Separation method of tree shrew SMSCs, immortalized SMSCs, construction method and application
Through mixed enzyme digestion method and SV40 overexpression lentivirus technology, immortalized cell lines of tree shrew SMSCs were isolated and constructed, and the technical difficulties of tree shrew SMSCs were solved, providing a foundation for subsequent research, and realizing tree shrew Efficient isolation and immortalization of SMSCs.
Patent Information
- Application Number
- CN202410236090.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-01
- Publication Date
- 2025-01-10
AI Technical Summary
The prior art has failed to effectively isolate and construct tree shrew skeletal muscle satellite cells (SMSCs), and there is a lack of methods for immortalizing tree shrew SMSCs.
SMSCs were isolated from adult tree shrew muscle tissue by mixed enzyme digestion method, and identified by cell immunofluorescence, RT-PCR, etc., and SV40 overexpression of lentivirus infected cells was used to establish immortalized cell lines of tree shrew SMSCs to construct tree shrew Skeletal muscle satellite cell line SQJC-HGY.
The efficient isolation and immortalization of tree shrew SMSCs was successfully achieved, providing the basis for muscle injury models and biological phylogenetic research, and preliminary exploration of the biological characteristics and functions of tree shrew SMSCs.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of tree shrew immortalized SMSCs construction, and more particularly to tree shrew immortalized SMSCs capable of stable passage, a construction method and an application thereof. Background Art
[0002] Tree shrew (Tupaia Belangeri Chinensis) is a new type of experimental animal, which has gradually attracted the attention and application of scholars at home and abroad in recent years. Tree shrews belong to the family Tupaiaceae of the order Shrews. There are abundant tree shrew resources in Guangxi Zhuang Autonomous Region, Yunnan, Guizhou and other places in my country. Tree shrews have many advantages such as small size, short reproductive and developmental cycle, easy to raise, low price, long life, short gestation period, and multiple births. Tree shrews have been used as experimental animals in the fields of life and medicine for more than 30 years. Tree shrews have a higher homology with primates. Phylogenetic tree analysis based on tree shrew skeletal muscle proteome data by the Kunming Institute of Zoology, Chinese Academy of Sciences, showed that tree shrews are closer to primates (humans) than rodents (mice and rats). Therefore, more and more studies are using tree shrews in muscle injury models.
[0003] Since Mauro A successfully isolated skeletal muscle satellite cells (SMSCs) from frog tibialis anterior muscle using electron microscopy in 1961, mature isolation and culture protocols have been established for SMSCs of various species, including humans, mice, cattle, rabbits, horses, sheep, chickens, and geese. At present, there are no relevant literature reports on the isolation and culture of tree shrew SMSCs, and there are no reports on the method of constructing immortalized tree shrew SMSCs cells.
[0004] Therefore, how to provide a method for isolating tree shrew SMSCs and construct immortalized tree shrew SMSCs is an urgent problem to be solved by those skilled in the art. Summary of the invention
[0005] In view of this, the present invention provides a method for isolating tree shrew SMSCs, immortalized tree shrew SMSCs, a construction method and application.
[0006] In order to achieve the above object, the present invention adopts the following technical solution:
[0007] A method for isolating tree shrew SMSCs, the process comprising:
[0008] 1) Take 3 g of isolated hamstring muscle tissue, disinfect it, and remove impurities;
[0009] 2) Cut the tissue into soybean size, rinse, centrifuge, cut into minced meat, add mixed enzyme digestion solution, digest for 70-80 minutes, and add an equal volume of growth medium to terminate the digestion;
[0010] 3) Centrifuge and remove impurities, use 6 ml of growth medium at 1.2×10 6 The cells / ml were inoculated into T25 culture flasks pre-coated with 0.1% gelatin and placed at 37°C and 5% CO 2 After culturing in a cell culture incubator with saturated humidity for 12-24 hours, the culture medium is transferred to a gelatin-precoated culture bottle. After continuing to culture for 4-5 days, the medium is changed for the first time. When the cell density reaches 70-80%, subculture is carried out. Differential adhesion is used for 30-40 minutes each time to remove most of the fibroblasts. After purification 2-3 times, SMSCs with higher purity can be obtained.
[0011] Preferably, the disinfection treatment is as follows: the popliteal muscle is placed in a centrifuge tube containing 75% alcohol and rinsed for 10 seconds, transferred to a centrifuge tube containing 2% tetraclonal antibody and 1% FBS in an HBSS solution, placed in an ice box and transferred to a sterile clean bench; the muscle tissue is placed on a 60 mm culture dish and repeatedly scrubbed 2-3 times with a D-Hanks washing solution containing 8% tetraclonal antibody and 1% FBS.
