Immortalized chicken skeletal muscle satellite cell line as well as construction method and application thereof

Abstract

The invention relates to an immortalized chicken skeletal muscle satellite cell line as well as a construction method and application thereof, and belongs to the technical field of biology. The immortalized chicken skeletal muscle satellite cell line Im MSLC has the preservation number of CCTCC NO: C2023115, and is preserved in the China Center for Type Culture Collection on July 5, 2023, and the address is Wuhan University, Wuhan University, No. 299, eight road, Wuchang District, Wuhan City, Hubei Province. After 10 generations of passage, the immortalized chicken skeletal muscle satellite cell line can still keep the growth speed similar to that of primary chicken skeletal muscle cells and keep the consistent growth trend, and after 22 generations of passage, the immortalized chicken skeletal muscle satellite cell line still keeps the same cellular morphology as the primary cells; and the immortalized chicken skeletal muscle satellite cell line still has differentiation capability after 10 generations of passage. According to the immortalized chicken skeletal muscle satellite cell line provided by the invention, the use of chick embryos is reduced, and the possibility that stable and reliable results are difficult to obtain due to different batches of chick embryos from skeletal muscle cells is reduced.

Description

Technical Field

[0001] The invention relates to an immortalized chicken skeletal muscle satellite cell line and a construction method and application thereof, belonging to the field of biotechnology. Background Art

[0002] Skeletal muscle is one of the important parts of livestock and poultry, and plays an important role in maintaining livestock and poultry movement and posture support. Muscle tissue is mainly composed of long fibers formed by multinucleated cells after mitosis, and its lifelong maintenance is mediated by satellite cells. Skeletal muscle satellite cells (MSCs) are a type of myogenic stem cells with proliferation and differentiation potential located between the myocyte membrane and the basement membrane, and play a vital role in the normal development and regeneration and repair of muscles. MSCs are generally in a relatively quiescent state. When muscle tissue is damaged or otherwise stimulated, a part of MSCs can be activated. After activation, satellite stem cells divide symmetrically to expand their number or divide asymmetrically to produce directional satellite cell queues, thereby producing progenitor cells. Myogenic progenitor cells proliferate and eventually rebuild fiber integrity and function by mutual fusion or differentiation with damaged fibers to maintain the regeneration needs of damaged muscle tissue.

[0003] In vitro studies of poultry skeletal muscle development are mainly based on myoblasts, but the isolation process of primary chicken myoblasts is complex and time-consuming, 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 have the advantages of self-renewal potential such as proliferation and differentiation, and can be directed to differentiate into myocytes, adipocytes, and osteocytes under different culture conditions. They have gradually become a research hotspot and commonly used cells for in vitro studies of poultry skeletal muscle development. At present, poultry skeletal muscle satellite cells with a certain purity can be obtained by collagenase digestion. However, these cells begin to show a large number of aging and apoptosis phenomena after 3-5 generations of in vitro culture, which cannot meet the experimental needs at all. If multiple separations are performed, the process is complicated and time-consuming, and the stability and reliability of cells derived from different batches of chicken embryos cannot be guaranteed. At the same time, the purity of poultry skeletal muscle satellite cells in passaged culture is not enough, and there is often contamination from other cells when they are separated 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 the in vitro differentiation of skeletal muscle satellite cells, the growth and development of muscle, fat deposition, and bone formation and reconstruction. Summary of the invention

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

[0005] The second object of the present invention is to provide a method for constructing an immortalized chicken skeletal muscle satellite cell line to solve the problem that immortalized chicken skeletal muscle satellite cells cannot be obtained in the prior art.

[0006] The third object of the present invention is to provide an immortalized chicken skeletal muscle satellite cell line for use in constructing an in vitro cell model, so as to solve the problem in the prior art that in constructing an in vitro cell model, primary chicken skeletal muscle satellite cells need to be separated multiple times, resulting in a waste of time and cost and difficulty in obtaining stable and reliable results.

[0007] In order to achieve the above object, a technical solution of an immortalized chicken skeletal muscle satellite cell line in the present invention is:

[0008] The invention discloses an immortalized chicken skeletal muscle satellite cell line, and its deposit number is CCTCC NO: C2023115.

