40-IRES-puromycin can be purchased from Shanghai Hanyin Biotechnology Co., Ltd. The methods involved in the present invention, unless otherwise specifically mentioned, are all conventional methods and will not be elaborated one by one here.
[0033] Example 1 Isolation and culture of primary chicken cecal epithelial cells
[0034] 1) Take 15-day-old SPF chicken embryos, isolate the cecum under sterile conditions, remove the mesentery, and wash it several times with PBS containing a mixture of penicillin and streptomycin.
[0035] 2) Cut the tissue into pieces about 1 mm 3 in size, add a thermolysin digestive solution with a final concentration of 50 μg / mL, place it in a 37°C constant-temperature shaking water bath for digestion for 2 h, then add 2 volumes of epithelial cell medium containing 10% FBS, and pipette several times. Centrifuge at 1000 rpm for 5 min and discard the supernatant.
[0036] 3) Resuspend the cell pellet with epithelial cell complete medium containing 10% FBS, count the cells, and adjust the cell concentration to 2×10 5 cells / mL. Uniformly inoculate them into a T25 cell culture flask coated with Matrigel, 5 mL per flask. Place it in an incubator at 37°C, saturated humidity, and 5% CO2 for 1 h to remove fibroblasts.
[0037] 4) Collect the cell culture medium, centrifuge at 1000 rpm for 5 min, resuspend the pellet with epithelial cell complete medium, and mix well. Uniformly inoculate it into a new T25 cell culture flask coated with Matrigel and continue culturing in the incubator. Observe under the microscope and change the medium the next day, and change the medium every 2 - 3 days thereafter.
[0038] The results are shown in the appendix Figure 1 , and the primary chicken cecal epithelial cells isolated and cultured grew in a "cobblestone paving" pattern in sheets, with uniform cell morphology and stable growth.
[0039] Example 2 Construction of an immortalized chicken cecal epithelial cell line
[0040] 1) Transfect cells with an SV40 overexpression lentivirus
[0041] Inoculate the stably growing chicken cecal epithelial cells into a 6-well plate, with 100,000 cells per well, and culture them in an incubator at 37°C, saturated humidity, and 5% CO2;
[0042] After the cells grow for 24 h, change the medium with epithelial cell complete medium, add 1 mL of epithelial cell complete medium and 20 μL of simian vacuolating virus 40 (EF1α - SV40 - IRES - puromycin), mix well and continue culturing;
[0043] Observe the cell status 12 h after inoculating simian vacuolating virus 40 (EF1α - SV40 - IRES - puromycin), and change to fresh medium and continue culturing;
[0044] When the cells cover the entire bottom of the plate, use 1 mL of trypsin - EDTA digestive solution (0.25%) to digest the cells and passage them to a T25 cell culture flask for standby passage.
[0045] The results are shown in the appendix Figure 2 , after transfection with the SV40 overexpression lentivirus, the cell morphology was basically the same as that of the primary cells, and the growth was stable, which could be used for subsequent experiments.
[0046] 2) Determination of puromycin killing curve
[0047] The stably growing primary chicken cecal epithelial cells were seeded in 24-well plates at a density of 50,000 cells per well and cultured in an incubator at 37 °C, saturated humidity, and 5% CO2.
[0048] After 24 h of culture, the medium in the culture plate was discarded, and puromycin was diluted with fresh complete epithelial cell medium to concentration gradients of 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, and 7 μg / mL, and then added to the 24-well plates seeded with cells.
[0049] The medium was changed every 48 h, and the cell status was observed daily to determine the survival ratio of the cells; the minimum puromycin concentration used was the lowest screening concentration that could kill all cells within 1 - 4 days from the start of puromycin screening. The results showed that the puromycin concentration used was 1 μg / mL and the action time was 2 days.
[0050] 3) Screening of transfected cells with puromycin
[0051] The transfected chicken cecal epithelial cells were seeded in 24-well plates at a density of 50,000 cells per well and cultured in an incubator at 37 °C, saturated humidity, and 5% CO2.
[0052] After 24 h of culture, the old medium was discarded, and puromycin screening medium containing 1 μg / mL was added and incubated in the medium.
[0053] The culture was continued by changing the fresh complete epithelial cell medium every 48 h, and the survival ratio of the cells was observed daily.
[0054] The cells that survived within 2 days were the successfully transfected cells, and the screened cells were expanded.
