Use of pdgf-c in muscle injury repair

Abstract

This invention discloses the application of PDGF-C in muscle injury repair. The study found that in CTX model mice with PDGF-C knockout, the proliferation of muscle satellite cells (Pax7)... + Ki67 + The number of muscle stem cells in CTX model mice was significantly lower than that in wild-type mice. AAV-mediated PDGF-C overexpression significantly increased the number of muscle stem cells, improved their activation level, and enhanced their self-renewal capacity. Furthermore, PDGF-C overexpression significantly increased the surface area of ​​muscle fibers in CTX model mice, enhancing their regenerative and repair capabilities, thereby promoting muscle injury healing and demonstrating a therapeutic effect on this disease. Moreover, PDGF-C's effect on promoting muscle injury repair was significantly higher than that of PDGF-B. Therefore, this invention provides a new method for treating muscle injuries and may open new avenues for the treatment of other muscle-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of PDGF-C in muscle injury repair. Background Technology

[0002] Muscle stem cells (also known as satellite cells) are a special type of adult stem cell in skeletal muscle. They possess the ability to self-renew and differentiate into mature muscle cells, acting as the executors of muscle regeneration. Under normal circumstances, muscle stem cells are in a quiescent state and do not divide. When muscle is injured, satellite cells are activated and proliferate and self-renew, replenishing the stem cell pool. The proliferated muscle stem cells can differentiate into new muscle cells needed for muscle regeneration to repair the damage. PAX7 (Paired Box 7) is a key transcription factor for muscle stem cells, expressed in the quiescent state. When muscle stem cells are activated, they begin to express MyoD (Myogenic Differentiation 1), co-expressing it with PAX7. KI67 (a proliferation marker) is commonly used to identify cells in an actively proliferating state. Good skeletal muscle regeneration capacity of muscle stem cells can maintain muscle homeostasis and promote damage repair; conversely, regenerative impairment will lead to muscle atrophy and functional decline. Therefore, muscle stem cells play a decisive role in muscle injury repair. A lack of muscle stem cells can exacerbate various diseases, such as congenital muscular dystrophy, age-related sarcopenia, permanent muscle damage and atrophy, etc. Treatment for these diseases typically focuses on symptom relief, improving quality of life, and attempting to restore the function of muscle stem cells. Currently, research in areas such as stem cell therapy is exploring new treatment methods in hopes of improving the prognosis for patients with these diseases.

[0003] The cardiotoxin (CTX) muscle injury model is a widely used experimental model for inducing skeletal muscle injury and regeneration in animals, especially mice. CTX works by disrupting the muscle cell membrane, leading to myofiber necrosis. This damage activates muscle stem cells, triggering a series of regenerative processes, including stem cell proliferation, differentiation, and the formation of new myofiber. The CTX model effectively mimics inflammatory muscle diseases and traumatic injuries and muscle atrophy, which are characterized by muscle inflammation, damage, and muscle shrinkage.

[0004] Platelet-derived growth factor-C (PDGF-C) is an important member of the PDGF family, playing a crucial role in angiogenesis and organismal development. Studies have shown that PDGF-BB can act as a regenerative factor in patients with Duchenne muscular dystrophy (DMD), activating the proliferation and migration of satellite cells to damaged muscle fibers to promote muscle regeneration. However, the muscle regeneration and repair function of PDGF-B cannot be directly inferred from the fact that PDGF-C also possesses this property, because the two differ fundamentally in their ligand-receptor interaction patterns and biological characteristics. Firstly, their receptor binding specificity differs; PDGF-C is a high-affinity ligand for PDGFR-α, while PDGF-B tends to bind more strongly to PDGFR-β. Secondly, they exhibit spatiotemporal heterogeneity in expression and structure; PDGF-C and PDGF-B show significant differences in developmental stages, tissue distribution, and protein domains. Furthermore, they differ in their interactions with cerebral ischemia, organ fibrosis, and estrogen receptor-positive (ER) processes. + In diseases such as breast cancer, the two exhibit heterogeneity in their molecular mechanisms within their microenvironment. Currently, little is known about the biological role of PDGF-C in regulating muscle stem cells, especially its role in muscle regeneration and repair, and its clear role in muscle regeneration has not yet been discovered. Summary of the Invention

[0005] The purpose of this invention is to explore a novel strategy for effective repair of muscle tissue damage. This invention discovers that AAV-mediated PDGF-C overexpression can promote the activation, proliferation, and self-renewal of muscle stem cells, enhance the regenerative capacity of muscle fibers, and significantly promote the repair of muscle injury.

