Preparation method and application of drug-loaded muscle fiber exosome for improving sarcopenia of pet dog

Abstract

The invention belongs to the technical field of exosome biological medicines, and particularly relates to a preparation method and application of a drug-loaded muscle fiber exosome for improving sarcopenia of a pet dog. The method is realized by the following steps: firstly, separating and culturing high-activity muscle stem cells: culturing and amplifying the high-activity muscle stem cells; then culturing, amplifying and differentiating the cultured primary muscle stem cells (MPCs) to form muscle fiber cells; the differentiated muscle fiber cells are loaded with SR12343 in several times, and the drug-loaded muscle fiber exosome is obtained. The muscle fiber exosome prepared by the invention contains specific bioactive factors of muscle functional cells, and can effectively improve the activity of an NF-kB signal channel for inhibiting mediating cell senescence and promote the activation and regeneration of senescent muscle cells in vivo under the action of cooperating with SR12343.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome biomedicine, and particularly relates to a preparation method and application of drug-loaded muscle fiber exosomes for improving sarcopenia in pet dogs. Background Art

[0002] In a healthy state, as pets enter old age, muscle mass will naturally and gradually decrease, which is called sarcopenia, also known as myopenia. Sarcopenia affects their quality of life, manifested as slow movement, movement disorders, weakness, and decreased immunity. Moreover, joint pain is prone to occur, and in severe cases, limb disability may occur. In addition to normal aging, diseases such as metabolic disorders, heart disease, kidney disease, and diabetes may also lead to muscle loss. As muscle mass decreases, fat mass will increase, further affecting their health. Muscle loss not only affects the daily activity ability of pets but also may pose a threat to their life and health. Currently, there is still a lack of effective and safe treatment methods for sarcopenia in pet dogs. Currently, for pet sarcopenia, drug treatment is an effective way to relieve muscle atrophy. Commonly used drug classifications include antioxidants, muscle relaxants, and hormonal drugs, etc. However, these drugs are prone to produce side effects during the treatment process. Therefore, seeking a safe and effective drug for improving sarcopenia in pet dogs has become an urgent problem to be solved. Summary of the Invention

[0003] Aiming at the problems existing in the prior art, the present invention provides a preparation method of drug-loaded muscle fiber exosomes for improving sarcopenia in pet dogs. Another object of the present invention is to provide the application of the drug-loaded muscle fiber exosomes prepared by the above preparation method in the preparation of drugs for improving and treating sarcopenia in pet dogs.

[0004] The technical solution adopted by the present invention to achieve the above object is as follows: The present invention provides a preparation method of drug-loaded muscle fiber exosomes for improving sarcopenia in pet dogs, comprising the following steps: (1) Isolating and culturing highly active muscle stem cells: Muscle stem cells (MPCs) from the hindlimb skeletal muscle of pets are screened and isolated for cells with high ALDH1 expression (ALDH1+ MPC) to obtain highly active muscle stem cells; The highly active muscle stem cells are cultured and amplified to obtain primary muscle cells; (2) When the cultured primary muscle stem cells (MPCs) are cultured and amplified to the 4th - 6th generation, DMEM basal culture medium containing horse serum is added to differentiate into muscle fiber cells; (3) Add the differentiated muscle fiber cells to a serum-free medium containing the drug SR12343 for culture. After the culture is completed, collect the culture medium and isolate the exosomes to obtain muscle fiber cell exosomes loaded with SR12343. (4) Add the muscle fiber cell exosome solution loaded with SR12343, add SR12343), then perform electroporation on the exosomes, and then perform water bath treatment to obtain drug-loaded muscle fiber exosomes.

[0005] Preferably, in step (1), the cells with high ALDH1 expression have an expression level of 40-50%.

[0006] Preferably, in step (1), the culture and amplification is to culture highly active muscle stem cells in a cell culture medium at 37 o °C for 4-5 days; the cell culture medium is DMEM + 20% fetal bovine serum.

[0007] Preferably, in step (2), the DMEM basal culture medium contains 4-6% horse serum; the differentiation time is 5 days.

[0008] Preferably, in step (3), the final concentration of the SR12343 drug in the culture medium is 5-20 μg / ml; the culture time is 48-72 h.

[0009] Preferably, in step (4), the concentration of the muscle fiber cell exosome solution loaded with SR12343 is 100-200 μg / m physiological saline; the concentration of SR12343 in the system is 50-150 μg / ml.

[0010] Preferably, in step (4), the electroporation is to perform 3-5 electric shocks using an electroporator at 200 - 400 V; the water bath treatment is to treat in a 37°C water bath for 20-40 min.

[0011] The present invention also provides a drug-loaded muscle fiber exosome prepared by the above preparation method.

[0012] The present invention further provides the application of the above drug-loaded muscle fiber exosomes in the preparation of drugs for improving and treating sarcopenia in pet dogs.

[0013] When the drug-loaded muscle fiber exosomes prepared by the present invention are used, the drug-loaded muscle fiber exosomes are suspended and dissolved in physiological saline, and intravenously injected at an equivalent of 5-20 mg / kg SR12343, and continuously injected 4-8 times.

[0014] The beneficial effects of the present invention are: (1) The muscle fiber exosomes prepared by the present invention contain bioactive factors unique to muscle functional cells. When acting synergistically with SR12343, they can effectively increase the activity of the NF-kB signaling pathway that inhibits cell senescence, and promote the activation and regeneration of senescent muscle cells in the body.

