Application of mouse muscle cell-derived apoptosis vesicles in muscle physiological regulation
By extracting and applying apoptotic vesicles during the myogenesis of mouse myocytes, the problem of muscle atrophy in muscle-related diseases is solved, the effect of promoting the differentiation and development of skeletal muscle cells is achieved, and the research and development of drugs for skeletal muscle disease is promoted.
Patent Information
- Application Number
- CN202510483858.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-17
- Publication Date
- 2025-08-19
AI Technical Summary
In the prior art, the regulatory function of apoptotic vesicles in the process of myocyte differentiation has not been fully studied, resulting in problems such as muscular atrophy, senile sarcopenia and diabetic muscular atrophy.
The apoptotic vesicles produced during the myogenesis and differentiation of mouse myocytes are extracted through specific centrifugation steps and applied to muscle physiological regulation to promote muscle development and delay muscle atrophy.
Apoptotic vesicles can promote the differentiation and development of skeletal muscle cells in and out of the body, significantly delay muscle atrophy caused by aging or diabetes, and help the development of drugs related to skeletal muscle diseases.
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Abstract
Description
Technical Field
[0001] The present invention relates to extracellular vesicles, and in particular to application of mouse muscle cell-derived apoptotic vesicles in muscle physiological regulation. Background Art
[0002] Skeletal muscle accounts for over 40% of the body weight of adult animals and is the body's largest metabolic and motor organ. Normal skeletal muscle development plays a key role in locomotion and metabolic homeostasis. During development, skeletal muscle formation depends on the directed differentiation of myoblasts, a process driven by the activation of a cascade of myogenic regulatory factors, such as MyoD and Myogenin, and involves key events such as cell cycle exit, myotube fusion, and contractile protein expression. Abnormalities in the skeletal muscle cell differentiation program can lead to muscle fiber atrophy associated with motor neuron degeneration in amyotrophic lateral sclerosis (ALS), or to impaired glucose metabolism caused by insulin resistance in type 2 diabetes. Even muscle loss during aging is associated with dysregulation of this process.
[0003] Studies have shown that apoptotic activity increases significantly during myogenic differentiation. Classical theory posits that apoptosis is the terminal event of programmed death and plays no role in regulating subsequent physiological processes. However, emerging evidence suggests that apoptotic cells can actively regulate various physiological and pathological processes by secreting apoptotic vesicles (apoVs), which act as important paracrine signals. However, no studies have yet demonstrated that apoptotic vesicles participate in myogenic differentiation. Summary of the Invention
[0004] Purpose of the invention: The purpose of the present invention is to provide an application of apoptotic vesicles produced during the myogenic differentiation of mouse muscle cells in the regulation of muscle physiology.
[0005] Technical solution: The application of the apoptotic vesicles derived from the myogenic differentiation process of mouse muscle cells described in the present invention in the regulation of muscle physiology.
[0006] Preferably, the application is an application for promoting muscle development.
[0007] Preferably, the application is an application for delaying muscle atrophy.
[0008] Preferably, the application is application in the preparation of drugs for treating muscle-related diseases.
[0009] Preferably, the muscle-related diseases include muscular dystrophy, disuse muscle atrophy, senile sarcopenia, and diabetic muscle atrophy.
[0010] Preferably, the steps of preparing the apoptotic vesicles include:
[0011] (1) Inducing mouse muscle cell differentiation using differentiation medium, and collecting the differentiation culture medium after culture;
[0012] (2) The differentiation culture medium obtained in step 1 is subjected to differential centrifugation, and the precipitate is collected to obtain mouse muscle cell-derived apoptotic vesicles.
[0013] Preferably, the mouse muscle cells in step 1 are mouse myoblast cell line C2C12 substrain.
[0014] Preferably, the differentiation medium in step 1 is a basal medium containing 2% horse serum.
[0015] Preferably, the horse serum is vesicle-removed horse serum.
[0016] Preferably, the culture time in step 1 is 40-50 hours.
[0017] Preferably, the differential centrifugation step in step 2 comprises:
[0018] (21) The collected differentiation culture medium was centrifuged at 2-8°C, 700-900 g for 8-12 min, and the cell pellet was removed to obtain the first supernatant;
[0019] (22) The first supernatant was centrifuged at 2-8°C, 1800-2200 g for 8-12 min to remove cell debris and obtain the second supernatant;
[0020] (23) The second supernatant was centrifuged at 14,000–18,000 g for 25–35 min at 2–8 °C, and the supernatant was removed. The resulting precipitate was crude apoptotic vesicles and resuspended in PBS.
