Kit for detecting AChR antibody based on CBA method

Abstract

The invention provides a kit for detecting an AChR antibody based on a CBA method. The kit comprises skeletal muscle primary cells of fetal mice and skeletal muscle primary cells of adult mice. The skeletal muscle primary cells of fetal rats and adult rats obtained through primary cell isolation culture can naturally express fetal and adult AChR target antigens respectively, and compared with a traditional cell transfection technology, the skeletal muscle primary cells of fetal rats and adult rats obtained through the method have natural AChR protein expression, and can be used for preparing the skeletal muscle primary cells of fetal rats and adult rats. The problems of high cytotoxicity and low detection sensitivity caused by transient expression of five subunits and polymeric protein in traditional cell transfection are successfully avoided, and the detection sensitivity of the AChR antibody is effectively improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a kit for detecting AChR antibodies based on a CBA method. Background Art

[0002] Myasthenia gravis (MG) is an autoimmune disease caused by antibodies targeting the neuromuscular junction. It has the characteristics of a wide age range, high disability rate, easy relapse, and poor prognosis, making it a major disease that seriously threatens human health. Based on the great heterogeneity of MG clinical manifestations, subgroup classification based on serum antibodies and clinical characteristics has guiding significance for the individualized treatment and prognosis assessment of MG. The detection of autoantibodies is a key auxiliary diagnostic indicator recommended by domestic and international MG diagnosis and treatment guidelines. Among them, acetylcholine receptor (AChR) antibodies are the most common MG pathogenic antibodies, which can be detected in the serum of approximately 80%-85% of MG patients.

[0003] The target antigen AChR of AChR antibody is a pentamer formed by α, β, δ and ε subunits (adult AChR-ε) or α, β, δ and γ subunits (fetal AChR-γ) under the clustering of Rapsyn protein. Simultaneous detection of adult and fetal AChR antibodies in serum can improve the sensitivity of detection. The commonly used method for detecting AChR antibodies is cell-based immunofluorescence assay (CBA). The cell model used by the common CBA method for detecting AChR antibodies on the market is HEK293 cells, which are co-transfected with exogenous cells or established cell lines co-expressing Rapsyn and AChR α, β, δ and ε subunits or γ subunits (fetal / adult AChR pentamers). However, when constructing cell lines or transiently expressing 5 subunits and aggregated proteins in cells, it is highly toxic to cells, and the overall level of each subunit protein is reduced, resulting in reduced detection sensitivity. Summary of the invention

[0004] In view of this, the present invention provides a kit for detecting AChR antibodies based on the CBA method. The primary skeletal muscle cells of fetal mice can stably express fetal AChR target antigens, and the primary skeletal muscle cells of adult mice can stably express adult AChR target antigens. The kit has high detection sensitivity.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: The invention provides a CBA-based kit for detecting AChR antibodies, comprising primary skeletal muscle cells of fetal mice and primary skeletal muscle cells of adult mice.

[0006] Preferably, a fluorescent secondary antibody is also included.

[0007] Preferably, the fetal mouse is a mouse that is 12.5-13.5 days old, and the adult mouse is a mouse that is 6-8 weeks old.

[0008] Preferably, the method for preparing primary skeletal muscle cells of fetal mice comprises the following steps: S10, obtaining muscle tissue from fetal mice at 12.5-13.5 days, digesting the muscle tissue, and obtaining muscle tissue fragments; S20, adding the muscle tissue fragments to the culture medium to terminate digestion, filtering, and obtaining a precipitate; S30, adding the precipitate to a culture medium for re-suspending, and then inoculating the precipitate into a culture dish containing rat tail collagen for culture, so as to obtain the fetal rat skeletal muscle primary cells.

[0009] Preferably, in step S10, the specific steps of obtaining the muscle tissue of the fetal mouse at 12.5-13.5 days are: peeling off the placental tissue of the fetal mouse, cutting off the head, neck, limbs, and internal organs, washing away the blood, and obtaining the muscle tissue; and / or, In step S10, the muscle tissue is digested to obtain muscle tissue fragments by chopping the muscle tissue into pieces, transferring the pieces into a centrifuge tube, and adding a mixed solution of type I collagenase and type II collagenase into the centrifuge tube for digestion to obtain muscle tissue fragments.