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

[0010] The beneficial effects of the above technical scheme are: the immortalized chicken skeletal muscle satellite cell line Im MSLC of the present invention has the following characteristics: first, the chicken TERT gene is introduced into the primary chicken skeletal muscle satellite cells by lentiviral infection, and the immortalized chicken skeletal muscle satellite cell line Im MSLC obtained by blasticidin screening can still maintain a growth rate similar to that of the primary chicken skeletal muscle cells after 10 generations, maintain a consistent growth trend, and still maintain the same cell morphology of the primary chicken skeletal muscle satellite cells after 22 generations; second, the immortalized chicken skeletal muscle satellite cell line Im MSLC still has differentiation ability after 10 generations. The immortalized chicken skeletal muscle satellite cell line Im MSLC of the present invention provides a new platform and cell model for the functional study of poultry skeletal muscle-related genes, which 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 stage of the present invention, a lentivirus with exogenous chicken TERT gene or SV40 large T antigen (SV40-LT) is constructed to infect primary chicken skeletal muscle satellite cells respectively, and after screening and identification, the cells are expanded and cultured. However, during the expansion culture process, it is found that the chicken skeletal muscle satellite cells infected with the SV40-LT lentivirus lose their original state and gradually lose their differentiation ability as the culture generations increase during the expansion culture, and immortalized chicken skeletal muscle satellite cells cannot be obtained.

[0012] In order to achieve the above object, the technical scheme of the method for constructing an immortalized chicken skeletal muscle satellite cell line in the present invention is:

[0013] A method for constructing an immortalized chicken skeletal muscle satellite cell line comprises the following steps: introducing 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 effect of the above technical solution is that 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, and can greatly reduce the time and cost of construction.

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

[0016] The beneficial effect of the above technical solution is that the use of digestive enzymes to separate and obtain primary chicken skeletal muscle satellite cells is a commonly used method for separating primary cells in the prior art, and has the advantages of being highly efficient, simple, and not requiring 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 to infect primary chicken skeletal muscle satellite cells with a lentivirus carrying exogenous chicken TERT gene.

[0019] The beneficial effect of the above technical solution is that the transfection efficiency of chicken skeletal muscle satellite cells is low, and the use of lentiviral infection helps to improve the transfection efficiency of the TERT gene, thereby increasing the probability of obtaining immortalized chicken skeletal muscle satellite cells.

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

[0021] The beneficial effect of the above technical solution is that the lentivirus carrying the exogenous TERT gene constructed by the present invention also contains a resistance gene for blasticidin. After the above lentivirus is infected with primary chicken skeletal muscle satellite cells, the primary chicken skeletal muscle satellite cells infected with the lentivirus are cultured using a complete culture medium containing blasticidin, which can effectively kill the cells that have not been successfully transfected, thereby further improving the probability of obtaining an immortalized chicken skeletal muscle satellite cell line.

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

[0023] The beneficial effect of the above technical solution is that: using the above concentration of blasticidin can kill the chicken skeletal muscle satellite cells that have not been successfully transfected to the greatest extent, while not affecting the morphology and growth state of the cells.

[0024] In order to achieve the above-mentioned purpose, the technical solution of the application of the immortalized chicken skeletal muscle satellite cell line in the construction of an in vitro cell model is as follows:

[0025] Application of an immortalized chicken skeletal muscle satellite cell line in constructing an in vitro cell model.

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

[0027] As a further improvement, the application is a study on the growth and development of skeletal muscle in vitro.

