An immortalized chicken skeletal muscle satellite cell line, its construction method and application

Abstract

This invention relates to an immortalized chicken skeletal muscle satellite cell line, its construction method, and its applications, belonging to the field of biotechnology. The immortalized chicken skeletal muscle satellite cell line Im MSLC of this invention, with accession number CCTCC NO: C2023115, was deposited on July 5, 2023, at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University. The immortalized chicken skeletal muscle satellite cell line of this invention maintains a similar growth rate and consistent growth trend to primary chicken skeletal muscle cells after 10 passages, and retains the same cell morphology as primary cells after 22 passages; moreover, the immortalized chicken skeletal muscle satellite cell line still possesses differentiation ability after 10 passages. The immortalized chicken skeletal muscle satellite cell line provided by this invention reduces the use of chicken embryos and lowers the possibility of difficulty in obtaining stable and reliable results due to different batches of chicken embryos from which skeletal muscle cells are derived.

Description

Technical Field

[0001] This invention relates to an immortalized chicken skeletal muscle satellite cell line, its construction method, and its application, belonging to the field of biotechnology. Background Technology

[0002] Skeletal muscle is a vital component of livestock and poultry, playing a crucial role in maintaining movement and postural support. Muscle tissue is primarily composed of long fibers formed from the mitotic division of multinucleated cells, and its lifelong maintenance is mediated by satellite cells. Skeletal muscle satellite cells (MSCs) are a type of myogenic stem cell located between the muscle cell membrane and the basement membrane, possessing the potential for proliferation and differentiation. They play a crucial role in the normal development, regeneration, and repair of muscle. MSCs are generally in a relatively quiescent state. When muscle tissue is injured or subjected to other stimuli, some MSCs can be activated. After activation, satellite stem cells undergo symmetrical division to increase their number or asymmetrical division, generating a directed satellite cell cohort, thereby producing progenitor cells. These myogenic progenitor cells proliferate and ultimately rebuild fiber integrity and function through mutual fusion or differentiation with damaged fibers, maintaining the regeneration needs of damaged muscle tissue.

[0003] In vitro studies of avian skeletal muscle development primarily focus on myoblasts. However, the isolation process of primary chicken myoblasts is complex and time-consuming, the cells cannot be passaged indefinitely, and it is difficult to obtain stable and reliable results from cells derived from different batches of chicken embryos. MSCs, on the other hand, possess the advantages of self-renewal potential through proliferation and differentiation, and can differentiate into myocytes, adipocytes, and osteoblasts under different culture conditions. They have gradually become a research hotspot and commonly used cell type for in vitro studies of avian skeletal muscle development. Currently, a certain purity of avian skeletal muscle satellite cells can be obtained through collagenase digestion. However, these cells begin to show significant senescence and apoptosis after 3-5 passages in vitro, which is insufficient to meet experimental requirements. Multiple isolation processes are complex and time-consuming, and the stability and reliability of cells derived from different batches of chicken embryos cannot be guaranteed. Furthermore, the purity of passaged avian skeletal muscle satellite cells is insufficient, and contamination from other cells is frequently encountered during isolation from skeletal muscle. However, there are currently no reports on immortalized poultry skeletal muscle satellite cells. Therefore, establishing immortalized poultry skeletal muscle satellite cells is of great significance for in vitro differentiation research of skeletal muscle satellite cells, growth and development of body muscles, fat deposition, and bone formation and reconstruction processes. Summary of the Invention

[0004] The first objective of this invention is to provide an immortalized chicken skeletal muscle satellite cell line to solve the problems in the prior art where primary chicken skeletal muscle satellite cells cannot be passaged indefinitely, cells from different batches of chicken embryos are difficult to obtain stable and reliable results, and the isolated chicken skeletal muscle satellite cells are contaminated by other cells.

[0005] The second objective of this invention is to provide a method for constructing an immortalized chicken skeletal muscle satellite cell line, thereby solving the problem that existing technologies cannot obtain immortalized chicken skeletal muscle satellite cells.

[0006] The third objective of this invention is to provide the application of immortalized chicken skeletal muscle satellite cell lines in the construction of in vitro cell models, in order to solve the problem that in the prior art, it is necessary to isolate primary chicken skeletal muscle satellite cells multiple times in the construction of in vitro cell models, which leads to waste of time and cost and difficulty in obtaining stable and reliable results.

[0007] To achieve the above objectives, the technical solution of the immortalized chicken skeletal muscle satellite cell line in this invention is as follows:

[0008] An immortalized chicken skeletal muscle satellite cell line, with accession number CCTCC NO: C2023115.