[0021] (24) Apoptotic vesicles derived from mouse muscle cells were obtained by centrifugation at 14,000–18,000 g for 25–35 min at 2–8°C.
[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:
[0023] 1. The apoptotic vesicles are derived from normal physiological conditions rather than chemical drug induction, thus avoiding abnormal vesicle contents caused by the disruption of cell homeostasis under drug stimulation;
[0024] 2. The apoptotic vesicles can promote the differentiation and development of skeletal muscle cells both in vivo and in vitro, and significantly delay muscle atrophy caused by aging or diabetes, which will help the development of drugs related to skeletal muscle diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 For the extraction and identification of apoptotic vesicles from mouse muscle cells, Figure 1 A is the extraction method of apoptotic vesicles (apoVs); Figure 1 B is the electron microscopic analysis result of apoptotic vesicles; Figure 1C is the nanoparticle size tracking analysis result of apoptotic vesicles; Figure 1 D is the result of Annexin V-FITC staining, the red curve is the growth medium vesicles, the green curve is the apoptotic vesicles, and the purple curve is the apoptotic vesicles induced by staurosporine; Figure 1 E is the western blot analysis results of vesicles in growth medium, apoptotic vesicles in differentiation medium, and apoptotic vesicles induced by staurosporine;
[0026] Figure 2 This is a diagram showing the effect of apoptotic vesicle treatment on promoting skeletal muscle cell differentiation, where: Figure 2 A is the western blot analysis results of myosin heavy chain (MyHC) after C2C12 was treated with benzamide (ZVAD) and growth medium vesicles; Figure 2 B is the western blot analysis result of myosin heavy chain (MyHC) after ZVAD and apoVs treatment of C2C12; Figure 2 C is the MyHC immunofluorescence results after C2C12 was treated with ZVAD and apoVs; Figure 2 D is the western blot analysis result of MyHC after apoVs treatment of C2C12; Figure 2 E is the MyHC immunofluorescence result after apoVs treatment of C2C12;
[0027] Figure 3 The results show that apoptotic vesicles processing promotes the development of mouse skeletal muscle, Figure 3 A is the in vivo imaging at different time points after PKH67-labeled apoVs were injected into the tibialis anterior muscle; Figure 3 B is the immunofluorescence image of the isolated tibialis anterior muscle after PKH26-labeled apoVs injection; Figure 3 C is the H&E staining result of the tibialis anterior muscle after apoVs injection; Figure 3 D is the western blot analysis result of MyHC in tibialis anterior muscle after apoVs injection;
[0028] Figure 4 The figure shows the comparison between the isolated tibialis anterior muscle and the control group after apoptotic vesicle treatment, as well as the statistical results of the ratio to body weight;
[0029] Figure 5 The results showed that the treatment of apoptotic vesicles delayed skeletal muscle atrophy in aged mice, Figure 5 A is the muscle strength test results of mice of different ages; Figure 5 B is the western blot analysis results of aging-related proteins; Figure 5 C is the statistical result of the ratio of tibialis anterior muscle to body weight after apoVs injection; Figure 5D is the H&E staining result of the tibialis anterior muscle after apoVs injection; Figure 5 E is the immunofluorescence result of dystrophin after apoVs injection; Figure 5 F is the western blot analysis result of MyHC in tibialis anterior muscle after apoVs injection; Figure 5 G is the muscle strength test results of aged mice after apoVs injection;
[0030] Figure 6 The results showed that apoptotic vesicle treatment delayed skeletal muscle atrophy in diabetic mice. Figure 6 A is the statistical result of the ratio of tibialis anterior muscle weight to body weight of mice after apoVs injection; Figure 6 B is the H&E staining result of skeletal muscle after apoVs injection; Figure 6 C is the muscle strength test results of diabetic mice after apoVs injection. DETAILED DESCRIPTION
[0031] The technical solution of the present invention is further described below.
[0032] Example 1: Extraction and identification of apoptotic vesicles from mouse muscle cells
[0033] 1. The extraction process of apoptotic vesicles from mouse muscle cells is as follows: Figure 1 As shown in A:
[0034] (1) Inducing differentiation of a mouse myoblast cell line C2C12 substrain using a differentiation medium containing 2% horse serum, and collecting the differentiation medium after 48 hours of culture. The horse serum was used to remove extracellular vesicles, and the basal medium of the differentiation medium was a high-glucose DMEM medium;
[0035] (2) The differentiation culture medium obtained in step 1 was centrifuged at 800 g for 10 min at 4°C to remove the cell pellet and obtain the first supernatant;
[0036] (3) The first supernatant was centrifuged at 2000 g for 10 min at 4°C to remove cell debris and obtain the second supernatant;
[0037] (4) The second supernatant was centrifuged at 16,000 g for 30 min at 4°C, and the supernatant was removed. The resulting precipitate was the crude apoptotic vesicles and resuspended in sterile PBS.