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

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

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] First, it was previously unknown whether skeletal muscle satellite cells could be immortalized. The present invention, for the first time, immortalized chicken skeletal muscle satellite cells by introducing exogenous TERT genes into them, and established an immortalized chicken skeletal muscle satellite cell line. It has now 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 studies on the immortalization of other cells with differentiation ability. Second, the establishment of an immortalized chicken skeletal muscle satellite cell line reduces the use of chicken embryos, reduces the possibility of obtaining stable and reliable results from myoblasts derived from chicken embryos in different batches, and improves animal welfare. Third, the cell line established by this method can be continuously passaged, has good growth performance, is serum-dependent, and has no malignant transformation phenomenon, which is conducive to in vitro research on the growth and development of chicken skeletal muscle. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0033] Figure 2The morphological 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 results of the immunofluorescence detection of the Im MSLC P10 marker gene PAX7 in Example 2 of the present invention (100X);

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

[0036] Figure 5 This is an analysis of the proliferation characteristics of the chicken skeletal muscle satellite cell line in Example 2 of the present invention (wherein, (left) Pri MSCs and Im MSLC P10 growth curves; (right) Pri MSCs and Im MSLC P10 cell cycles);

[0037] Figure 6 This is the Im MSLC P10 and Im MSLC P22 serum dependence analysis in Example 2 of the present invention;

[0038] Figure 7 Schematic diagram of the soft agar analysis results in Example 2 of the present invention (100X) (wherein, (A) Im MSLC P10 cultured in soft agar for 2 days (100X); (B) Im MSLC P10 cultured in soft agar for 5 days (100X); (C) Im MSLCP10 cultured in soft agar for 2 weeks (100X); (D) HEK293T cells cultured in soft agar for 2 days (100X); (E) HEK293T cells cultured in soft agar for 5 days (100X); (F) HEK293T cells cultured in soft agar for 2 weeks (100X));

[0039] Figure 8Schematic diagram of the results of induced differentiation of Pri MSCs, Im MSLC P10 and Im MSLC P22 in Example 3 of the present invention (wherein, (A) changes in expression of PAX7 gene in Pri MSCs, Im MSLC P10 and Im MSLC P22; (B) changes in expression of MyOD gene in Pri MSCs, Im MSLC P10 and Im MSLC P22; (C) changes in expression of MyHC gene in Pri MSCs, Im MSLCP10 and Im MSLC P22). DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with specific implementation methods, but the protection scope of the present invention is not limited thereto; unless otherwise specified, various reagents, instruments, etc. used in the examples are commercially available products.

[0041] Some of the biological materials, experimental reagents, experimental equipment, etc. involved in the following examples are briefly introduced as follows:

[0042] Experimental materials: 15-year-old AA broiler embryos.

[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 (SourthernBiotech, 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); SYBRGreen qPCR Master (Roche, 25325100); Agarose (Qingke Biotechnology, 1GA21B02); Type I collagenase (Solarbio, C8140); Bovine serum albumin (Solarbio, A8020); Red blood cell lysis buffer (Solarbio, R1010); Blasticidin (Solarbio, B9300); TERT lentivirus was synthesized from Shanghai Bioengineering Company.

[0045] Main instruments and equipment:

[0046] Carbon dioxide incubator (Thermo, USA); biological safety cabinet (Antai, Suzhou); fluorescence inverted microscope (Nikon, Japan); high-speed refrigerated centrifuge (Thermo, USA); electric constant temperature shaking water tank (Shanghai Jinghong, China); cell counter (Shanghai Ruiyu, China); PCR instrument (Thermo, USA); trace nucleic acid protein detector (Thermo, USA); qPCR quantitative instrument (Thermo, USA); vertical autoclave MLS-3751L-PC (Panasonic, Japan); micropipette (Eppendorf, Germany); 1.5ml and 2ml centrifuge tubes (Nikon, Japan).

[0047] Data processing and analysis:

[0048] The data of the embodiment of the present invention were statistically analyzed using One-way ANOVA and Duncan in SPSS27.0 software, and the results were expressed in the form of Mean±SEM. 0.01<P<0.05 indicated a significant difference, and P<0.01 indicated an extremely significant difference.