[0006] According to one aspect of the present invention, the use of PDGF-C in the preparation of a medicament that promotes the repair of muscle damage is provided.

[0007] Preferably, in the above applications, the PDGF-C can promote the activation, proliferation and self-renewal of muscle stem cells and enhance the regenerative capacity of muscle fibers.

[0008] Preferably, the muscle injury includes: traumatic muscle injury or non-traumatic muscle injury.

[0009] Preferably, the non-traumatic muscle injury includes any one or more of the following: myogenic muscle atrophy, disuse muscle atrophy, muscle dystrophy, sarcopenia, myopathy, and myasthenia gravis.

[0010] According to a second aspect of the invention, a medicament for promoting muscle damage repair is provided, comprising a vector overexpressing the PDGF-C gene and pharmaceutically acceptable excipients.

[0011] Preferably, the vector overexpressing the PDGF-C gene is an adeno-associated virus vector overexpressing the PDGF-C gene.

[0012] Preferably, the dosage form of the drug is an injection.

[0013] Preferably, the injectable includes an injection solution and a lyophilized powder for injection.

[0014] Preferably, the excipient is physiological saline.

[0015] According to a third aspect of the invention, the application of PDGF-C in promoting the proliferation of muscle stem cells is provided.

[0016] Preferably, in the above-described applications, the step of increasing the expression level of the PDGF-C gene in cells is included.

[0017] This invention found that in CTX model mice with Pdgf-c knockout, the proliferating muscle satellite cells (Pax7) + Ki67 + The number of muscle stem cells in CTX model mice was significantly lower than that in wild-type mice. AAV-mediated PDGF-C overexpression significantly increased the number of muscle stem cells, improved their activation level, and enhanced their self-renewal capacity. Furthermore, PDGF-C overexpression significantly increased the surface area of ​​muscle fibers in CTX model mice, enhancing their regenerative and repair capabilities, thereby promoting muscle injury healing and demonstrating a therapeutic effect on this disease. Moreover, PDGF-C's effect on promoting muscle injury repair was significantly higher than that of PDGF-B. Therefore, this invention provides a novel method for treating muscle injuries and may open new avenues for the treatment of other muscle-related diseases. Attached Figure Description

[0018] Figure 1 This is a flowchart of the mouse CTX model. In the diagram, A represents high expression of PDGF-C (red) in muscle stem cells. BC represents muscle satellite cells (yellow) that remain quiescent in healthy muscle fibers but are activated after injury and participate in regeneration or self-renewal. D shows the knockout efficiency of PDGF-C in the tibialis anterior muscle as displayed by Western blot, where n=3.

[0019] Figure 2 PDGF-C knockout significantly inhibited the proliferation of muscle stem cells. In the image, A shows representative immunofluorescence staining of PAX7 (red) and Ki67 (white) in muscle tissue on day 7 post-injury, with cell nuclei counterstained with DAPI (blue). Scale bar: 50 μm. B shows PAX7... + KI67 + Quantitative results of cells, n=5 per group.

[0020] Figure 3 PDGF-C overexpression significantly increased the number of proliferating muscle stem cells. A shows a schematic diagram of the AAV-induced PDGF-C overexpression and CTX-induced muscle injury model in the mouse tibialis anterior muscle. B shows Western blot results demonstrating PDGF-C overexpression in the tibialis anterior muscle via AAV, where n=3. C shows representative immunofluorescence staining of PAX7 (red) and Ki67 (white) in muscle tissue on day 7 post-injury, with cell nuclei counterstained with DAPI (blue). Scale bar: 50 μm. D shows PAX7... + KI67 + Quantitative results of cells, n=5 per group.