[0038] (5) The resulting suspension was centrifuged at 16,000 g for 30 min at 4°C, and PBS was removed. The resulting precipitate was mouse muscle cell-derived apoptotic vesicles (apoVs).
[0039] 2. Perform transmission electron microscopy analysis on the obtained apoptotic vesicles. The specific process is as follows:
[0040] (1) 20 μL of apoptotic vesicles were aspirated and dropped onto a copper grid for 1 min, and the supernatant was removed by filter paper.
[0041] (2) 20 μL of uranyl acetate was added dropwise to a copper grid for 1 min, and the supernatant was removed by filter paper.
[0042] (3) Dry at room temperature for several minutes; perform electron microscopy imaging at 100 kV.
[0043] The results are as follows Figure 1 As shown in Figure B, apoptotic vesicles derived from mouse skeletal muscle cells are biconcave disc-shaped with a diameter of approximately 100 to 200 nm.
[0044] 3. Perform nanoparticle tracking analysis (NTA) on the obtained apoptotic vesicles:
[0045] (1) Using a nanoparticle tracking analyzer to record the trajectory of apoptotic microvesicles under Brownian motion;
[0046] (2) Analyze using NTA analysis software.
[0047] The results are as follows Figure 1 As shown in C, the particle size distribution of apoVs derived from mouse skeletal muscle cells is 100-1000 nm.
[0048] 4. FITC staining of the obtained apoptotic vesicles and flow cytometry analysis:
[0049] (1) According to the above method, the culture medium was replaced to prepare growth medium vesicles and staurosporine-induced apoptosis vesicles;
[0050] (2) Resuspend the growth medium vesicles (GM.EVs), differentiation medium apoptotic vesicles (DM.apoVs), and staurosporine-induced apoptotic vesicles (STS.apoVs) pellets in PBS and stain with Annexin V-FITC staining solution at room temperature for 10 min;
[0051] (2) Centrifuge at 16,000 g for 30 min at 4°C to remove the staining solution;
[0052] (3) Wash the vesicles with PBS and centrifuge at 16,000 g for 30 min at 4°C. Remove the PBS and resuspend in PBS.
[0053] (4) Detect Annexin V-FITC staining on the machine.
[0054] The results are as follows Figure 1As shown in D, in contrast to the vesicles in the growth medium, the apoptotic vesicles in the differentiation medium and the staurosporine-induced apoptotic vesicles were both positively stained with Annexin V-FITC.
[0055] 5. Perform western blot analysis on the obtained apoptotic vesicles to detect marker proteins:
[0056] (1) The protein concentrations of the three vesicle precipitates obtained above were measured and then lysed using RIPA lysis buffer;
[0057] (2) After denaturation by adding SDS loading buffer, protein immunoblotting was performed to analyze the expression of Alix, TSG101, and Cleaved Caspase3.
[0058] The results are as follows Figure 1 As shown in E, apoptotic vesicles derived from mouse skeletal muscle cells can express the extracellular vesicle marker proteins TSG101 and Alix. In addition, compared with vesicles in the growth medium, apoptotic vesicles significantly overexpress the apoptosis-related marker protein cleaved Caspase 3.
[0059] Example 2: Apoptotic vesicle treatment promotes skeletal muscle cell differentiation
[0060] 1. Set up DMSO control group, ZVAD treatment group, GM.EVs rescue group, STS.apoVs rescue group and DM.apoVs rescue group.
[0061] Among them, the cells in the control group were pretreated with medium containing 0.1% DMSO for 2 h after the confluence reached 90%, and then replaced with differentiation medium to induce cell differentiation;
[0062] After the cells in the ZVAD-treated group reached 90% confluence, they were treated with 0.1%
[0063] The culture medium was pretreated with DMSO for 2 h and then replaced with differentiation medium to induce cell differentiation;
[0064] After the cells in the GM.EVs, STS.apoVs, and DM.apoVs rescue groups reached 90% confluence, they were pretreated with a medium containing 0.1% DMSO and a final concentration of 50 μM ZVAD for 2 h, and then replaced with differentiation medium containing GM.EVs, STS.apoVs, or DM.apoVs at a final concentration of 20 ng / mL to induce cell differentiation;
[0065] After 3 days of differentiation, RIPA lysis buffer was used to extract proteins, and SDS loading buffer was added for denaturation before Western blotting to analyze the expression of MyHC. At the same time, immunofluorescence staining was performed to directly observe the expression of MyHC.