[0049] Example 1 An immortalized chicken skeletal muscle satellite cell line

[0050] An immortalized chicken skeletal muscle satellite cell line in this embodiment is an immortalized chicken skeletal muscle satellite cell line Im MSLC, with a preservation number of CCTCC NO: C2023115, which was preserved in the China Center for Type Culture Collection on July 5, 2023, address: 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 example, digestive enzymes were used to separate primary chicken skeletal muscle satellite cells, and then differential digestion was used to purify primary chicken skeletal muscle satellite cells. Finally, exogenous TERT gene was introduced into primary chicken skeletal muscle satellite cells, and after blasticidin screening, the immortalized chicken skeletal muscle satellite cell line Im MSLC in Example 1 was obtained. The construction method is described in detail as follows:

[0053] 1. Preparation of Chicken Skeletal Muscle Satellite Cells

[0054] (a) After routine disinfection of 15-day-old chicken embryos, separate the chest muscles in a clean bench, wash them three times with PBS buffer containing double antibodies, remove blood vessels, fat, and connective tissue, and add a small amount of digestive enzyme (the digestive enzyme is prepared as follows: 0.1g type I collagenase, 2g bovine serum albumin, add to 100mL DMEM / F12 medium and mix well). Then use curved forceps to clamp the chest muscle tissue into a 1.5mL centrifuge tube and cut it into minced meat with ophthalmic scissors.

[0055] (b) Transfer the chopped tissue to a 50 mL centrifuge tube and add sufficient digestive enzyme solution to achieve a tissue to collagenase volume ratio of about 1:5. Seal the tube with sealing film and digest in a 37°C water bath for 45 to 60 min until the liquid becomes viscous and no large pieces of tissue remain.

[0056] (c) The mixed solution was passed through 100-mesh, 200-mesh, and 500-mesh cell sieves, respectively. The filtrate was collected and centrifuged at 1000 r for 5 min. The supernatant was discarded.

[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 / min for 5 min, and discard the supernatant.

[0058] (e) Complete culture medium (1% penicillin-streptomycin, 15% fetal bovine serum and DMEM / F12 culture medium) was added, the suspension was resuspended by pipetting, and the suspension was centrifuged at 1000 r / min for 5 min, and the supernatant was discarded.

[0059] (f) Complete culture medium was added to the flask, and the cells were cultured in a 37°C, 5% CO2 incubator. After 4 hours, the cells were allowed to adhere to the wall at a differential rate, thereby obtaining primary chicken skeletal muscle satellite cells.

[0060] 2. Identification of Chicken Skeletal Muscle Satellite Cells

[0061] The primary chicken skeletal muscle satellite cells isolated in step 1 were identified as follows:

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

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

[0064] (c) Membrane permeabilization: Aspirate the fixative on the slide and wash with PBS three times, 5 minutes each time. Remove the water from the slide and place it on a culture dish support. Mix 0.5% Trition X-100 and PBS in a ratio of 1:1. Take 50 μL of the membrane permeabilization solution (0.5% Trition X-100 and PBS in a ratio of 1:1) and drop it on the waterproof film. Cover the side of the slide with cells and permeabilize the membrane at room temperature for 20 to 30 minutes.

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

[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 the waterproof film (in a wet box), cover the glass slide (the side with cells) and place it at 4°C.

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

[0068] (g) Nuclear staining: stain with DAPI for 5 min and wash three 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.

[0071] The identification results are as follows Figure 1 As shown by Figure 1 It can be seen that in the cells separated in step 1, the Pax7 gene is positively expressed in the cell nucleus, indicating that the separated 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 mainly expressed in skeletal muscle satellite cells, while it is expressed at a lower level or not at all in other cells. Pax7 is mainly expressed in the late embryonic stage and after birth in poultry. When the muscle is damaged, the expression level of Pax7 increases. The expression of the Pax7 gene 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 blasticidin

[0074] (a) Primary chicken skeletal muscle satellite cells isolated in step 1 were plated at 3×10 5 The cells were plated into 24-well plates and placed in a 37°C, 5% CO2 incubator for continued culture.

[0075] (b) When the cells grew to 80%, the original culture medium in the 24-well plate was removed, and fresh culture medium containing different concentrations of blasticidin (0 μg / mL, 2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, 10 μg / mL, 12.5 μg / mL) was added to the 24-well plate with cells.