[0010] Preferably, in step S20, the muscle tissue fragments are added to the culture medium to terminate digestion, filtered, and the precipitate is obtained by adding DMEM complete culture medium to the muscle tissue fragments to terminate digestion, filtering through a cell sieve, and obtaining a precipitate; and / or, In step S20, a 70 μm cell sieve is specifically used for filtering.

[0011] Preferably, the method for preparing primary skeletal muscle cells of adult mice comprises the following steps: SA, obtaining muscle tissue of adult mice, and mincing the muscle tissue to obtain muscle tissue fragments; SB, digesting the muscle tissue fragments, filtering and obtaining a precipitate; SC, adding the precipitate to the culture medium for re-suspending, and then inoculating it into a culture dish containing rat tail collagen for culture, so as to obtain the primary skeletal muscle cells of the adult mouse.

[0012] Preferably, in step SA, the mouse strain used is BALB / C adult mice; and / or, In step SA, the specific steps of obtaining the muscle tissue of the adult mouse are: after killing the adult mouse by dislocating the neck, the gastrocnemius muscle, soleus muscle, plantar muscle and tibialis anterior muscle are removed from the hind limbs of the mouse to obtain the muscle tissue of the adult mouse; and / or, In step SA, the specific steps of mincing the muscle tissue to obtain muscle tissue fragments are: placing the muscle tissue in a centrifuge tube filled with pre-cooled PBS solution, weighing the muscle tissue and transferring it to a culture dish under a biosafety hood, mincing the muscle into a fine paste with a scalpel to obtain muscle tissue fragments.

[0013] Preferably, in step SC, the precipitate is added to a culture medium and resuspended, and then inoculated into a culture dish containing rat tail collagen for culture until the cells reach a growth density of 80-90%, thereby obtaining primary skeletal muscle cells of adult mice.

[0014] Preferably, in step SC, the precipitate is added to the culture medium and resuspended, and then inoculated into a culture dish containing rat tail collagen and cultured at a temperature of 37-39°C and a CO 2 The concentration is 4-6%.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The fetal rat and adult rat skeletal muscle primary cells obtained by primary cell separation and culture in the present invention can naturally express fetal and adult AChR target antigens, respectively. Compared with traditional cell transfection technology, it successfully avoids the problems of high cytotoxicity and low detection sensitivity caused by transient expression of five subunits and aggregated proteins by traditional cell transfection, and effectively improves the detection sensitivity of AChR antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The cell morphology diagram of the primary skeletal muscle cells of fetal rats provided for the cell morphology observation experiment of the present invention; Figure 2 The cell morphology diagram of primary skeletal muscle cells of adult mice provided for the cell morphology observation experiment of the present invention; Figure 3 The fluorescence identification diagram of the primary skeletal muscle cells of fetal rats and adult rats provided in the immunofluorescence identification experiment of the present invention; Figure 4 A display diagram of the results of detecting human serum without AChR antibodies provided by the present invention under fluorescence microscope observation; Figure 5 This is a graph showing the results of detecting human serum containing AChR antibodies under fluorescence microscope observation provided by the present invention. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0018] Sources and physical and chemical parameters of key test materials: DMEM complete medium includes: DMEM + 10% FBS + 1% P / S Fluorescent secondary antibody: anti-human IgG-Alex594 secondary antibody (Invitrogen, A-11012) Myasthenia gravis (MG) is an autoimmune disease caused by antibodies targeting the neuromuscular junction. It has the characteristics of a wide age range, high disability rate, easy relapse, and poor prognosis, making it a major disease that seriously threatens human health. Based on the great heterogeneity of MG clinical manifestations, subgroup classification based on serum antibodies and clinical characteristics has guiding significance for the individualized treatment and prognosis assessment of MG. The detection of autoantibodies is a key auxiliary diagnostic indicator recommended by domestic and international MG diagnosis and treatment guidelines. Among them, acetylcholine receptor (AChR) antibodies are the most common MG pathogenic antibodies, which can be detected in the serum of approximately 80%-85% of MG patients.