[0021] Figure 4 PDGF-C overexpression enhances the activation and self-renewal of muscle stem cells. In the image, A shows the MyoD (white), PAX7 (red), and DAPI (blue) immunofluorescence images of PDGF-C-overexpressing muscle in a CTX injury model, scale bar: 50 μm. B shows the PAX7... + MyoD - Quantitative results of cells; C represents PAX7. + MyoD + Quantitative results of cells. n=5 per group.

[0022] Figure 5 PDGF-C overexpression increases muscle fiber area and repairs muscle fibers. In figure A, HE staining shows enlarged muscle fiber regions in muscles overexpressing PDGF-C (scale bar: 50 μm). Figure B shows the quantitative analysis results of muscle fiber cross-sectional area. Each group had n = 5. Detailed Implementation

[0023] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents and materials used are commercially available unless otherwise specified.

[0025] Example 1

[0026] I. Materials

[0027] AAV2 / 9-PDGF-C and AAV2 / 9-GFP were purchased from Heyuan Biotechnology.

[0028] Information on the antibodies used in the experiment is shown in Table 1:

[0029] Table 1 Antibodies

[0030]

[0031] II. Methods

[0032] 1. Pdgf-c systemic knockout mice

[0033] The construction of Pdgf-c whole-body knockout mice was performed using a gene targeting strategy, as detailed in (Ding et al., Nature Genetics, 2004, PMID 15361870), and the mice have been bred for more than six generations on the C57BL / 6J background. The primers used for genotyping Pdgf-c defective mice were: 5'-CTGATGTTCTCGTGACTCTGA-3'; 5'-TAGCTAGTCGATACCGTCGA-3'; 5'-AGCTGACATTTGATGAGAGAT-3'; and 5'-AGTAGGTGAAATAAGAGGTGAACA-3'. A 200bp electrophoresis band represents a wild-type mouse, and a 350bp band represents a Pdgf-c knockout mouse. The steps for establishing muscle damage in Pdgf-c whole-body knockout mice are described in step 2 below.

[0034] 2. Cardiac toxin-induced muscle injury model

[0035] Eight-week-old C57 / BL6J mice were injected with 50 μL of AAV2 / 9-PDGF-C or AAV2 / 9-GFP (5 × 10⁻⁶) into the anterior tibialis muscle of both legs. 10 (vg / side). Muscle injury was induced two weeks later. To induce muscle injury, mice were anesthetized with 1% sodium pentobarbital and injected with 50 μL of cardiotoxin (CTX, 50 μM) into the right tibialis anterior muscle. An equal volume of saline was injected into the left leg. Seven days after injury, the tibialis anterior muscle was collected for weighing, Western blotting, HE staining, and immunofluorescence staining.

[0036] 3. Total protein extraction and BCA protein quantification.

[0037] 1) Add an appropriate amount of RIPA to the tissue to be tested, then add steel balls and grind in a grinder.

[0038] 2) Prepare reference samples of known concentrations by serially diluting the standard with 1×PBS according to the BCA kit; for protein samples, dilute the supernatant prepared in step 1) 10 times with 1×PBS to prepare the test sample, wherein the final volume of the test sample and the reference sample solution is 20μL.

[0039] 3) Preparation of working solution: Prepare the working solution according to the ratio of solution A: solution B = 50:1.

[0040] 4) Add the working solution prepared in step 3) to the test sample and the reference sample solution, with 200 μL of working solution for each tube, and mix thoroughly.

[0041] 5) Take 200 μL of the sample prepared in step 4) and add it to each well of a 96-well plate. Incubate at 37°C for 30 min.

[0042] 6) After incubation, use an ELISA reader to measure the absorbance of the sample at 562 nm and plot a standard curve (where the R value must be greater than 0.99), and calculate the protein concentration of each sample to be tested.

[0043] 4. Western blot experiment

[0044] 1) Preparing liquids:

[0045] ①10×Running Buffer: Add 144g glycine, 10g SDS powder, and 30.3g Tris powder sequentially, using ddH2O as the solvent, and bring the volume to 1L to prepare a storage solution. Dilute to 1×Running Buffer to prepare the electrophoresis working solution for use.