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

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

[0078] 4. Immortalization of Chicken Skeletal Muscle Satellite Cells

[0079] (a) Remove the original culture medium of primary chicken skeletal muscle satellite cells, wash twice with PBS, add 0.25% trypsin to the culture flask, shake the flask slowly to allow the trypsin to completely cover the cells, observe the cell shedding under an inverted microscope, add 3 times the volume of complete culture medium to terminate digestion when more than 60% of the cells fall off the bottom of the flask, centrifuge at 1000r for 5min, and discard the supernatant. Subsequently, the cells were inoculated into a 6-well plate.

[0080] (b) The next day, after the cells adhere to the wall, change the medium. First add 1 mL of complete culture medium to a 1.5 mL centrifuge tube, then add TERT lentivirus, mix well and add to the well plate (the primary chicken skeletal muscle satellite cells with TERT lentivirus added are the experimental group, and a control group is set up at the same time. The control group is the primary chicken skeletal muscle satellite cells inoculated in a 6-well plate in step (a) without adding TERT lentivirus, and the subsequent culture operations of the control group and the experimental group are the same), and continue to culture in a 37 ° C, 5% CO2 incubator. Observe the cell state after 12 hours and replace with fresh culture medium.

[0081] (c) When the cells reached 80% of their growth rate, they were subcultured into T25 culture flasks.

[0082] (d) When the cells grew to 80%, the infected cells were plated at 3×10 5 Plate into 24-well plates and incubate overnight.

[0083] (e) When the cells grow to 80%, remove the old medium in the 24-well plate and add screening medium (add blasticidin to the screening medium at a final concentration of 2.5 μg / mL). Replace the fresh screening medium every 2 days and observe the survival rate of cells every day until the cells in the control group die completely. Then continue to subculture the experimental group with complete medium until a positive cell line is obtained.

[0084] Note: After obtaining the positive cell lines, the present invention performed subclone screening of the 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 and requires further research.

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

[0086] As can be seen from the figure, Figure 2 A shows the morphology of primary chicken skeletal muscle satellite cells. Figure 2B shows the cell 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 are the cell morphologies of the 10th, 15th, and 22nd generations of chicken skeletal muscle satellite cell lines infected with TERT lentivirus, respectively. It can be seen that compared with primary cells, the cell morphology of the chicken skeletal muscle satellite cell line infected with TERT lentivirus did not change during continuous subculture and remained fibrous.

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

[0088] The immortalized chicken skeletal muscle satellite cell lines cultured to the 8th and 15th generations were taken for identification. The experimental process is detailed in step 2. The results of the identification of the immortalized chicken skeletal muscle satellite cell lines of the 8th and 15th generations are shown in FIG. Figure 3 , Figure 4 As shown in the figure, in Im MSLC P10 and P15, PAX7 was positively expressed in the cell nucleus, and the cells infected, screened, and expanded and cultured with TERT lentivirus were 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 to draw cell growth curves using CCK-8 assay. 2 The cells were evenly inoculated into 96-well plates at a density of cells / well and placed in a 37°C, 5% CO2 incubator for 8 days. The complete medium and CCK8 reagent were mixed evenly at a ratio of 9:1 from 1 to 8 days, the original medium was discarded, and the complete medium with CCK8 reagent was replaced, and the plates were returned to the incubator for 2 hours of incubation. The absorbance value was measured at a wavelength of 450nm using an enzyme marker and the cell growth curve was drawn.

[0091] The results are as follows Figure 5 As shown by Figure 5 As can be seen in the figure on the left, primary chicken skeletal muscle satellite cells and 10th generation immortalized chicken skeletal muscle satellite cells were cultured continuously for 8 days. The cells were in the latent period for 1 to 2 days, in the logarithmic growth period for 3 to 6 days, and then entered the plateau period. The two maintained a consistent growth trend.

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

[0093] Primary chicken skeletal muscle satellite cells and 10th generation immortalized chicken skeletal muscle satellite cell lines were used for cell cycle analysis. The specific experimental procedures are as follows:

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

[0095] (b) When the cells were more than 80% confluent, they were washed three times with PBS and digested with 0.25% trypsin at 37°C for 1 min.

[0096] (c) Add 3 volumes of complete medium to terminate digestion, collect cells in a 1.5 mL centrifuge tube, and centrifuge at 2000 rpm 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 rpm 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 rpm for 4 min.