[0019] The antigen AChR of AChR antibody is a pentamer formed by α, β, δ and ε subunits (adult AChR-ε) or α, β, δ and γ subunits (fetal AChR-γ) under the clustering of Rapsyn protein. Simultaneous detection of adult and fetal AChR antibodies in serum can improve the sensitivity of detection. The commonly used method for detecting AChR antibodies is cell-based immunofluorescence assay (CBA). The cell model used by the common CBA method for detecting AChR antibodies on the market is HEK293 cells, which are co-transfected with exogenous cells or established cell lines co-expressing Rapsyn and AChR α, β, δ and ε subunits or γ subunits (fetal / adult AChR pentamers). However, when constructing cell lines or transiently expressing 5 subunits and aggregated proteins in cells, it is highly toxic to cells, and the overall level of each subunit protein is reduced, resulting in reduced detection sensitivity.

[0020] In order to solve this technical problem, the present invention provides a kit for detecting AChR antibodies based on the CBA method, comprising primary skeletal muscle cells of fetal mice and primary skeletal muscle cells of adult mice.

[0021] In some embodiments, the method of using the kit of the present invention comprises the following steps: (1) Primary cells of fetal and adult mouse skeletal muscles were cultured in a 1:1 mixture of complete DMEM medium in six-well plates with cover slips, and placed in an incubator at 37 °C and 5% CO 2 for cultivation; (2) When the cell growth density reached 80%-90%, the liquid was discarded and the cells were washed with PBS for 3 min; (3) Then, formaldehyde was added to the six-well plate and the cell slides were fixed at 4 °C for 10 min; (4) The liquid was discarded and the cell slides were washed with PBS for 3 min; (5) The diluted clinical serum sample was added to the cell slides and incubated for 45 min; (6) The liquid was discarded and the cell slides were washed with PBS for 3 min; (7) Fluorescent secondary antibody diluted 1:100 was added and incubated for 45 min; (8) The liquid was discarded and the cells were washed with PBS 2-3 times. After washing off the excess fixing solution, the cell slides were placed under a fluorescence microscope for microscopic examination.

[0022] Among them, the determination of the above test results is to determine whether the target antibody is contained in the sample by observing the position and fluorescence intensity of the red fluorescence under the microscope. Specifically, the red fluorescence was observed at a wavelength of EX BP 470 / 40, and the red fluorescence represents the antibody fluorescence in the test sample; it was judged whether the test antibody was contained in the sample by the fluorescence morphology, position and fluorescence intensity; as Figure 5 shown, if clustered positive fluorescence staining was observed under the microscope, it was a positive result; as Figure 4 shown, if no red fluorescence was observed under the microscope, it was a negative result.

[0023] First of all, the present invention abandons the traditional method of constructing an exogenous expression AChR cell model by lentiviral transfection technology, and adopts the primary cell isolation and culture technology to obtain primary cells of fetal and adult mouse skeletal muscles respectively, so as to obtain a cell model that endogenously and stably expresses fetal and adult AChR target antigens. Moreover, the primary cells can undergo several passages, retaining the cell morphology and target antigen conformation, without the need to undergo multiple transfections of multiple subunits, saving time and labor costs. Secondly, the cell model provided by the present invention is suitable for the detection of serum AChR antibodies by the CBA technique, retaining the advantages of the CBA method for detecting AChR antibodies. At the same time, the cell model endogenously and naturally expresses fetal and adult AChR target antigens, effectively improving the sensitivity of serum AChR antibody detection.

[0024] Furthermore, it also includes fluorescent secondary antibody. Furthermore, the fetal mouse is a mouse at 12.5-13.5 days of growth, and the adult mouse is a mouse at 6-8 weeks of age.