[0009] The immortalized chicken skeletal muscle satellite cell line Im MSLC of the present invention was deposited on July 5, 2023 at the China Center for Type Culture Collection, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China (Wuhan University).

[0010] The beneficial effects of the above technical solution are as follows: The immortalized chicken skeletal muscle satellite cell line ImMSLC of the present invention has the following characteristics: First, the chicken TERT gene is introduced into primary chicken skeletal muscle satellite cells through lentiviral infection. After screening with blast fungicides, the immortalized chicken skeletal muscle satellite cell line ImMSLC maintains a similar growth rate and consistent growth trend as primary chicken skeletal muscle cells after 10 passages, and retains the same cell morphology as primary chicken skeletal muscle satellite cells after 22 passages. Second, the immortalized chicken skeletal muscle satellite cell line ImMSLC still has differentiation ability after 10 passages. The immortalized chicken skeletal muscle satellite cell line ImMSLC of the present invention provides a new platform and cell model for functional research on poultry skeletal muscle-related genes, and is of great significance for studying the in vitro differentiation of skeletal muscle satellite cells and the growth and development of poultry skeletal muscle.

[0011] In the early stages of this invention, lentiviruses carrying exogenous chicken TERT gene or SV40 large T antigen (SV40-LT) were constructed and used to infect primary chicken skeletal muscle satellite cells. After screening and identification, these cells were then cultured on a large scale. However, during the large-scale culture process, it was found that chicken skeletal muscle satellite cells infected with SV40-LT lentiviruses lost their original state and gradually lost their differentiation ability as the culture passages increased, making it impossible to obtain immortalized chicken skeletal muscle satellite cells.

[0012] To achieve the above objectives, the technical solution for constructing an immortalized chicken skeletal muscle satellite cell line in this invention is as follows:

[0013] A method for constructing an immortalized chicken skeletal muscle satellite cell line includes the following steps: introducing an exogenous chicken TERT gene into primary chicken skeletal muscle satellite cells, and establishing an immortalized cell line with normal skeletal muscle satellite cell biological characteristics after screening and identification.

[0014] The beneficial effects of the above technical solution are as follows: the method for constructing the immortalized chicken skeletal muscle satellite cell line of the present invention has the advantages of high success rate and simple operation, which can greatly reduce the construction time and cost.

[0015] As a further improvement, the primary chicken skeletal muscle satellite cells are obtained by separating chicken embryo breast muscle using digestive enzymes.

[0016] The beneficial effects of the above technical solution are as follows: using digestive enzymes to separate and obtain primary chicken skeletal muscle satellite cells is a commonly used method for separating primary cells in existing technologies, which has the advantages of being efficient, simple, and requiring no complex instruments.

[0017] As a further improvement, the digestive enzyme includes type I collagenase and bovine serum albumin.

[0018] As a further improvement, the introduction is performed by infecting primary chicken skeletal muscle satellite cells with a lentivirus carrying an exogenous chicken TERT gene.

[0019] The beneficial effects of the above technical solution are as follows: the transfection efficiency of chicken skeletal muscle satellite cells is relatively low. Using lentivirus infection helps to improve the transfection efficiency of the TERT gene, thereby increasing the chance of obtaining immortalized chicken skeletal muscle satellite cells.

[0020] As a further improvement, the screening involves culturing primary chicken skeletal muscle satellite cells infected with lentivirus using a complete culture medium containing blastomycin.

[0021] The beneficial effects of the above technical solution are as follows: The lentivirus with exogenous TERT gene constructed in this invention also contains the resistance gene of blast fungicide. After infecting primary chicken skeletal muscle satellite cells with the above lentivirus, the primary chicken skeletal muscle satellite cells infected with lentivirus are cultured in a complete culture medium containing blast fungicide, which can effectively kill the cells that have not been successfully transfected, and further increase the probability of obtaining immortalized chicken skeletal muscle satellite cell lines.

[0022] As a further improvement, the concentration of the blast fungicide is 2.5 μg / mL.

[0023] The beneficial effects of the above technical solution are that using the above concentration of blast fungicide can kill chicken skeletal muscle satellite cells that have not been successfully transfected to the greatest extent, while not affecting the morphology and growth status of the cells.

[0024] To achieve the above objectives, the technical solution for the application of the immortalized chicken skeletal muscle satellite cell line in constructing in vitro cell models in this invention is as follows:

[0025] Application of an immortalized chicken skeletal muscle satellite cell line in the construction of in vitro cell models.