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

[0100] (g) The fixed cell suspension was centrifuged at 1200 rpm 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 rpm for 5 min.

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

[0103] The results of cell cycle analysis are shown in Figure 5 ,Depend on Figure 5 As shown in the figure on the right, the number of cells in the S phase of the 10th generation immortalized chicken skeletal muscle satellite cells is more than that of the primary chicken skeletal muscle satellite cells, and the difference between the two is extremely significant (P < 0.01). Im MSLC P10 has a higher proliferation activity.

[0104] 8. Analysis of serum dependence of immortalized chicken skeletal muscle satellite cell lines at passages 10 and 22

[0105] Immortalized chicken skeletal muscle satellite cells of passage 10 and 22 were cultured at 5×10 2Cells were inoculated into 96-well plates at 100 μL / well. After 12 hours of culture, the culture medium was replaced with DMEM / F12 culture medium containing 0%, 5%, 10%, and 20% fetal bovine serum, respectively. At least 3 biological replicates were performed for each concentration. After returning to the incubator and continuing to culture for 72 hours, 10 μL CCK-8 reagent was added to each well and cultured in a 37°C incubator for 2 hours. The absorbance value was measured at a wavelength of 450 nm using an ELISA reader and a bar graph was drawn.

[0106] The results of serum-dependent analysis are shown in Figure 6 As shown in the figure, the immortalized chicken skeletal muscle satellite cell line cannot grow normally in the absence of serum, and can proliferate when the serum concentration is 5%, 10%, and 20%. The ability of ImMSLC P10 and Im MSLC P22 to promote proliferation at a serum concentration of 15% is significantly higher than that at a serum concentration of 0% (P<0.0001). This shows that the immortalized chicken skeletal muscle satellite cells still maintain serum dependence during in vitro passage, indicating that the introduction of the TERT gene did not cause malignant transformation of chicken skeletal muscle satellite cells.

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

[0108] The 10th generation immortalized chicken skeletal muscle satellite cells and 293T cells were used for soft agar analysis. The specific experimental procedures are as follows:

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

[0110] (b) Slowly mix 5 mL of 1.2% Agarose medium and 5 mL of 20% FBS DMEM / F12 medium in a 50 mL centrifuge tube, add 1.5 mL of each well and slowly spread on a six-well plate without creating 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 medium to terminate digestion, and centrifuge at 1000 rpm for 5 min.

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

[0113] (e) Immortalized skeletal muscle satellite cells were diluted to 1000 cells per 1 mL, and HEK293T cells were diluted to 200 cells per 1 mL.

[0114] (f) Slowly mix 3 mL of 0.7% Agarose medium and 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, layer on the lower layer of gel, and return to the incubator for continued culture.

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

[0116] (h) After culturing for 2 weeks, HEK293T cells grew into clones and were photographed and preserved.

[0117] Another important indicator for evaluating whether cells have become cancerous is soft agar analysis. Tumor cells can usually grow in soft agar and form cell clones, while normal cells cannot grow in soft agar and cannot form cell clones. The results of soft agar analysis of immortalized chicken skeletal muscle satellite cells at the 10th generation are shown in Figure 7 As can be seen from the figure, after culturing for 2 days, 5 days, and 2 weeks, the chicken skeletal muscle satellite cell line was a single cell in the soft agar and no cell clones were formed ( Figure 7 A, B, C), while 293T cells formed distinct independent cell clones ( 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 model

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

[0120] Primary chicken skeletal muscle satellite cells, 10th and 22nd generation immortalized chicken skeletal muscle satellite cells were plated in 24-well plates and cultured for 12 hours until the cells were completely attached. The culture medium was replaced with 2% horse serum induction differentiation medium, and cells grown to 70% and differentiated 1 to 4 days later were collected. The primers in Table 1 were used to detect marker genes of skeletal muscle satellite cells, myoblasts, and muscle fibers.