[0025] In some embodiments, the method for preparing primary cells from fetal mice comprises the following steps: S10, obtaining muscle tissue from fetal mice at 12.5-13.5 days, digesting the muscle tissue, and obtaining muscle tissue fragments; S20, adding the muscle tissue fragments into the culture medium to terminate the digestion, filtering and obtaining a precipitate; S30. After the precipitate is added to the culture medium for re-suspending, it is inoculated into a culture dish containing rat tail collagen for culture to obtain primary skeletal muscle cells of fetal rats.

[0026] In some embodiments, in step S10, the specific steps of obtaining the muscle tissue of the 12.5-13.5 day old fetus are: peeling off the placental tissue of the fetus, cutting off the head, neck, limbs, and internal organs, washing away the blood, and obtaining the muscle tissue; and / or, In step S10, the muscle tissue is digested to obtain muscle tissue fragments by chopping the muscle tissue into pieces, transferring the pieces into a centrifuge tube, and adding a mixed solution of type I collagenase and type II collagenase into the centrifuge tube for digestion to obtain muscle tissue fragments.

[0027] In some embodiments, in step S20, the steps of adding the muscle tissue fragments to the culture medium to terminate digestion, filtering, and obtaining a precipitate are: adding the muscle tissue fragments to DMEM complete culture medium to terminate digestion, filtering through a cell sieve, and obtaining a precipitate; and / or, In step S20, a 70 μm cell sieve is specifically used for filtering.

[0028] In some embodiments, the specific steps for preparing primary skeletal muscle cells from fetal mice are: (1) Use 12.5-13.5 day old fetal mice, peel off the embryonic tissue, cut off the head, neck, limbs, and internal organs, and wash away the blood.

[0029] (2) Mince the muscle tissue, transfer it to a centrifuge tube, add a mixture of collagenases (type I collagenase and type II collagenase), and digest with pancreatic enzymes at room temperature until the embryos are dispersed into pieces of about 2 mm.

[0030] (3) Add DMEM complete medium to terminate digestion and filter the cells through a 70 μm cell sieve into a new centrifuge tube.

[0031] (4) After centrifugation, the collected cells were discarded, and the supernatant was added with 10 mL of DMEM complete medium for resuspending. The cells were then inoculated into a 10 cm culture dish pretreated with rat tail collagen and incubated at 38.5°C and 5% CO. 2 Under the above conditions, primary skeletal muscle cells of fetal rats were obtained and passaged when the cell confluence reached 80-90%.

[0032] Culture and passaging of primary cells from fetal mice: (1) When the cell growth density reaches about 90%, rinse the cells with PBS buffer, add 2 mL of 0.25% trypsin solution and digest for 2 min, then add 2 mL of DMEM complete medium to terminate the digestion.

[0033] (2) Centrifuge at 300 x g for 10 min at room temperature to collect the cell pellet; (3) The collected cell pellet can be subcultured at a ratio of 1:1 or 1:2. When the growth density reaches 80% to 90%, it can be expanded to the third generation.

[0034] Among them, the identification of fetal mouse skeletal muscle primary cells mainly includes cell morphology observation and immunofluorescence identification.

[0035] like Figure 1 As shown, cell morphology observation: (1) After primary cells were cultured in a six-well plate covered with a coverslip until the cell density reached 80%, the cell morphology was observed under an inverted microscope; (2) Skeletal muscle cells are fibrous, unbranched, have obvious transverse striations, and are multinucleated, all of which are located below the cell membrane. The cells are mostly spindle-shaped and have a certain directionality.

[0036] (3) Each myofibril has light bands (I bands) and dark bands (A bands) arranged alternately. The light bands are stained lighter, while the dark bands are stained darker. There is a brighter line in the middle of the dark bands called the H line, and there is an M line in the middle of the H line; there is a darker line in the middle of the light bands called the Z line, and the section between the two Z lines is called a sarcomere.