[0026] The beneficial effects of the above technical solution are as follows: The immortalized chicken skeletal muscle satellite cell line Im MSLC of the present invention can avoid the tedious and lengthy isolation and culture process of skeletal muscle cells, and greatly reduce the research time; on the other hand, the cell line has excellent homogeneity, which can reduce the differences in cell activity between batches, and help to obtain more accurate experimental results. It can be used to construct in vitro cell models and conduct skeletal muscle-related research.

[0027] As a further improvement, the application is for in vitro skeletal muscle growth and development studies.

[0028] As a further improvement, the application is for in vitro fat deposition studies.

[0029] As a further improvement, the application is for in vitro bone formation and reconstruction studies.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] First, the immortalization of skeletal muscle satellite cells was previously unknown. This invention, for the first time, immortalized chicken skeletal muscle satellite cells by introducing an exogenous TERT gene, thus establishing an immortalized chicken skeletal muscle satellite cell line. This line has been successfully passaged to the 22nd generation and still maintains the same cell morphology as the primary skeletal muscle satellite cells, providing a theoretical basis for subsequent research on the immortalization of other differentiated cell types. Second, the establishment of the immortalized chicken skeletal muscle satellite cell line reduces the use of chicken embryos and lowers the possibility of obtaining unstable and unreliable results due to the different batches of myoblasts derived from chicken embryos, thereby improving animal welfare. Third, the cell line established by this method can be continuously passaged, exhibits good growth performance, is serum-dependent, and shows no malignant transformation, which is beneficial for in vitro studies of chicken skeletal muscle growth and development. Attached Figure Description

[0032] Figure 1 The immunofluorescence detection results (100X) of the primary chicken skeletal muscle satellite cell marker gene PAX7 in Example 2 of this invention;

[0033] Figure 2Morphological characteristics of immortalized chicken skeletal muscle satellite cells of different generations in Example 2 of the present invention (100X) (wherein, (A) primary chicken skeletal muscle satellite cells (Pri MSCs) (100X); (B) third generation chicken skeletal muscle satellite cells (100X); (C) 10th generation immortalized chicken skeletal muscle satellite cells (Im MSLC P10) (100X); (D) 15th generation immortalized chicken skeletal muscle satellite cells (Im MSLC P15) (100X); (E) 22nd generation immortalized chicken skeletal muscle satellite cells (Im MSLC P22) (100X));

[0034] Figure 3 The result of immunofluorescence detection (100X) of the PAX7 marker gene of Im MSLC P10 in Example 2 of this invention;

[0035] Figure 4 The result of immunofluorescence detection (100X) of the PAX7 marker gene of Im MSLC P15 in Example 2 of this invention;

[0036] Figure 5 Analysis of the proliferation characteristics of chicken skeletal muscle satellite cell lines in Example 2 of the present invention (wherein, (left) growth curves of Pri MSCs and Im MSLC P10; (right) cell cycle of Pri MSCs and Im MSLC P10);

[0037] Figure 6 Serum dependence analysis of Im MSLC P10 and Im MSLC P22 in Example 2 of this invention;

[0038] Figure 7 This is a schematic diagram (100X) of the soft agar analysis results in Example 2 of the present invention (wherein, (A) Im MSLC P10 cells cultured on soft agar for 2 days (100X); (B) Im MSLC P10 cells cultured on soft agar for 5 days (100X); (C) Im MSLC P10 cells cultured on soft agar for 2 weeks (100X); (D) HEK293T cells cultured on soft agar for 2 days (100X); (E) HEK293T cells cultured on soft agar for 5 days (100X); (F) HEK293T cells cultured on soft agar for 2 weeks (100X)).

[0039] Figure 8This is a schematic diagram of the differentiation results induced by Pri MSCs, Im MSLC P10, and Im MSLC P22 in Example 3 of the present invention (wherein, (A) changes in PAX7 gene expression in Pri MSCs, Im MSLC P10, and Im MSLC P22; (B) changes in MyOD gene expression in Pri MSCs, Im MSLC P10, and Im MSLC P22; (C) changes in MyHC gene expression in Pri MSCs, Im MSLC P10, and Im MSLC P22). Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto; unless otherwise specified, all reagents, instruments and other items used in the embodiments are commercially available products.

[0041] The following is a brief introduction to some of the biological materials, experimental reagents, and experimental equipment involved in the examples:

[0042] Experimental material: AA broiler chicken embryos at 15 embryonic ages.