[0046] ②5×SDS Loading Buffer: Add 4g of SDS powder, 20mg of bromophenol blue, 3.085g of DTT, 10mL of Tris-HCl (1M pH 6.8), and 20mL of glycerol in sequence, and use ddH2O as a solvent to make up to 40mL.

[0047] ③ 10× Transfer Buffer: Add 30.3g Tris powder and 144g glycine sequentially, using ddH2O as the solvent, and bring the volume to 1L to prepare a storage solution. Dilute 10× Transfer Buffer 100mL + methanol 200mL + ddH2O 700mL to prepare 1× Transfer Buffer as the working solution for membrane transfer.

[0048] 2) Preparation of separating gel and stacking gel

[0049] ① Prepare 10% separating gel (10 mL):

[0050] Table 2 shows the preparation of 10% separating gel.

[0051]

[0052] ②Prepare a 5% concentrate (5 mL):

[0053] Table 3 shows the preparation of 5% concentrated gel.

[0054]

[0055] 3) Protein gel electrophoresis: Assemble the prepared gel and add the newly prepared electrophoresis solution to the tank. Check for leakage. Remove the comb and add the marker and sample to the gel wells in sequence. Add electrophoresis solution to the electrophoresis tank and set the program to 80V for 30 minutes of constant voltage electrophoresis. Then switch to 120V for 1 hour of constant voltage electrophoresis.

[0056] 4) Transfer: First, activate the PVDF membrane with methanol, and remove the gel from step 3). Assemble the transfer clamps in a "sandwich" structure, taking care not to leave air bubbles during assembly. Assemble the transfer apparatus, add the transfer working solution, set the program to a constant current of 250 mA for 2 hours, and place it on ice for transfer.

[0057] 5) Blocking: Take out the membrane after the transfer is completed, add 1×TBST and place it on a shaker at room temperature for 5 minutes to clean it. Discard the solution and add 5% skim milk as the blocking solution. Place it on a shaker at room temperature for blocking for 1 hour.

[0058] 6) Primary antibody incubation: Discard the blocking solution, add 1×TBST and place on a shaker at room temperature to wash until the blocking solution is clean. Discard the liquid, add the primary antibody prepared with 5% BSA as antibody diluent, and incubate overnight on a shaker in a 4°C cold storage.

[0059] 7) Wash the membrane: Wash the membrane three times with 1×TBST, 10 min each time.

[0060] 8) Secondary antibody incubation: Discard the solution, add secondary antibody prepared with 5% skim milk at a ratio of 1:5000, and incubate on a shaker at room temperature for 1 hour.

[0061] 9) Same as step 7).

[0062] 10) Exposure and development: Prepare the developer (A solution: B solution = 1:1 ratio, prepare and use immediately). Place the film on the plate, add developer solution to the film, shake well, expose and develop on the machine, and save the results.

[0063] 5. Immunofluorescence staining

[0064] Mouse muscle tissue was fixed with 4% polyoxymethylene (PFA, Sigma) for 2 hours at room temperature, then embedded overnight in 30% sucrose (Sigma) at 4°C. The tissue was then frozen using OCT (SAKURA) and sectioned into 10 μm sections. Sections were blocked for 1 hour at room temperature with a blocking solution of 5% BSA and 0.5% Triton X-100 prepared in PBS, followed by incubation overnight with primary antibody at 4°C. Secondary antibody (Life Technologies, Waltham, MA, USA) was incubated at room temperature for 1 hour. The sections were imaged using a fluorescence microscope (Zeiss, Oberkosen, Germany). Primary antibodies included Ki67 (AF7649-SP, R&D System), P63 (ET1610-44, HUABIO), PAX7 (AB-528428, DSHB), and MyoD (18943-1-AP, Proteintech). Secondary antibody included donkey anti-rabbit IgG (Alexa Fluor). TM 555, Life Technologies, A-31572), Goat anti-mouse IgG (Alexa Fluor) TM 488, Life Technologies, A-10680) and goat anti-mouse IgG (Alexa Fluor TM 488, Life Technologies, A-11006).