[0037] Immunofluorescence identification: (1) After observing the cell morphology, discard the culture medium in the well plate and wash with PBS three times, 3 minutes each time; (2) Fix the slides in the six-well plate with 4% paraformaldehyde for 15 min, and wash the slides with PBS three times, each time for 3 min; (3) 0.5% Triton X-100 (prepared in PBS) at room temperature for 20 min; (4) Afterwards, wash the slides with PBS three times, 3 min each time, absorb the PBS with absorbent paper, add normal goat serum on the slides, and block at room temperature for 30 min. (5) After the blocking solution is removed, add a sufficient amount of diluted primary antibody (Vimentin antibody) to each slide without washing and place it in a wet box and incubate overnight at 4°C; (6) Then discard the liquid, wash the slides with PBS three times, 3 min each time, dry the excess liquid on the slides with absorbent paper, add the diluted fluorescent secondary antibody, and incubate in a wet box at 20-37°C for 1 h. (7) Then discard the liquid and wash the slides with PBST three times, each time for 3 minutes; (8) Finally, add DAPI and incubate in the dark for 5 minutes to stain the nucleus of the specimen. Wash off the excess DAPI with PBST for 5 minutes × 4 times; use absorbent paper to absorb the liquid on the slide, seal the slide with a sealing solution containing an anti-fluorescence quencher, and then observe and collect images under a fluorescence microscope. Figure 3 shown.

[0038] If 90% of the cells are labeled with Vimentin under a fluorescence microscope, it means that the purity of the primary cells is high.

[0039] In some embodiments, the method for preparing primary skeletal muscle cells from adult mice comprises the following steps: SA, obtaining muscle tissue of adult mice, mincing the muscle tissue, and obtaining muscle tissue fragments; SB, digest the muscle tissue fragments, filter and obtain the precipitate; SC. The precipitate was added to the culture medium for resuspending and then inoculated into a culture dish containing rat tail collagen to obtain primary skeletal muscle cells of adult mice.

[0040] Further, in step SA, the mouse strain used is BALB / C adult mice; and / or, In step SA, the specific steps of obtaining the muscle tissue of the adult mouse are: after killing the adult mouse by dislocating the neck, the gastrocnemius muscle, soleus muscle, plantar muscle and tibialis anterior muscle are removed from the hind limbs of the mouse to obtain the muscle tissue of the adult mouse; and / or, In step SA, the specific steps of mincing the muscle tissue and obtaining muscle tissue fragments are as follows: placing the muscle tissue in a centrifuge tube filled with a pre-cooled PBS solution, weighing the muscle tissue and transferring it to a culture dish under a biosafety hood, mincing the muscle with a scalpel into a fine paste, and obtaining muscle tissue fragments. Further, in step SC, the precipitate is added to the culture medium and resuspended, and then inoculated into a culture dish containing rat tail collagen and cultured until the cells are grown at a density of 80-90%, thereby obtaining primary skeletal muscle cells of adult mice.

[0041] Further, in step SC, the precipitate is added to the culture medium and resuspended, and then inoculated into a culture dish containing rat tail collagen and cultured at a temperature of 37-39°C. During the culture, CO 2 The concentration is 4-6%.

[0042] In some embodiments, primary skeletal muscle cells from adult mice are isolated: (1) Select adult BALB / C mice, kill them by dislocating their necks, and then soak them in 75% ethanol solution for 5 minutes for disinfection; (2) Remove the gastrocnemius, soleus, plantar and tibialis anterior muscles from the mouse hind limbs and place them in a centrifuge tube containing pre-cooled PBS solution; (3) Weigh the muscle and transfer it to a culture dish under a biosafety hood and mince the muscle into a fine paste using a scalpel; (4) Transfer the minced tissue paste to a 15 mL centrifuge tube and add 5 mL of a mixture of 2 mg / mL type I collagenase and 2 mg / mL type II collagenase, and incubate at 37 °C for 45 min. (5) Add 5 mL of DMEM complete medium to the cell suspension to inactivate the digestion solution; (6) Filter the cell suspension through a 70 μm cell sieve into a 50 mL centrifuge tube. After filtration, rinse the cell sieve with 5 mL of DMEM complete medium. (7) Centrifuge the collected cell suspension at 300 × g for 10 min at room temperature and discard the supernatant; (8) Add 10 mL of DMEM complete medium to the centrifuge tube and resuspend the cells. Then inoculate the cells into a 10 cm culture dish and place it at 37°C and 5% CO. 2 The cells are cultured in an incubator to obtain primary skeletal muscle cells of adult mice. When the growth density reaches about 80-90%, subculture can be performed.