[0043] Main reagents:

[0044] DMEM: F12 (Solarbio, C3130-0500); Fetal bovine serum (Gibco, 1414426); Paraformaldehyde (PFA) (Solarbio, P1110); MYHC (Proteintech, 22281-1-AP); Fluoromount-G fluorescent mounting medium (Sourthern Biotech, 0100-01); Plasmid endotoxin-free extraction kit (TIANGEN, DP118-02); Animal total RNA rapid extraction kit (GENERay, GK3016); 5×All-In-One RT MasterMix (Abm, G490); SYBR Green qPCR Master (Roche, 25325100); Agarose (Qingke Biotechnology, 1GA21B02); Type I collagenase (Solarbio, C8140); Bovine serum albumin (Solarbio, A8020); Red blood cell lysate (Solarbio, R1010); Rice blast fungicide (Solarbio, B9300); TERT lentivirus synthesized from Shanghai Sangon Biotech Co., Ltd.

[0045] Main instruments and equipment:

[0046] CO2 incubator (Thermo, USA); Biosafety cabinet (Antai, Suzhou); Fluorescence inverted microscope (Nikon, Japan); High-speed refrigerated centrifuge (Thermo, USA); Electric thermostatic shaking water bath (Shanghai Jinghong, China); Cell counter (Shanghai Ruiyu, China); PCR instrument (Thermo, USA); Micro-volume nucleic acid and protein analyzer (Thermo, USA); qPCR quantitative instrument (Thermo, USA); Vertical autoclave MLS-3751L-PC (Panasonic, Japan); Micropipettes (Eppendorf, Germany); 1.5ml and 2ml centrifuge tubes (Nikon, Japan).

[0047] Data processing and analysis:

[0048] The data from the embodiments of this invention were statistically analyzed using One-way ANOVA and Duncan in SPSS 27.0 software, and the results are expressed in Mean ± SEM form. 0.01 < P < 0.05 indicates a significant difference, and P < 0.01 indicates a highly significant difference.

[0049] Example 1: An Immortalized Chicken Skeletal Muscle Satellite Cell Line

[0050] This embodiment describes an immortalized chicken skeletal muscle satellite cell line, Im MSLC, with accession number CCTCC NO: C2023115. It was deposited on July 5, 2023, at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University.

[0051] Example 2: A method for preparing an immortalized chicken skeletal muscle satellite cell line

[0052] In this embodiment, primary chicken skeletal muscle satellite cells were isolated using digestive enzymes, then purified using differential digestion, and finally the exogenous TERT gene was introduced into the primary chicken skeletal muscle satellite cells. After screening with blastomycin, the immortalized chicken skeletal muscle satellite cell line ImMSLC of Example 1 was obtained. The construction method is described in detail below:

[0053] 1. Preparation of chicken skeletal muscle satellite cells

[0054] (a) After routine disinfection of 15-day-old chicken embryos, the pectoral muscle was separated in a laminar flow hood. The embryos were washed three times with PBS buffer containing antibiotics and bismuth subsalicylate. Blood vessels, fat, and connective tissue were removed. A small amount of digestive enzyme was added (the digestive enzyme was prepared by adding 0.1g type I collagenase and 2g bovine serum albumin to 100mL DMEM / F12 medium and mixing well). The pectoral muscle tissue was then picked up with curved forceps and placed in a 1.5mL centrifuge tube and cut into a paste-like consistency using ophthalmic scissors.

[0055] (b) Transfer the minced tissue to a 50mL centrifuge tube, add sufficient digestive enzyme solution to ensure that the volume ratio of tissue to collagenase is about 1:5, seal with sealing film, and digest in a 37℃ water bath for 45-60 minutes until the liquid is viscous and no large pieces of tissue remain.

[0056] (c) Pass the mixture through cell sieves of 100 mesh, 200 mesh and 500 mesh respectively, collect the filtrate, centrifuge at 1000 r for 5 min and discard the supernatant.

[0057] (d) After centrifugation, discard the culture medium, add red blood cell lysis buffer, resuspend and let stand for 5 min, centrifuge at 1000 r for 5 min, and discard the supernatant.

[0058] (e) Add complete culture medium (1% penicillin and streptomycin, 15% fetal bovine serum and DMEM / F12 medium), resuspend by pipetting, centrifuge at 1000 r for 5 min, and discard the supernatant.

[0059] (f) Add complete culture medium, seed bottle, and place the cells in a 37°C, 5% CO2 incubator. After 4 hours, allow differential adhesion to obtain primary chicken skeletal muscle satellite cells.

[0060] 2. Identification of satellite cells in chicken skeletal muscle

[0061] The primary chicken skeletal muscle satellite cells isolated in step 1 were identified using the following procedures:

[0062] (a) Cell climbing: Take 3 glass slides into a 24-well plate, add cells, and continue to culture in a 37°C, 5% CO2 incubator.