[0055] As shown in the appendix Figure 3 At the initial stage of subculture of the constructed immortalized chicken cecal epithelial cell line, the cell morphology showed a typical "cobblestone paving" growth characteristic of epithelial cells, with uniform cell morphology and stable growth.
[0056] As shown in the appendix Figure 4 When the constructed immortalized chicken cecal epithelial cell line was subcultured to the 15th generation, the cell status was the same as that of the cells at the beginning of subculture (appendix Figure 3 ), indicating that the immortalized chicken cecal epithelial cell line constructed in the present invention was successfully constructed.
[0057] Example 3: Immunofluorescence Identification of Immortalized Chicken Cecal Epithelial Cells
[0058] 1) Take the constructed immortalized chicken cecal epithelial cells, wash them 3 times with 1×PBS, then add 1 mL of trypsin-EDTA digestive solution (0.25%) for digestion. After digestion is completed, add an appropriate amount of complete epithelial cell medium to terminate digestion and centrifuge at 1000 r / min for 5 min. Add an appropriate medium to adjust the cell concentration to 1×10 5 cell / mL to obtain a cell suspension for standby;
[0059] 2) Take 3 pieces of glass slides for cell culture inserts in 24-well plates and place them in the 24-well plates. Add 1 mL of the above cell suspension to each well and place it in an incubator for 2 h;
[0060] 3) After the cells adhere to the wall, take out the above 24-well plates, discard the medium, wash once with 1×PBS, and add 4% paraformaldehyde to fix at room temperature for 30 min;
[0061] 4) After fixation is completed, discard the fixing solution and wash 3 times with 1×PBS; Take 50 μL of permeabilization blocking solution (mix 0.5% Triton X-100 and 1×PBS at a ratio of 1:1, add 10% goat serum and mix well to obtain the permeabilization blocking solution), drop it on the waterproof membrane, cover the side with the cell slide for 2 h;
[0062] 5) After permeabilization blocking is completed, discard the permeabilization blocking solution and wash 3 times with 1×PBS. Prepare the primary antibody solution by mixing the rabbit PCK primary antibody solution and 1×PBS at a ratio of 1:100. After mixing, take 50 μL and drop it on the waterproof membrane, cover the side with the cell slide, and place it in a wet box at 4°C overnight;
[0063] 6) After the primary antibody incubation is completed, discard the primary antibody solution and wash 3 times with 1×PBS. Prepare the secondary antibody by mixing the goat anti-rabbit secondary antibody (HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L)) and 1×PBS at a ratio of 1:500. After mixing, take 50 μL and drop it on the waterproof membrane, and incubate at room temperature in the dark for 2 h;
[0064] 7) After the secondary antibody incubation is completed, wash 3 times with 1×PBS in the dark. Prepare DAPI by mixing it with 1×PBS at a ratio of 1:1000. After mixing, take 50 μL and drop it on the waterproof membrane and incubate at room temperature for 5 min;
[0065] 8) After nuclear staining is completed, discard the DAPI staining solution and wash 3 times with 1×PBS. Drop a drop of Fluoromount-G on the slide, cover the side with the cells, and take pictures with a laser confocal microscope.
[0066] The results are shown in the appendixFigure 4 Indirect immunofluorescence can detect the cell PCK protein signal (an epithelial cell-specific marker), demonstrating that the immortalized cells constructed are chicken cecal epithelial cells.
[0067] Example 4: Experiment on the invasion of immortalized chicken cecal epithelial cells by coccidia
[0068] 1) The sporulated oocysts of the virulent Eimeria tenella preserved in potassium dichromate were repeatedly washed with sterile water and then ground and digested. Digestion was stopped after 80% of the sporozoites were released.
[0069] 2) The sporozoites obtained by digestion were centrifuged and resuspended, and then filtered through 800-mesh, 1400-mesh, and 1800-mesh sieves three times. The sporozoites were diluted to 20×10 4 per mL and inoculated into the complete epithelial cell medium for standby.
[0070] 3) The immortalized chicken cecal epithelial cells were digested with trypsin, centrifuged, and resuspended with the complete epithelial cell medium. The cells were counted using a cell counter, and the cell density was diluted to 30×10 4 per mL for standby.
[0071] 4) The cells were inoculated into a 6-well plate. After the cells adhered to the wall, the medium was discarded, and 2 mL of the complete epithelial cell medium containing sporozoites was added. The ratio of sporozoites to cells was 1:0.9.