[0043] Cultivation and passaging of primary cells: (1) When the cell growth density reaches about 90%, wash the cells with PBS buffer, then add 2 mL of 0.25% trypsin solution for digestion for 2 min, and then add 2 mL of DMEM complete medium to terminate the digestion; (2) Centrifuge at 300 × g for 10 min at room temperature to collect the cell pellet; (3) The collected cell pellet can be subcultured at a ratio of 1:1 or 1:2. When the growth density reaches 80% to 90%, it can be expanded to the third generation.

[0044] Among them, the identification of primary skeletal muscle cells of adult mice mainly includes cell morphology observation and immunofluorescence identification.

[0045] like Figure 2 As shown, cell morphology observation: (1) After primary cells were cultured in a six-well plate covered with a coverslip until the cell density reached 80%, the cell morphology was observed under an inverted microscope; (2) Skeletal muscle cells are fibrous, unbranched, have obvious transverse striations, and are multinucleated, all of which are located below the cell membrane. The cells are mostly spindle-shaped and have a certain directionality.

[0046] (3) Each myofibril has light bands (I bands) and dark bands (A bands) arranged alternately. The light bands are stained lighter, while the dark bands are stained darker. There is a brighter line in the middle of the dark bands called the H line, and there is an M line in the middle of the H line; there is a darker line in the middle of the light bands called the Z line, and the section between the two Z lines is called a sarcomere.

[0047] Immunofluorescence identification: (1) After observing the cell morphology, discard the culture medium in the well plate and wash with PBS three times, 3 minutes each time; (2) Fix the slides in the six-well plate with 4% paraformaldehyde for 15 min, and wash the slides with PBS three times, each time for 3 min; (3) 0.5% Triton X-100 (prepared in PBS) permeabilization at room temperature for 20 min; (4) Afterwards, wash the slides with PBS three times, 3 min each time, absorb the PBS with absorbent paper, add normal goat serum on the slides, and block at room temperature for 30 min. (5) After the blocking solution is removed, add a sufficient amount of diluted primary antibody (Vimentin antibody) to each slide without washing and place it in a wet box and incubate overnight at 4°C; (6) Then discard the liquid, wash the slides with PBS three times, 3 min each time, dry the excess liquid on the slides with absorbent paper, add the diluted fluorescent secondary antibody, and incubate in a wet box at 20-37°C for 1 h. (7) Then discard the liquid and wash the slides with PBST three times, each time for 3 minutes; (8) Finally, add DAPI and incubate in the dark for 5 min to stain the nucleus of the specimen. Wash off the excess DAPI with PBST for 5 min × 4 times. Use absorbent paper to absorb the liquid on the slide, seal the slide with a sealing solution containing an anti-fluorescence quencher, and then observe and collect images under a fluorescence microscope. Figure 3 If 90% of the cells are labeled with Vimentin under a fluorescence microscope, it means that the purity of the primary cells is high.

[0048] Embodiment and performance test thereof 25 serum samples with medium to high titer (1:32-1000) of AChR antibody detected by this kit were tested with RIAP, the internationally recognized "gold standard" for AChR antibody detection (results in Table 1). RIAP results indicated that 25 samples were AChR antibody positive. This shows that for medium to high titer positive samples, the test results of this kit and RIAP are in good agreement.

[0049] Table 1

[0050] For serum samples from 7 patients with clinical diagnosis or suspected myasthenia gravis, the test results of this kit were all low titer (1:10), of which 2 cases detected AChR antibody titer of 1:10 based on the CBA method of traditional HEK-293 cell transfection, and the remaining 5 cases did not detect antibodies; RIAP and ELISA tests of 7 cases were all negative (results in Table 2). This shows that for low titer positive samples of AChR antibodies, the detection sensitivity of this kit is higher than that of traditional CBA kits and other detection methods.