[0012] Preferably, the removing of impurities comprises: removing connective tissue, fascia, blood vessels, and fat impurities with sterile ophthalmic scissors and ophthalmic forceps.
[0013] Preferably, the mixed enzyme digestion solution includes: 0.2% collagenase II + 0.01% hyaluronidase
[0014] IV+0.005% DNase I+1% FBS+DMEM / F12 culture medium.
[0015] As an inventive concept identical to the above technical solution, the present invention also seeks to protect the use of the isolated tree shrew SMSCs in constructing immortalized tree shrew SMSCs.
[0016] As an inventive concept identical to the above-mentioned technical solution, the present invention also requests protection for a kind of tree shrew immortalized SMSCs, wherein the immortalized SMSCs are the tree shrew skeletal muscle satellite cell line SQJC-HGY, which is deposited in Guangdong Microbiological Culture Collection Center, with a deposit date of January 31, 2024, a deposit number of GDMCC No: 64339, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and is classified and named as tree shrew skeletal muscle satellite cell line.
[0017] As an inventive concept similar to the above technical solution, the present invention also claims a method for constructing immortalized SMSCs of tree shrews, the process comprising: taking purified P3 generation tree shrew SMSCs and inoculating them into a 6-well plate, with the number of cells in each well being approximately 1×10 5 Then, the cells were transfected with SV40 overexpression lentivirus and screened with puromycin 1ug / mL for 3 days. The immortalized cells were screened.
[0018] As an inventive concept identical to the above-mentioned technical solution, the present invention also requests protection for the application of immortalized SMSCs of tree shrews in constructing an in vitro model of tree shrew skeletal muscle, a pathological model, and in exploring the system development of tree shrew organisms.
[0019] It can be seen from the above technical scheme that compared with the prior art, the present invention can efficiently separate a large number of SMSCs from the muscle tissue of adult tree shrews through a mixed enzyme digestion method, identify SMSCs by cell immunofluorescence, RT-PCR and other detections, induce myogenic and adipogenic differentiation by different inducers, and infect cells with a lentivirus carrying the SV40T gene, successfully establish a method for in vitro isolation, identification and immortalization of adult tree shrew SMSCs, preliminarily explore the biological characteristics of tree shrew SMSCs, construct immortalized tree shrew SMSCs, and provide a basis for subsequent research on the function and molecular mechanism of tree shrew SMSCs. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.
[0021] Figure 1 The attached figure is a technical roadmap for constructing immortalized SMSCs in tree shrews;
[0022] Figure 2 The attached figure shows the morphological observation results of the purified P3 generation cells and the cell growth curve detected by CCK8;
[0023] Figure 3 The attached figure shows the HE staining result;
[0024] Figure 4 The accompanying drawings are flow cytometry test results;
[0025] Figure 5 The attached figure shows the results of immunofluorescence detection;
[0026] Figure 6The attached figure is a diagram showing the expression of Desmin, Pax7, MyoD1 and GAPDH genes in SMSCs detected by RT-PCR;
[0027] Figure 7 The attached figure shows the expression result of MyHC, a specific marker of myogenic differentiation, detected by immunofluorescence staining;
[0028] Figure 8 The attached figure shows the results of adipogenic differentiation of tree shrew SMSCs induced by adipogenic differentiation medium;
[0029] Fig. 9 The attached figure is a diagram for constructing an SV40 overexpression lentiviral expression vector with a puromycin resistance gene;
[0030] Fig.10 The attached figure shows the cell morphology of tree shrew SMSCs before and after transfection with sv40;
[0031] Fig.11 The attached figures show the proliferation of CCK8 before transfection, after screening the P1 generation, and after screening the P13 generation;
[0032] Fig.12 The attached figure shows the RT-PCR amplification results of SMSCsSV40; M represents DL500 DNA Marker, 1 represents P3 generation SMSCs before transfection, 2 represents P13 generation SMSCs after transfection, and 3 represents P30 generation SMSCs after transfection;
[0033] Fig.13 The attached figure shows the immunofluorescence results of α-actin protein of P13 tree shrew SMSCs; DAPI is the staining of cell nucleus, Cy3 is the staining of α-actin protein, Merge is the superposition of DAPI and Cy3 images, scale bar = 200 μm (100x, 200x);