[0072] 5) After 4 hours of sporozoite invasion, the medium was discarded, and the cells were fixed with 4% paraformaldehyde. Then, the cell slides were taken out for HE staining, and the state of sporozoite invasion into the cells was observed under an optical microscope.
[0073] The results are shown in the appendix Figure 5 , and sporozoites entered most of the immortalized chicken cecal epithelial cells. One to multiple sporozoites were seen in most cells, indicating that the constructed immortalized chicken cecal epithelial cells have the ability to be invaded by coccidia and can carry out subsequent coccidia drug screening tests.
[0074] The immortalized chicken cecal epithelial cells can be passaged multiple times in vitro culture, and the cell morphology remains stable. Compared with primary cell culture, large-scale coccidia drug screening tests can be carried out, which can not only reduce the culture cost in the test but also ensure the consistency of test samples, providing an adequate sample basis for the research and screening of chicken coccidia drugs in the later stage.
[0075] The various examples in this specification are described in a progressive manner. Each example focuses on the differences from other examples. For the same or similar parts among the examples, reference can be made to each other.
[0076] The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not 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. A method for constructing immortalized chicken cecal epithelial cells, characterized in that: The isolated and cultured primary chicken cecal epithelial cells were inoculated into a culture plate, cultured until the cells adhered to the wall, and then inoculated with simian vacuolating virus 40 for transfection. The transfected cells were then selected and identified with puromycin to obtain immortalized chicken cecal epithelial cells.
2. The method for constructing immortalized chicken cecal epithelial cells according to claim 1, characterized in that: The separation and cultivation process is specifically as follows: taking 15-day-old SPF chicken embryos, isolating the cecum under sterile conditions, and cleaning it; The tissue was minced and digested with thermolysin; The cell pellet was resuspended in complete epithelial cell culture medium containing 10% FBS, inoculated into a Matrigel-coated T25 cell culture flask, cultured for 1 h, and fibroblasts were removed; The cell culture fluid was collected and centrifuged, the precipitated cells were resuspended in complete epithelial cell culture medium, inoculated into a new matrix gel-coated T25 cell culture flask, and placed in an incubator for continued culture.
3. The method for constructing immortalized chicken cecal epithelial cells according to claim 1, characterized in that: The process of inoculating simian vacuolating virus 40 for transfection is specifically as follows: primary chicken cecal epithelial cells are inoculated into a 6-well plate and placed in an incubator at 37° C., saturated humidity, and 5% CO 2 for culture; After the cells have grown for 24 h, the medium is replaced with complete epithelial cell medium, and 1 mL of complete epithelial cell medium and 20 μL of simian vacuolating virus 40 are added, mixed and continued to be cultured; After 12 h, observe the cell status and replace with fresh medium to continue culturing; When the cells cover the entire bottom of the plate, use 1 mL of trypsin-EDTA digestion solution to digest the cells and subculture them into a T25 cell culture flask for future use.
4. The method for constructing immortalized chicken cecal epithelial cells according to claim 1, characterized in that: The process of using puromycin to select transfected cells is specifically as follows: cells transfected with simian vacuolating virus 40 are inoculated into a 24-well plate and placed in an incubator at 37° C., saturated humidity, and 5% CO 2 ; After culturing for 24 h, the culture medium in the culture plate was discarded, and complete epithelial cell culture medium containing 1 μg / mL puromycin was added and incubated; After 48 hours, fresh complete epithelial cell culture medium was replaced for culture, and the survival rate of cells was observed every day; Cells that survived within 2 days were considered to be successfully transfected cells.
5. The method for constructing immortalized chicken cecal epithelial cells according to claim 1, characterized in that: The identification process is specifically as follows: using indirect immunofluorescence method to detect whether there is a PCK protein signal in the cell, and if there is, it is successful; in the indirect immunofluorescence method, the primary antibody is rabbit PCK, and the secondary antibody is goat anti-rabbit secondary antibody.
6. An immortalized chicken cecal epithelial cell line, characterized in that: The method is constructed by any one of claims 1 to 5.
7. Use of the chicken cecal epithelial cell line constructed by the construction method of any one of claims 1 to 5 or the chicken cecal epithelial cell line according to claim 6 in preparing a cell model of chicken coccidia invasion.