[0063] (b) Fixation: After the cells crawl onto the slide, the culture medium was aspirated, the cells were washed once with PBS, and 4% paraformaldehyde was added and fixed at 4°C for 30 min.

[0064] (c) Membrane disruption: Discard the fixative on the slide, wash three times with PBS for 5 min each time. Remove the water from the slide, place it on a culture dish support, mix 0.5% Trition X-100 with PBS at a 1:1 ratio, and drop 50 μL of the membrane disruption solution (0.5% Trition X-100 and PBS at a 1:1 ratio) onto the waterproof membrane. Cover the slide with the side containing cells and disrupt the membrane at room temperature for 20–30 min.

[0065] (d) Blocking: Discard the membrane rupture solution, wash with PBS 3×5min / time, add goat serum to the slide, and block at room temperature for 30min.

[0066] (e) Primary antibody incubation: Discard the blocking solution, do not wash, take 50 μL of MYHC primary antibody (antibody and PBS 1:100) on a waterproof membrane (in a humidified chamber), cover with a glass slide (the side with cells) and place at 4°C.

[0067] (f) Secondary antibody incubation: Incubate the secondary antibody (secondary antibody: PBS = 1:500) at room temperature in the dark for 1 hour, then wash 3 times with PBS.

[0068] (g) Nuclear staining: stain with DAPI for 5 min, then wash 3 times with PBS.

[0069] (h) Embedding: Place 1 drop of Fluoromount-G on each slide and cover the side with cells.

[0070] (i) Photography: Observe under a fluorescence microscope and take pictures for preservation.

[0071] The results of the identification are as follows Figure 1 As shown, by Figure 1 It can be seen that the Pax7 gene is positively expressed in the cell nucleus in the cells isolated in step 1, indicating that the isolated cells are indeed chicken skeletal muscle satellite cells. The positive expression rate of the Pax7 gene is 92%, which can be used for subsequent experimental research.

[0072] The Pax7 gene is primarily expressed in skeletal muscle satellite cells, while its expression is low or absent in other cells. Pax7 is mainly expressed in late embryonic development and after birth in poultry. Its expression level increases during muscle injury, and its expression indicates the presence of skeletal muscle satellite cells. Therefore, the Pax7 gene is often used to identify skeletal muscle satellite cells.

[0073] 3. Determination of the killing curve of blast fungicide

[0074] (a) The primary chicken skeletal muscle satellite cells separated in step 1 were divided into groups of 3 × 10⁻⁶ cells per well. 5 The plates were laid into 24-well plates and placed in an incubator at 37°C with 5% CO2 for further incubation.

[0075] (b) When the cells reach 80%, remove the original culture medium from the 24-well plate and add fresh culture medium containing different concentrations of blast fungicide (0 μg / mL, 2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, 10 μg / mL, 12.5 μg / mL) to the 24-well plate that has been coated with cells.

[0076] (c) Replace the screening medium with fresh medium every 2 days and observe the cell survival rate daily. The minimum concentration of blast fungicide used is the lowest screening concentration that kills all cells within 1 to 4 days from the start of blast fungicide screening.

[0077] Results: The concentration of blast fungicide used was 2.5 μg / mL, and the action time was 4 days.

[0078] 4. Immortality of chicken skeletal muscle satellite cells

[0079] (a) Remove the original culture medium from the primary chicken skeletal muscle satellite cells, wash twice with PBS, add 0.25% trypsin to the culture flask, and gently shake the flask to ensure the trypsin completely covers the cells. Observe the cell detachment under an inverted microscope. When more than 60% of the cells have detached from the bottom of the flask, add 3 times the volume of complete culture medium to stop digestion, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Subsequently, seed the cells into 6-well plates.

[0080] (b) On the second day, after the cells adhered, the medium was changed. 1 mL of complete culture medium was added to a 1.5 mL centrifuge tube, followed by TERT lentivirus. After mixing, the mixture was added to wells (the primary chicken skeletal muscle satellite cells with TERT lentivirus were used as the experimental group; a control group was also set up, consisting of primary chicken skeletal muscle satellite cells seeded in 6-well plates in step (a) without TERT lentivirus, and the subsequent culture procedures for the control and experimental groups were the same). The cells were then incubated at 37°C in a 5% CO2 incubator. After 12 hours, the cell status was observed, and the medium was replaced with fresh medium.

[0081] (c) When the cells reach 80% confluence, passage them to T25 culture flasks.