[0066] The results are as follows Figure 2 As shown in AC, after skeletal muscle cells were treated with ZVAD, the expression level of MyHC was significantly reduced. The addition of GM.Evs to the culture medium had no rescue effect, while the addition of apoVs could rescue the decrease in MyHC expression level caused by ZVAD treatment, indicating that apoVs can rescue the impaired skeletal muscle cell differentiation caused by inhibition of apoptosis activity.
[0067] 2. Set up a control group and an apoVs-treated group. When the confluence of the control group cells reached 90%, the cells were replaced with differentiation medium to induce differentiation. When the confluence of the apoVs-treated group cells reached 90%, the cells were replaced with differentiation medium containing DM.apoVs at a final concentration of 20 ng / mL to induce cell differentiation.
[0068] After 3 days of differentiation, RIPA lysis buffer was used to extract proteins, and SDS loading buffer was added for denaturation before Western blotting to analyze the expression of MyHC. At the same time, immunofluorescence staining was performed to directly observe the expression of MyHC.
[0069] like Figure 2 As shown in Figures DE, under normal differentiation conditions, DM.apoVs can also significantly increase the expression level of MyHC and promote skeletal muscle cell differentiation.
[0070] Example 3: Apoptotic vesicle treatment promotes skeletal muscle development in mice
[0071] Eight-week-old male C57BL / 6J mice were randomly divided into three groups: control group (Con), PKH26-labeled apoVs-treated group (apoVs), with 6 mice in each group. Five eight-week-old male C57BL / 6J mice were used for PKH67-labeled apoVs-treated group.
[0072] The DM.apoVs obtained in Example 1 were stained with PKH67 or PKH26 at a final concentration of 4 μM at room temperature for 20 min, and then washed twice with PBS, each time at 4°C. The precipitates were collected by centrifugation at 16,000 g for 30 min to obtain PKH67- or PKH26-labeled apoVs precipitates, which were then resuspended in PBS.
[0073] PKH26-labeled apoVs were injected into the tibialis anterior muscle of mice in the apoVs-treated group at two points in the muscle, with an injection dose of 2 μg per gram of body weight in a volume of 100 μL. The control group mice were injected with an equal volume of PBS; the injections were administered once a week for 8 consecutive weeks; PKH67-labeled apoVs were also injected into the tibialis anterior muscle of mice, with the same injection method, dose, and volume as before, and injected once.
[0074] 1. In vivo imaging of PKH67-labeled apoVs-treated mice was performed using a small animal in vivo imaging system before injection and 1, 2, and 3 days after injection, with a detection wavelength of 465 nm.
[0075] The results are as follows Figure 3 As shown in A, apoVs can be absorbed by the tibialis anterior muscle and the fluorescence intensity reaches the maximum on the second day after injection.
[0076] 2. After 8 weeks of continuous injection, the tibialis anterior muscles of the mice in the control group and the PKH26-labeled apoVs-treated group were isolated, and the body weights of the mice and the tibialis anterior muscles were weighed and recorded. The ratio of tibialis anterior muscle to body weight was calculated.
[0077] Tibialis anterior muscles were fixed with 4% paraformaldehyde, dehydrated, embedded, sectioned, stained with DAPI, and the intensity of PKH26 red fluorescence in skeletal muscle was detected;
[0078] Tibialis anterior muscle was lysed using RIPA lysis buffer to extract protein, and after denaturation by adding SDS loading buffer, the expression of MyHC was analyzed by western blotting.
[0079] The results are as follows Figure 3 B. Figure 4 As shown in Figure 2, apoVs can be taken up by mouse skeletal muscle cells, promote the development of tibialis anterior muscle, and significantly increase the tibialis anterior muscle-body weight ratio; H&E staining results are shown in Figure 2. Figure 3 As shown in C, apoVs can increase the cross-sectional area of skeletal muscle. The results of western blot analysis are shown in Figure 3 As shown in D, apoVs can significantly increase the expression level of MyHC and promote skeletal muscle development.
[0080] Example 4: Apoptotic vesicle treatment delays skeletal muscle atrophy in mice
[0081] 1. Apoptotic vesicle treatment delays skeletal muscle atrophy in aged mice:
[0082] Male C57BL / 6J mice aged 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 months were selected, with 5 mice per month. Grip strength of mice at each age was measured and statistically analyzed. At the same time, tibialis anterior muscles of mice aged 6 and 17 months were isolated, and protein was extracted to detect the expression of aging-related proteins.
[0083] The results are as follows Figure 5 As shown in AB, the muscle strength of mice began to decrease significantly at 17 months of age, and the expression levels of aging-related proteins P21 and P53 increased significantly.