[0034] Fig.14 The attached figures show the β-galactosidase staining of cell senescence of tree shrew SMSCs at p10 before transfection, P13 after transfection, and p30 after transfection. DETAILED DESCRIPTION
[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0036] The technical route for the isolation of tree shrew SMSCs and the construction of immortalized SMSCs is shown in Figure 1 , the specific process is Example 1 to Example 2:
[0037] Example 1 Isolation of Tree Shrew SMSCs
[0038] After the tree shrews were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (65 mg / kg), they were fixed on the experimental operation table, the skin of both legs was prepared and disinfected, the outer skin of the legs was cut off with sterile instruments, and about 3g of hamstring muscle tissue was peeled off with new instruments, placed in a centrifuge tube containing 75% alcohol and rinsed for 10 seconds, transferred to a centrifuge tube containing 2% tetraclonal antibody and 1% FBS in HBSS solution, placed in an ice box and quickly transferred to a sterile clean bench. The whole process was performed on ice. First, the muscle tissue was placed on a 60mm culture dish, and repeatedly scrubbed 2-3 times with D-Hanks' washing solution containing 8% tetraclonal antibody and 1% FBS. Sterile ophthalmic scissors and ophthalmic forceps were used to remove impurities such as connective tissue, fascia, blood vessels, and fat as much as possible. The tissue was cut into soybean size, rinsed with washing solution 2-3 times, and then transferred to a 2ml EP tube and continued to be cut into 1-2mm. 3 The minced meat was then transferred to a 15ml tube and washed twice with washing solution at 4℃300g for 5min. Depending on the volume of the tissue, 5-10ml of 37℃ preheated mixed enzyme digestion solution (0.2% collagenase II + 0.01% hyaluronidase IV + 0.005% DNase I + 1% FBS + DMEM / F12 culture medium) was added, and the digestion solution was placed in a 50ml centrifuge tube, sealed with sealing glue, and digested on a shaker at 37℃ for 70-80min. After complete digestion, an equal volume of growth medium (DMEM / F12 + 10% FBS + 10% HS + 1% triple antibody) was added to terminate the digestion, and after repeated blowing and mixing, the mixture was filtered through a 100-mesh cell sieve, centrifuged at 4℃300g for 10min with high centrifugal force, the supernatant was discarded, and the mixture was filtered through a 400-mesh cell sieve, centrifuged at 4℃300g for 5min, large pieces of debris were discarded, and then centrifuged at 4℃100g for 10min with low centrifugal force to discard fine impurities. The cell number was counted by an automatic cell counter, and the cell yield was approximately 7.60×10 6 Use 6 ml of growth medium to calculate 1.2 × 10 6 The cells / ml were inoculated into T25 culture flasks pre-coated with 0.1% gelatin and placed at 37°C and 5% CO 2 , in a cell culture incubator with saturated humidity.
[0039] After 12-24 hours of cell culture, the culture medium is transferred to a gelatin-precoated culture bottle. After 4-5 days of continuous culture, the medium is changed for the first time. When the cell density reaches 70-80%, subculture is performed. Each subculture uses differential adhesion for 30-40 minutes to remove most of the fibroblasts. After purification 2-3 times, SMSCs with higher purity can be obtained.
[0040] The purified P3 cells were taken for morphological observation, and the results are shown in Figures 2A and Figure 2B; The newly separated SMSCs are translucent and plump, showing long spindle and spindle shapes ( Figure 2 A), a large number of proliferating cells fuse to form multinucleated elongated myotubes, and there is a phenomenon of self-differentiation ( Figure 2 B) CCK8 detection cell growth curve, see Figure 2 C, indicating that the SMSCs growth curve is a standard "S" shape, and the proliferation process goes through a latent period, a logarithmic growth period, and a plateau period ( Figure 2 C);
[0041] HE observation of cell morphology: HE staining shows that the cells are long spindle-shaped or polygonal, the nucleus is lavender, and is a regular oval. The cytoplasm is eosinophilic, so the cytoplasm is stained light pink ( Figure 3 ).
[0042] Flow cytometry detection of apoptosis and cycle: Flow cytometry detection of apoptosis of P3, P5, and P7 SMSCs showed that the activity of P3 and P5 cells was better, both above 85%, while the activity of P7 cells decreased significantly, below 80%, and the apoptosis rate increased significantly ( Figure 4 A). Flow cytometry detection of the P3, P5, and P7 SMSCs cycles indicated that the majority of P3 and P5 cells were in the quiescent phase, while the P7 generation had a decrease in quiescent cells and an increase in dividing cells due to cell aging ( Figure 4 B).