[0082] (d) When the cells reach 80% confluence, the infected cells are divided into groups of 3 × 10⁶ cells per well. 5 Lay the incubator in a 24-well plate and incubate overnight.

[0083] (e) When the cells reached 80% confluence, remove the old culture medium from the 24-well plate and add selection medium (with blastomycin added to a final concentration of 2.5 μg / mL). Replace the selection medium with fresh medium every 2 days and observe the cell survival rate daily until the control group cells die completely. Then, continue to passage the experimental group in complete culture medium until positive cell lines are obtained.

[0084] Note: After obtaining positive cell lines, this invention performed subclonal screening of positive cell lines. However, during the screening process, it was found that individual positive cells could not grow normally, and cell clones of single positive cells could not be obtained. This may be related to the characteristics of skeletal muscle satellite cells, which needs to be studied further.

[0085] (f) After the positive cell clones have expanded, the positive cell clones are digested and passaged into 12-well plates for large-scale culture. Figure 2 ).

[0086] As can be seen from the figure, Figure 2 A represents the morphology of primary chicken skeletal muscle satellite cells. Figure 2B shows the morphology of primary chicken skeletal muscle satellite cells after three passages. It can be seen that the morphology of the third-generation cells has changed and their proliferation is very slow. Figure 2 C, D, and E show the cell morphology of the 10th, 15th, and 22nd generations of the TERT lentivirus-infected chicken skeletal muscle satellite cell line, respectively. It can be seen that, compared with the primary cells, the TERT lentivirus-infected chicken skeletal muscle satellite cell line did not change in cell morphology during continuous passage culture and remained fibrous.

[0087] 5. Identification of immortalized chicken skeletal muscle cell lines infected with TERT lentivirus

[0088] Immortalized chicken skeletal muscle satellite cell lines cultured to the 8th and 15th generations were used for identification. The experimental procedure is detailed in step 2. The results of the identification of the 8th and 15th generation immortalized chicken skeletal muscle satellite cell lines are as follows: Figure 3 , Figure 4 As shown in the figure, PAX7 is positively expressed in the nucleus in Im MSLC P10 and P15 cells. The cells infected, screened, and expanded by TERT lentivirus are chicken skeletal muscle satellite cells.

[0089] 6. Proliferation analysis of the 10th generation immortalized chicken skeletal muscle cell line

[0090] Primary chicken skeletal muscle satellite cells and 10th generation immortalized chicken skeletal muscle satellite cell lines were used, and cell growth curves were plotted using the CCK-8 assay. Cell growth was measured at a rate of 5 × 10⁻⁶ cells / year. 2 Cells were evenly seeded into 96-well plates at a density of [number] cells per well and cultured at 37°C in a 5% CO2 incubator for 8 days. From day 1 to day 8, complete culture medium and CCK8 reagent were mixed at a ratio of 9:1, the original medium was discarded, and the mixture was replaced with complete culture medium containing CCK8 reagent. The plates were then returned to the incubator for another 2 hours. The absorbance was measured at 450 nm using a microplate reader, and cell growth curves were plotted.

[0091] The results are as follows Figure 5 As shown, by Figure 5 As shown in the left figure, primary chicken skeletal muscle satellite cells and 10th generation immortalized chicken skeletal muscle satellite cells were continuously cultured for 8 days. The cells were in the latent phase from 1 to 2 days, in the logarithmic growth phase from 3 to 6 days, and then entered the plateau phase. Both showed the same growth trend.

[0092] 7. Cell cycle analysis of the 10th generation immortalized chicken skeletal muscle cell line

[0093] Cell cycle analysis was performed on primary chicken skeletal muscle satellite cells and the 10th generation immortalized chicken skeletal muscle satellite cell line. The specific experimental procedures are as follows:

[0094] (a) Mix Pri MSCs and Im MSLC P10 at 2×10 6 Cells per well were seeded in 6-well plates.

[0095] (b) Once the cells have reached 80% confluence, wash them three times with PBS, digest them with 0.25% trypsin at 37°C for 1 min.

[0096] (c) Add 3 times the volume of complete culture medium to stop digestion, collect the cells in a 1.5 mL centrifuge tube, and centrifuge at 2000 r / min for 4 min.

[0097] (d) Discard the supernatant, add 1 mL of pre-cooled PBS to resuspend the cells, transfer the cell suspension to a new 1.5 mL EP tube, and centrifuge at 2000 r / min for 4 min.

[0098] (e) Discard the supernatant, add 1 mL of pre-cooled PBS to resuspend the cells, transfer the cell suspension to a new EP tube, and centrifuge at 2000 r / min for 4 min.