[0084] Fifteen-month-old male C57BL / 6J mice were randomly divided into two groups: control group (Con) and apoVs-treated group (apoVs), with 7 mice in each group.
[0085] The DM.ApoVs obtained in Example 1 were resuspended in PBS to a final concentration of 1 mg / mL and injected into the tibialis anterior muscle of mice in the apoVs-treated group at two points in the muscle with an injection dose of 100 μL. The control group mice were injected with an equal volume of PBS. The injections were repeated once a week for 8 consecutive weeks.
[0086] After 8 consecutive weeks of injection at 17 months of age, the tibialis anterior muscles of the mice were isolated, and the body weights of the mice and the tibialis anterior muscles were weighed and recorded, and the ratio of tibialis anterior muscle to body weight was calculated;
[0087] The tibialis anterior muscle was fixed with 4% paraformaldehyde, and the cross-sectional area of the tibialis anterior muscle was examined by H&E staining;
[0088] The expression of dystrophin was detected by immunofluorescence.
[0089] At the same time, RIPA lysis buffer was used to lyse the tibialis anterior muscle to extract protein, and SDS loading buffer was added to denature it before Western blotting to analyze the expression of MyHC. The grip strength of the mice was also statistically analyzed.
[0090] The results are as follows Figure 5 As shown in Figures CD, apoVs significantly increased the ratio of tibialis anterior muscle weight to body weight in aged mice and increased the cross-sectional area of skeletal muscle; Figure 5 As shown in E, apoVs significantly increased the expression of dystrophin; Figure 5 As shown in FG, apoVs significantly promoted MyHC expression and improved muscle strength in mice.
[0091] 2. Apoptotic vesicle treatment delays skeletal muscle atrophy in diabetic mice:
[0092] Eight-week-old male C57BL / 6J mice were randomly divided into three groups: control group (Con), high-fat diet group (HFD-Con), and apoVs-treated group (HFD-apoVs), with 6 mice in each group. The control group was fed with normal mouse chow, while the high-fat diet and apoVs groups were fed with high-fat diet.
[0093] The DM.ApoVs obtained in Example 1 were resuspended in PBS to a final concentration of 1 mg / mL and injected into the tibialis anterior muscle of mice in the apoVs-treated group at two points in the muscle with an injection dose of 100 μL. The control group and the high-fat group were injected with an equal volume of PBS. The injections were repeated once a week for 8 consecutive weeks.
[0094] After 8 weeks of continuous injection, the grip strength of mice was analyzed.
[0095] The tibialis anterior muscles of mice were isolated and the ratio of tibialis anterior muscles to body weight was calculated. The collected tibialis anterior muscles were fixed with 4% paraformaldehyde and the cross-sectional area of the tibialis anterior muscles was detected by H&E staining.
[0096] The results are as follows Figure 6 As shown in A, apoVs can significantly increase the ratio of tibialis anterior muscle to body weight; Figure 6 The H&E staining results in B showed that compared with the HFD group, apoVs could significantly increase the cross-sectional area of skeletal muscle and alleviate muscle atrophy; Figure 6 As shown in C, apoVs significantly improved the muscle strength of mice compared with the HFD group.
Claims
1. Application of apoptotic vesicles derived from the myogenic differentiation process of mouse muscle cells in the regulation of muscle physiology.
2. The use according to claim 1, characterized in that The application is an application for promoting muscle development.
3. The use according to claim 1, characterized in that The application is an application for delaying muscle atrophy.
4. The use according to claim 1, characterized in that The application is the application in preparing medicines for treating muscle-related diseases.
5. The use according to claim 4, characterized in that The muscle-related diseases include muscular dystrophy, disuse muscle atrophy, senile sarcopenia, and diabetic muscle atrophy.
6. The use according to any one of claims 1 to 5, characterized in that The steps of preparing the apoptotic vesicles include: (1) Inducing mouse muscle cell differentiation using differentiation medium, and collecting the differentiation culture medium after culture; (2) The differentiation culture medium obtained in step 1 is subjected to differential centrifugation, and the precipitate is collected to obtain mouse muscle cell-derived apoptotic vesicles.
7. The use according to claim 6, characterized in that The mouse muscle cells described in step 1 are the mouse myoblast cell line C2C12 substrain.
8. The use according to claim 6, characterized in that The differentiation medium in step 1 is a basic medium containing 2% horse serum.
9. The use according to claim 8, characterized in that The horse serum is horse serum from which vesicles are removed.
10. The use according to claim 6, characterized in that The culture time in step 1 is 40-50 hours.