[0065] 6. Hematoxylin and eosin staining

[0066] All mice were anesthetized with isoflurane and euthanized. Separated muscles were fixed with 4% paraformaldehyde. Fixed tissues were dehydrated, embedded in paraffin, and sectioned. Frozen tissue blocks for hematoxylin and eosin (H&E) staining were cut into 10 μm thin sections. H&E staining was performed by Wuhan Saiwei Biotechnology Co., Ltd. according to standard protocols.

[0067] III. Results

[0068] All data are presented as mean ± standard deviation (SD). Statistical significance was assessed using a one-sample t-test. *p<0.05, **p<0.01, ***p<0.001.

[0069] 1. We found in publicly available databases that, among the PDGF family, PDGF-C expression is highest in muscle satellite cells (i.e., muscle stem cells) (Machado, Esteves de Lima et al., 2017) Figure 1 (A) To investigate the repair effect of PDGF-C on muscle injury, we established a CTX-induced mouse model of acute muscle injury (A). Figure 1In the process of CTX-induced damage, previously resting muscle stem cells are activated, thus initiating proliferation, self-renewal, and differentiation into new muscle fibers. We first constructed Pdgf-c gene knockout mice, then established the model, and Western blot analysis confirmed that PDGF-C was knocked out in muscle. Figure 1 (D in the middle).

[0070] 2. Immunofluorescence staining showed that in the Pdgf-c knockout mouse model of injury ( Figure 2 In A), proliferating muscle satellite cells (Pax7) + Ki67 + The number was significantly less than that of wild-type mice. Figure 2 (B in the middle).

[0071] 3. To explore the therapeutic effects of PDGF-C, we overexpressed PDGF-C in the anterior tibialis muscle of wild-type mice by injecting AAV-PDGF-C. Fourteen days later, we injected CTX into the anterior tibialis muscle to establish the model. Seven days later, we collected the anterior tibialis muscle tissue. Figure 3 In section A), Western blot analysis demonstrated that PDGF-C was significantly overexpressed in the tibialis anterior muscle. Figure 3 (B) Immunofluorescence staining showed that PDGF-C overexpression significantly increased the number of proliferating muscle stem cells, namely Pax7. + Ki67 + The proportion of double-positive muscle stem cells has increased. Figure 3 (CD in the middle).

[0072] 4. In addition, PDGF-C overexpression also increased the activation of PAX7. + MyoD + , Figure 4 (A and C in the middle) and self-renewing muscle stem cells (PAX7) + MyoD - , Figure 4 The number of A and B in the data indicates that PDGF-C overexpression not only improves the activation level of muscle stem cells, but also enhances their self-renewal capacity.

[0073] 5. HE staining further revealed that PDGF-C overexpression significantly increased the area of ​​muscle fibers in a mouse model of muscle injury. Figure 5 Figures A and B in the table show that PDGF-C overexpression significantly enhances the regenerative and repair capacity of muscle fibers. Therefore, these data fully demonstrate that mice overexpressing PDGF-C exhibit better muscle fiber repair performance than the control group.

[0074] The above detailed description is a specific description of the embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included in the patent scope of this case.

Claims

1. The application of a vector overexpressing the PDGF-C gene in the preparation of drugs that promote skeletal muscle injury repair, characterized in that, The skeletal muscle injury is a non-traumatic skeletal muscle injury.

2. The application according to claim 1, characterized in that, The PDGF-C can promote the activation, proliferation and self-renewal of muscle stem cells and enhance the regenerative capacity of muscle fibers.

3. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

4. The application according to claim 1, characterized in that, The vector overexpressing the PDGF-C gene is an adeno-associated virus vector overexpressing the PDGF-C gene.

5. The application according to claim 3, characterized in that, The drug is in the form of an injection.

6. The application according to claim 5, characterized in that, The injectables include injection solutions and lyophilized powder injections.

7. The application according to claim 3, characterized in that, The excipient is physiological saline.

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