[0099] (f) Discard the supernatant, add 1 mL of pre-cooled 70% ethanol, blow well, and fix at 4°C for 12–24 h.

[0100] (g) Centrifuge the fixed cell suspension at 1200 r / min for 5 min.

[0101] (h) Discard the supernatant, add 1 mL of pre-cooled PBS to resuspend the cells, and centrifuge the cell suspension at 1200 r / min for 5 min.

[0102] (i) Discard the supernatant, add 0.5 mL of propidium iodide staining solution to each tube, slowly and thoroughly resuspend the cells, incubate at 37°C in the dark for 30 min, and store at 4°C. Detect red fluorescence at 488 nm using flow cytometry and perform data analysis.

[0103] Cell cycle analysis results are shown below Figure 5 ,Depend on Figure 5 As shown in the right figure, the number of chicken skeletal muscle satellite cells in the S phase in the 10th generation immortalized chicken skeletal muscle satellite cells was more than that in the primary generation chicken skeletal muscle satellite cells, and the difference was extremely significant (P<0.01). Im MSLC P10 had higher proliferative activity.

[0104] 8. Serum-dependent analysis of 10th and 22nd generation immortalized chicken skeletal muscle satellite cell lines

[0105] Satellite cells of skeletal muscle from the 10th and 22nd generations of immortalized chickens were divided into groups of 5 × 10⁻⁶. 2Cells were seeded per well in 96-well plates and cultured for 12 hours until fully adhered. The culture medium was then replaced with DMEM / F12 medium containing 0%, 5%, 10%, and 20% fetal bovine serum, respectively, with at least three biological replicates for each concentration. After 72 hours of incubation, 10 μL of LCK-8 reagent was added to each well, and the plates were incubated at 37°C for 2 hours. The absorbance was measured at 450 nm using a microplate reader, and a bar chart was plotted.

[0106] The results of the serum dependence analysis are shown below. Figure 6 As shown in the figure, the immortalized chicken skeletal muscle satellite cell line could not grow normally in the absence of serum, but it could proliferate at serum concentrations of 5%, 10%, and 20%. The ability of ImMSLC P10 and ImMSLC P22 to promote proliferation at a serum concentration of 15% was significantly higher than that at a serum concentration of 0% (P<0.0001). This indicates that the immortalized chicken skeletal muscle satellite cells maintained serum dependence during in vitro passage, suggesting that the introduction of the TERT gene did not induce malignant transformation in the chicken skeletal muscle satellite cells.

[0107] 9. Soft agar analysis of the 10th generation immortalized chicken skeletal muscle cell line

[0108] Satellite cells of skeletal muscle from the 10th generation of immortalized chickens and 293T cells were analyzed on soft agar. The specific experimental procedures are as follows:

[0109] (a) Prepare 100 mL of 1.2% and 0.7% agarose culture medium, autoclave and place in an oven;

[0110] (b) Slowly mix 5 mL of 1.2% Agarose medium with 5 mL of 20% FBS DMEM / F12 medium in a 50 mL centrifuge tube. Add 1.5 mL to each well of a six-well plate and slowly spread the mixture, being careful not to generate air bubbles. After solidification, store in a 37°C, 5% CO2 incubator.

[0111] (c) Wash the cells twice with PBS, digest the cells with 0.25% trypsin for about 1 min, add complete culture medium to stop digestion, and centrifuge at 1000 r / min for 5 min.

[0112] (d) Discard the supernatant, add 1 mL of complete culture medium to resuspend the cells, and take 10 μL for cell counting.

[0113] (e) Dilute immortalized skeletal muscle satellite cells to 1000 per mL and HEK293T cells to 200 per mL.

[0114] (f) Slowly mix 3 mL of 0.7% Agarose medium with 3 mL of 20% FBS DMEM / F12 medium in a 50 mL centrifuge tube, add 6 mL of cells and mix slowly. Add 1 mL to each well, spread it on the lower gel, and return it to the incubator for further culture.

[0115] (g) Add 200 μL of complete culture medium every 2 days to prevent the well plate from drying out too much.

[0116] (h) Culture for 2 weeks, and after HEK293T cells grow into clones, take a picture and save it.

[0117] Another important indicator for evaluating whether cells have become cancerous is soft agar analysis. Tumor cells can typically grow in soft agar and form cell clones, while normal cells do not grow in soft agar and do not form cell clones. The soft agar analysis results of the 10th generation immortalized chicken skeletal muscle satellite cells are shown below. Figure 7 As shown in the figure, after culturing for 2 days, 5 days, and 2 weeks, the chicken skeletal muscle satellite cell lines remained as isolated cells in soft agar, without forming cell clones. Figure 7 A, B, C), while 293T cells formed distinct independent cell clone clusters ( Figure 7 (D, E, F). Therefore, the immortalized chicken skeletal muscle satellite cell line did not undergo malignant transformation.