[0121] Table 1 Detection primer sequences

[0122]

[0123] Chicken skeletal muscle satellite cells can differentiate into myoblasts and muscle fibers after being induced by 2% horse serum. PAX7, MyOD, and MyHC are used as marker genes for chicken skeletal muscle satellite cells, myoblasts, and muscle fibers, respectively. Figure 8As shown in the figure, the PAX7 gene expression of primary chicken skeletal muscle cells first decreased and then increased when the cells proliferated to 70% and induced differentiation for 1 to 2 days, while the PAX7 gene expression of 10th generation immortalized chicken skeletal muscle cells began to decrease after 1 day of induction of differentiation, and then increased ( Figure 8 A); MyOD gene expression in primary chicken skeletal muscle cells first increased and then decreased when the cells proliferated to 70% and induced differentiation for 1-2 days, while PAX7 gene expression in 10th generation immortalized chicken skeletal muscle cells began to increase after 1 day of differentiation induction and then decreased ( Figure 8 B); MyHC gene expression in primary chicken skeletal muscle cells began to increase after 1 day of cell differentiation induction and then decreased, while MyHC gene expression in 10th generation immortalized chicken skeletal muscle cells began to increase after 1 day of 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 had differentiation ability. The expression trend of PAX7 in primary chicken skeletal muscle cells and 22nd generation immortalized chicken skeletal muscle cells when they proliferated to 70% and induced differentiation for 1 to 4 days was consistent, first decreasing and then increasing ( Figure 8 A); The expression trend of MyOD in primary chicken skeletal muscle cells and 22nd generation immortalized chicken skeletal muscle cells was consistent when they proliferated to 70% and induced differentiation for 1 to 4 days, 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 was consistent when they proliferated to 70% and induced differentiation for 1 to 4 days, both of which increased first and then decreased ( Figure 8 C).

[0124] The above takes the TERT-transfected chicken skeletal muscle satellite cells of the 10th and 22nd generations as an example. The identification results and biological function verification prove that the immortalized chicken skeletal muscle satellite cell line obtained by the present invention maintains normal cell morphology and serum dependence during the culture process. Soft agar analysis proves that the immortalized chicken skeletal muscle satellite cell line does not deteriorate during the culture process. The construction and analysis of the induced differentiation model prove that the immortalized chicken skeletal muscle satellite cell line basically maintains the same differentiation trend as the primary chicken skeletal muscle cells during the culture process.

[0125] At present, the immortalized chicken skeletal muscle satellite cell line has been propagated to the 22nd generation, and the above characteristics have been maintained and continued. The experimental results of the in vitro cell model constructed by it have no significant difference 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 an in vitro cell model, thereby drastically shortening the model construction time, and utilizing its homogeneity to make the relevant research results more objective and practical.

[0126] Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 deposit number is CCTCC NO: C2023115.

2. A method for constructing an immortalized chicken skeletal muscle satellite cell line as claimed in claim 1, characterized in that: The following steps are involved: The exogenous chicken TERT gene was introduced into primary chicken skeletal muscle satellite cells, and an immortalized cell line with normal biological characteristics of skeletal muscle satellite cells was established after screening and identification.

3. The method for constructing an immortalized chicken skeletal muscle satellite cell line according to claim 2, characterized in that: The primary chicken skeletal muscle satellite cells are obtained by separating chicken embryo pectoral muscles using digestive enzymes.

4. The method for constructing an immortalized chicken skeletal muscle satellite cell line according to claim 3, characterized in that: The digestive enzymes include type I collagenase and bovine serum albumin.

5. The method for constructing an immortalized chicken skeletal muscle satellite cell line according to claim 2, characterized in that: The introduction is to infect primary chicken skeletal muscle satellite cells with a lentivirus carrying an exogenous TERT gene.

6. The method for constructing an immortalized chicken skeletal muscle satellite cell line according to claim 2, characterized in that: The screening is performed by culturing primary chicken skeletal muscle satellite cells infected with lentivirus using a complete culture medium containing blasticidin.

7. The method for constructing an immortalized chicken skeletal muscle satellite cell line according to claim 6, characterized in that: The use concentration of the blasticidin is 2.5 μg / mL.

8. Use of the immortalized chicken skeletal muscle satellite cell line according to claim 1 in constructing an in vitro cell model.

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