[0051] Table 2

[0052] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0053] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit for detecting AChR antibodies based on the CBA method, characterized in that: Includes fetal mouse skeletal muscle primary cells and adult mouse skeletal muscle primary cells.

2. The kit according to claim 1, characterized in that Fluorescent secondary antibodies are also included.

3. The kit according to claim 1, characterized in that The fetal mice are mice that are 12.5-13.5 days old, and the adult mice are mice that are 6-8 weeks old.

4. The kit according to claim 1, characterized in that The method for preparing the primary skeletal muscle cells of fetal mice comprises the following steps: S10, obtaining muscle tissue from fetal mice at 12.5-13.5 days, digesting the muscle tissue, and obtaining muscle tissue fragments; S20, adding the muscle tissue fragments to the culture medium to terminate digestion, filtering, and obtaining a precipitate; S30, adding the precipitate to a culture medium for re-suspending, and then inoculating the precipitate into a culture dish containing rat tail collagen for culture, so as to obtain the fetal rat skeletal muscle primary cells.

5. The kit according to claim 4, characterized in that In step S10, the specific steps of obtaining the muscle tissue of the fetal mouse at 12.5-13.5 days are: peeling off the placental tissue of the fetal mouse, cutting off the head, neck, limbs, and internal organs, washing away the blood, and obtaining the muscle tissue; and / or, In step S10, the muscle tissue is digested to obtain muscle tissue fragments by chopping the muscle tissue into pieces, transferring the pieces into a centrifuge tube, and adding a mixed solution of type I collagenase and type II collagenase into the centrifuge tube for digestion to obtain muscle tissue fragments.

6. The kit according to claim 4, characterized in that In step S20, the muscle tissue fragments are added to the culture medium to terminate digestion, filtered, and the precipitate is obtained by adding DMEM complete culture medium to the muscle tissue fragments to terminate digestion, filtering through a cell sieve, and obtaining a precipitate; and / or, In step S20, a 70 μm cell sieve is specifically used for filtering.

7. The kit according to claim 1, characterized in that The method for preparing primary skeletal muscle cells of adult mice comprises the following steps: SA, obtaining muscle tissue of adult mice, and mincing the muscle tissue to obtain muscle tissue fragments; SB, digesting the muscle tissue fragments, filtering and obtaining a precipitate; SC, adding the precipitate to the culture medium for re-suspending, and then inoculating it into a culture dish containing rat tail collagen for culture, so as to obtain the primary skeletal muscle cells of the adult mouse.

8. The kit according to claim 7, characterized in that In step SA, the mouse strain used is BALB / C adult mice; and / or, In step SA, the specific steps of obtaining the muscle tissue of the adult mouse are: after killing the adult mouse by dislocating the neck, the gastrocnemius muscle, soleus muscle, plantar muscle and tibialis anterior muscle are removed from the hind limbs of the mouse to obtain the muscle tissue of the adult mouse; and / or, In step SA, the specific steps of mincing the muscle tissue to obtain muscle tissue fragments are: placing the muscle tissue in a centrifuge tube filled with pre-cooled PBS solution, weighing the muscle tissue and transferring it to a culture dish under a biosafety hood, mincing the muscle into a fine paste with a scalpel to obtain muscle tissue fragments.

9. The kit according to claim 7, characterized in that In step SC, the precipitate is added to a culture medium and resuspended, and then inoculated into a culture dish containing rat tail collagen for culture until the cells reach a growth density of 80-90%, thereby obtaining primary skeletal muscle cells of adult mice.

10. The kit according to claim 7, characterized in that In step SC, the precipitate is added to the culture medium and resuspended, and then inoculated into a culture dish containing rat tail collagen for culture at a temperature of 37-39° C. and a CO 2 concentration of 4-6% during the culture.

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