[0118] Example 3: Application of Immortalized Chicken Skeletal Muscle Satellite Cell Line in Constructing In Vitro Cell Models

[0119] This embodiment uses the immortalized chicken skeletal muscle satellite cell line from Example 1 to construct and analyze an induced differentiation model. The specific procedures are as follows:

[0120] Primary chicken skeletal muscle satellite cells, as well as 10th and 22nd generation immortalized chicken skeletal muscle satellite cells, were seeded in 24-well plates and cultured for 12 hours until complete cell adhesion. The culture medium was then replaced with 2% horse serum-inducing differentiation medium, and cells that had grown to 70% and were 1–4 days post-differentiation were harvested. The primers listed in Table 1 were used to detect marker genes in skeletal muscle satellite cells, myoblasts, and myofibrils.

[0121] Table 1 Detection primer sequences

[0122]

[0123] Chicken skeletal muscle satellite cells, after induction with 2% horse serum, can differentiate into myoblasts and myofibrils. PAX7, MyOD, and MyHC were used as marker genes for chicken skeletal muscle satellite cells, myoblasts, and myofibrils, respectively. The detection results are as follows... Figure 8As shown in the figure, the expression of the PAX7 gene in primary chicken skeletal muscle cells first decreases and then increases from cell proliferation to 70% to 1-2 days after induction of differentiation. However, the expression of the PAX7 gene in 10th generation immortalized chicken skeletal muscle cells begins to decrease 1 day after induction of differentiation, and then increases. Figure 8 A); The expression of the MyOD gene in primary chicken skeletal muscle cells first increases and then decreases from cell proliferation to 70% to 1-2 days after induction of differentiation, while the expression of the PAX7 gene in 10th generation immortalized chicken skeletal muscle cells begins to increase 1 day after induction of differentiation, and then decreases. Figure 8 B); The expression of the MyHC gene in primary chicken skeletal muscle cells began to increase 1 day after cell differentiation induction, and then decreased, while the expression of the MyHC gene in 10th generation immortalized chicken skeletal muscle cells began to increase 1 day after differentiation induction, and then decreased. Figure 8 C). Therefore, although the 10th generation immortalized chicken skeletal muscle cells differentiated one day later than the primary cells, they still possessed differentiation capacity. The expression trend of PAX7 in primary chicken skeletal muscle cells and 22nd generation immortalized chicken skeletal muscle cells at 70% proliferation and 1-4 days after induction of differentiation was consistent, first decreasing and then increasing. Figure 8 A); The expression trends of MyOD in primary chicken skeletal muscle cells and 22nd generation immortalized chicken skeletal muscle cells at 70% proliferation and induced differentiation 1-4 days were consistent, first increasing and then decreasing. Figure 8 B); The expression trend of MyHC in primary chicken skeletal muscle cells and 22nd generation immortalized chicken skeletal muscle cells after proliferation to 70% and induced differentiation 1-4 days was consistent, both showing an initial increase followed by a decrease. Figure 8 C).

[0124] The above examples are chicken skeletal muscle satellite cells transfected with TERT at passages 10 and 22. Their identification results and biological function verification prove that the immortalized chicken skeletal muscle satellite cell line obtained by this invention maintains normal cell morphology and serum dependence during culture. Soft agar analysis proves that the immortalized chicken skeletal muscle satellite cell line did not deteriorate during culture. The construction and analysis of the induced differentiation model proves that the immortalized chicken skeletal muscle satellite cell line basically maintains the same differentiation trend as the primary chicken skeletal muscle cells during culture.

[0125] Currently, the immortalized chicken skeletal muscle satellite cell line has been passaged to the 22nd generation, and the above characteristics have been maintained and continued. The experimental results of the in vitro cell model constructed with it are not significantly different from the primary cell model, which proves that the TERT transfected chicken skeletal muscle satellite cell line of the present invention is very suitable for constructing in vitro cell models, thereby drastically shortening the model construction time. Furthermore, by utilizing its homogeneity, the relevant research results are made more objective and in line with reality.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An immortalized chicken skeletal muscle satellite cell line, characterized in that: Its preservation number is CCTCC NO: C2023115.

2. Use of the immortalized chicken skeletal muscle satellite cell line of claim 1 in constructing an in vitro model of cells.

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