[0043] Immunofluorescence and RT-PCR were used to detect the expression of marker genes and proteins, immunofluorescence identification after myogenic differentiation induction, and O staining identification after adipogenic differentiation induction: Immunofluorescence staining detected the positive expression of Pax7, a specific marker protein of SMSCs in the resting phase, MyoD1, a marker protein of myogenic differentiation, and Desmin, a marker protein of myogenicity. Figure 5 ). RT-PCR was used to detect the expression of Desmin, Pax7, MyoD1 and GAPDH genes in SMSCs. The expression of Desmin, Pax7, MyoD1 and GAPDH genes could be detected in SMSCs and the positive control, and the PCR products were 75, 138, 101 and 121 bp, respectively; which were consistent with the expected target fragment size, while the negative control results only detected the expression of GAPDH gene, indicating that there was no expression of Desmin, Pax7 and MyoD1 genes ( Figure 6 AC), the primer sequences are shown in Table 1.
[0044] Table 1 Primer sequences
[0045]
[0046] In this study, SMSCs were induced to differentiate for 6 days using low-concentration horse serum, and immunofluorescence staining was used to detect the positive expression of the myogenic differentiation marker MyHC ( Figure 7 ), the results suggest that P3 generation SMSCs have strong differentiation ability, and the induced differentiation method is feasible. Adipogenic differentiation of tree shrew SMSCs was induced using adipogenic differentiation medium. After 12 days of culture, a large number of orange-red lipid droplets were deposited in the cells of the differentiation medium group (DM group), while no lipid droplets were formed in the growth medium group (GM group). Figure 8 ), indicating that SMSCs adipogenic differentiation was successfully induced.
[0047] Example 2 Establishment of Immortalized Cells
[0048] The purified P3 cells were used to establish an immortalized cell line. The cells were inoculated into a 6-well plate, with the number of cells per well being approximately 1×10 5 Then, the cells were transfected with SV40 overexpression lentivirus and screened with puromycin 1ug / mL for 3 days. The immortalized cells were screened.
[0049] The specific process is:
[0050] Transfection
[0051] (1) Tree shrew skeletal muscle cells were inoculated into a 6-well plate, with the number of cells per well being approximately 1×10 5 indivual,
[0052] (2) The next day, after the cells have attached to the wall, change the medium.
[0053] (3) Add 1 mL of complete culture medium and 20 μL of SV40 overexpression lentivirus.
[0054] (4) After mixing, continue culturing.
[0055] (5) After 12 hours, observe the cell status and replace with fresh culture medium.
[0056] (6) When the cells have grown to the bottom of the plate, they are subcultured into T25 culture flasks.
[0057] After transfection, cells were expanded for 3-4 generations before screening.
[0058] Screening of tree shrew skeletal muscle cells
[0059] Determination of the kill curve
[0060] (1) Untransfected tree shrew skeletal muscle cells were plated into 24-well plates at a density of 0.05 million per well and incubated overnight.
[0061] (2) On the second day, remove the old culture medium in the 24-well plate.
[0062] (3) Fresh culture medium containing different concentrations of puromycin (1ug / mL, 2ug / mL, 3ug / mL, 4ug / mL, 5ug / mL, 6ug / mL, 7ug / mL) was added to the 24-well plate containing cells.
[0063] (4) Replace fresh screening medium every 2 days.
[0064] (5) Observe the survival rate of cells every day.
[0065] (6) The minimum puromycin concentration used is the lowest screening concentration that kills all cells within 1-4 days from the start of puromycin screening.
[0066] Results: The concentration of puromycin was 1ug / mL, and the action time was 3d.
[0067] Puromycin selection of transfected cells
[0068] (1) On the first day, 0.05 million transfected tree shrew skeletal muscle cells were plated into a 24-well plate and incubated overnight;
[0069] (2) On the second day, remove the old culture medium in the 24-well plate.
[0070] (3) Add screening medium containing puromycin (1 ug / mL) and incubate.
[0071] (4) Replace fresh screening medium every 2 days.
[0072] (5) Observe the survival rate of cells every day.
[0073] (6) Cells that survived at the same time point were successfully transfected tree shrew skeletal muscle cells.
[0074] (7) Expanding the screened cells.
[0075] After the immortalized cells were expanded, P13 and P30 were used for subsequent cell identification. Cell activity was detected by CCK8. Tree shrew SMSCs at P3 before transfection, P13 after transfection, and P30 after transfection were taken at 3×10 per well. 3 Each cell was inoculated in a 96-well plate and tested 24 hours after inoculation for 7 consecutive days. Five replicate wells and one blank control well were tested every day. 90 μL complete medium + 10 μL CCK-8 were added to each well and placed in an incubator for 1 hour before being taken out. The OD value was measured at a wavelength of 450 nm using a microplate reader, and a growth curve was drawn with the cell culture time as the horizontal axis and the OD value as the vertical axis.
[0076] Immunofluorescence identification of α-actin protein expression:
[0077] (1 cell slide
[0078] Take 3 glass slides and place them in a 24-well plate. Add 1 mL of culture medium to each well and add 5×10 cells. 4 Place in an incubator overnight.
[0079] (2) Fixed
[0080] After the cells have crawled onto the slide, remove the culture medium, wash once with PBS, add 4% PFA and fix at 4°C for 30 min. Wash 3×5 min / times with PBS. Alternatively, do not remove PBS for the last time and place at 4°C overnight.
[0081] (3) Membrane rupture and closure
[0082] Remove moisture from the glass slide and place it on a culture dish support. Prepare the glass slide blocking solution: 0.5% Trition X-100 mixed with PBS 1:1, then add 10% serum. Take 50uL of the membrane-breaking blocking solution and drop it on the waterproof film. Cover the side of the glass slide with cells for 2h.
[0083] (4) Primary antibody incubation
[0084] Primary antibody preparation: dilute the antibody with PBS 1:100(200)
[0085] After the membrane is broken and blocked, take 50uL of primary antibody on the waterproof film (in a wet box), cover the glass slide (the side with cells) and place it at 4℃ (can be placed for up to one week)
[0086] (5) Secondary antibody incubation
[0087] After incubation in secondary antibody (secondary antibody: PBS = 1:500) for 2 h at room temperature in the dark, the sections were washed 3×5 min / times with PBS, stained with DAPI (DAPI: PBS = 1:1000) for 5 min, and washed 3×5 min / times with PBS.
[0088] (6) Embedding
[0089] One drop of Fluoromount-G was placed on each slide, and the side with cells was covered to identify the cells as P13 cells.
[0090] RT-PCR detection of SV40 gene expression:
[0091] The SMSCs of tree shrews at P3 before transfection, P13 after transfection, and P30 after transfection were taken and the total RNA of cells was extracted using RNA simlpe Total RNA Kit. The total RNA of 1 μg was reverse transcribed into CDNA according to HiScript III RT SuperMix for qPCR (+gDNA wiper) kit. The expression of SMSCs marker gene SV40 was detected by RT-PCR with β-actin as the internal reference gene. All primers were designed by NCBI Primer and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences are shown in Table 1. PCR was performed according to Premix Taq TM (TaKaRa Taq TM Version 2.0plus dye) instruction manual, reaction system: Premix Taq (TaKaRa Taq Version 2.0plus dye) 25μL, 20μM upstream and downstream primers 1μL each, cDNA 2μL, ddH20 21μL. PCR reaction conditions: 94℃ denaturation 30sec, 60℃ annealing 30sec, 72℃ extension 1min, a total of 30 cycles. 2.0% agarose gel electrophoresis to detect amplification products.
[0092] Cell senescence detection:
[0093] 2×10 SMSCs of untransfected tree shrews at P10, transfected at P13, and transfected at P30 were collected per well. 4 Cells were seeded in 24-well plates. After overnight culture, the cell culture medium was removed, washed once with PBS or HBSS, and 1 ml of β-galactosidase staining fixative was added, and fixed at room temperature for 15 minutes.
[0094] Remove the cell fixative and wash the cells 3 times with PBS or HBSS for 3 minutes each time. Remove the PBS or HBSS and add 1 ml of staining solution to each well. Incubate overnight at 37°C with 10 μl of β-galactosidase staining solution A, 10 μl of β-galactosidase staining solution B, 930 μl of β-galactosidase staining solution C, and 50 μl of X-Gal solution. Seal the well plate with plastic wrap to prevent evaporation. Observe under a normal optical microscope.
[0095] The results showed that by constructing an SV40 overexpression lentiviral expression vector with a puromycin resistance gene ( Fig. 9 ) and transfected with SV40 overexpression lentivirus. After 3-4 generations of amplification, 1ug / ml puromycin was used for screening. After 13 generations of screening and amplification, the cell morphology changed significantly and the proliferation ability became faster, indicating that the initial construction of immortalization was successful ( Fig.10). The CCK8 results showed that the cell growth curves all showed an "S" shape. Compared with the P3 generation before transfection, the cell proliferation ability after screening the P13 generation and the P30 generation was significantly improved ( Fig.11 ). RT-PCR was used to detect the expression of SV40 and β-actin genes in SMSCs. The expression of SV40 and β-actin genes was detected in SMSCs, and the PCR products were 96 and 113 bp, respectively; consistent with the expected target fragment size ( Fig.12 ). After immunofluorescence screening, the α-actin protein in the immortalized tree shrew skeletal muscle satellite cells of the P13 generation showed positive expression, proving that its purity was as high as more than 90% ( Fig.13 ). β-galactosidase indicated that the P10 tree shrew SMSCs primary cells that were not transfected with SV40 lentivirus expressed blue β-galactosidase, while the P13 and P30 tree shrew SMSCs that were transfected with SV40 lentivirus did not express β-galactosidase, indicating that the transfected SMSCs showed no signs of cell senescence ( Fig.14 ). In summary, this study successfully constructed a tree shrew SMSCs immortalized cell line that can be stably propagated. The immortalized SMSCs are tree shrew skeletal muscle satellite cell line SQJC-HGY, which is deposited in Guangdong Microbial Culture Collection Center, with a deposit date of January 31, 2024, a deposit number of GDMCC No: 64339, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and is classified and named as tree shrew skeletal muscle satellite cell line.
[0096] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.
[0097] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A method for isolating tree shrew SMSCs, characterized in that the process include: 1) Take 3 g of isolated hamstring muscle tissue, disinfect it, and remove impurities; 2) Cut the tissue into soybean size, rinse, centrifuge, cut into minced meat, add mixed enzyme digestion solution, digest for 70-80 minutes, and add an equal volume of growth medium to terminate the digestion; 3) Centrifuge and remove impurities, use 6 ml of growth medium at 1.2×10 6 Cells / ml were inoculated in a T25 culture flask pre-coated with 0.1% gelatin, and cultured in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 12-24 hours. The culture medium was then transferred to a culture flask pre-coated with gelatin, and the culture medium was changed for the first time after continuing to culture for 4-5 days. When the cell density reached 70-80%, subculture was performed; each subculture used differential adhesion for 30-40 minutes to remove most of the fibroblasts, and after purification 2-3 times, SMSCs with higher purity were obtained.
2. The method for isolating tree shrew SMSCs according to claim 1, characterized in that: The disinfection treatment is as follows: the popliteal muscle is placed in a centrifuge tube containing 75% alcohol and rinsed for 10 seconds, transferred to a centrifuge tube containing 2% tetraclonal antibody and 1% FBS in an HBSS solution, placed in an ice box and transferred to a sterile clean bench; the muscle tissue is placed on a 60 mm culture dish and repeatedly scrubbed 2-3 times with a D-Hanks washing solution containing 8% tetraclonal antibody and 1% FBS.
3. The method for isolating tree shrew SMSCs according to claim 2, characterized in that: The impurities removal includes: removing connective tissue, fascia, blood vessels, and fat impurities with sterile ophthalmic scissors and ophthalmic forceps.
4. The method for isolating tree shrew SMSCs according to claim 3, characterized in that: The mixed enzyme digestion solution includes: 0.2% collagenase II+0.01% hyaluronidase IV+0.005% DNase I+1% FBS+DMEM / F12 culture medium.
5. Use of the tree shrew SMSCs isolated by any one of the methods of claims 1 to 4 in constructing immortalized tree shrew SMSCs.
6. A tree shrew immortalized SMSCs, characterized in that: The immortalized SMSCs are the tree shrew skeletal muscle satellite cell line SQJC-HGY, which is deposited in the Guangdong Provincial Microbiological Culture Collection Center. The deposit date is January 31, 2024, the deposit number is GDMCC No: 64339, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. It is classified and named as the tree shrew skeletal muscle satellite cell line.
7. A method for constructing immortalized SMSCs from tree shrews, characterized in that: The process includes: taking the purified P3 generation tree shrew SMSCs and inoculating them into a 6-well plate, with the number of cells per well being approximately 1×10 5 Then, the cells were transfected with SV40 overexpression lentivirus and screened with puromycin 1ug / mL for 3 days. The immortalized cells were screened.
8. Application of immortalized SMSCs of tree shrews in constructing in vitro skeletal muscle models and pathological models of tree shrews and exploring the systematic development of tree shrew organisms.