[0015] (2) The exosomes prepared by the preparation method provided by the present invention can be used to prepare biomedical products that promote the healthy growth and post-injury repair of the muscles of pets suffering from sarcopenia, and have excellent application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Morphology of isolated and cultured dog muscle stem cells and muscle fiber cells; among them, A. Cultured dog muscle stem cells. B. Muscle fiber cells (myotubes) induced and differentiated from muscle stem cells, red: myosin heavy chain (MHC); blue: DAPI (nuclear DNA); Figure 2 Effects of exosomes produced from muscle fiber cells and after drug loading on senescent cells; among them, A. Electron micrograph of exosomes from dog muscle fiber cells; B. Improvement effect of exosomes loaded with SR12343 on senescent dog muscle cells; Figure 3 Muscle pathological improvement effect of the drug-loaded muscle fiber exosomes prepared in Example 1 on the senile animal model; A. H.E staining of muscle sections after exosome treatment (5 weeks, once a week); B. Trichrome masson staining of muscle sections after exosome treatment (5 weeks, once a week); Figure 4 Improvement of muscle mass of the drug-loaded muscle fiber exosomes prepared in Example 2 on the senile animal model. DETAILED DESCRIPTION OF THE INVENTION

[0017] The technical solutions of the present invention will be further explained and illustrated below through specific examples.

[0018] Example 1 1. Isolate and culture highly active muscle stem cells: Extract muscle stem cells (MPCs) from the hindlimb skeletal muscles of a dog (male, 2 months old) to obtain approximately 5x10 4 cells. Use a flow cytometer to screen and isolate cells with high ALDH1 expression (ALDH1+ MPC), and isolate approximately 50% of ALDH1 to obtain approximately 2.5x10 4 cells; Then, add 10 ml of cell culture medium (DMEM + 20% fetal bovine serum) to the highly active muscle stem cells for large-scale culture and amplification, at 37 oAfter 5 days of culture, about 1x10 6 Muscle stem cells.

[0019] 2. Differentiate muscle stem cells into muscle fiber cells: When the cultured dog primary muscle stem cells (MPCs) were expanded to the fourth generation ( Figure 1 A), add DMEM basal medium containing 4% horse serum (1x10 6 cells / 10ml culture medium) differentiated into muscle fiber cells for 5 days ( Figure 1 B).

[0020] 3. Obtain muscle fiber cell exosomes loaded with SR12343: Serum-free DMEM culture medium (2x10 5 cells / 10 ml culture medium) and the culture medium was collected after 60 hours to isolate exosomes ( Figure 2 A). The final concentration of exosomes in cell culture medium can reach 14 μg / ml.

[0021] Figure 2 As can be seen in A, the exosomes from dog muscle fiber cells present a standard exosomal vesicle morphology.

[0022] 4. Continue to increase the loading of SR12343 in muscle fiber exosomes: The muscle fiber cell exosomes loaded with SR12343 were electroporated with SR12343 (50 μg / ml) in physiological saline solution (100 μg exosomes / mL) using an electroporator at 200 V (4 electric shocks), and then placed in a 37°C water bath for 20 minutes to load more SR12343 to obtain drug-loaded muscle fiber exosomes. The final content of SR12343 was about 2.5 μg SR12343 / 1 mg exosomes.

[0023] In vitro cell-level tests showed that after 4 days of treatment of D-galactose-induced aging dog muscle cells with drug-loaded exosomes (20 μg exosomes / ml culture medium), exosomes had a significant anti-aging effect on aging muscle cells (high expression of aging-related marker β-galactosidase), that is, the expression level of β-galactosidase was reduced ( Figure 2 B).

[0024] Effect Example 1 Use of exosomes on aging animal models The drug-loaded muscle fiber exosomes prepared in Example 1 were suspended and dissolved in physiological saline (10 mg / ml). The animal model was an 8-year-old dog, and an equivalent of 5 mg / kg SR12343 was intravenously injected once a week for 5 times. The effects were as Figure 3 .

[0025] As can be seen from Figure 3 , H.E. staining of muscle sections after exosome treatment (5 weeks, once a week) showed a reduction in the invasion of monocytes (such as macrophages) in senescent muscle.

[0026] Trichrome masson staining of muscle sections after exosome treatment (5 weeks, once a week) showed a reduction in fibrotic tissue (blue) in senescent muscle.

[0027] Example 2 1. Isolate and culture highly active muscle stem cells: Muscle stem cells (MPCs) were extracted from the hindlimb skeletal muscle of a dog (female, 4 months old), and about 7x10 4 cells were obtained. Flow cytometry was used to screen and isolate cells with high ALDH1 expression (ALDH1+ MPC), and about 40% of ALDH1 was isolated, resulting in about 2.8x10 4 cells; Then, the highly active muscle stem cells were rapidly and massively cultured and amplified. The cell culture medium (DMEM + 20% fetal bovine serum), and after 4 days of culture, about 1.2x10 6 muscle stem cells could be obtained.

[0028] 2. Differentiate muscle stem cells into muscle fiber cells: When the cultured primary dog muscle stem cells (MPCs) were cultured and amplified to the 6th generation, DMEM basal medium containing 6% horse serum was added and differentiated for 5 days to form muscle fiber cells.

[0029] 3. Obtain muscle fiber cell exosomes loaded with SR12343: Serum-free medium containing the SR12343 drug (final concentration of 10 μg / ml) was added for culture, and the culture medium was collected after 72 hours to isolate exosomes. The final concentration of exosomes in the cell culture medium could reach 18 μg / ml.

[0030] 4. Continue to increase the loading of SR12343 in muscle fiber exosomes: Muscle fiber cell exosome solution loaded with SR12343 (100 μg / mL), add SR12343 (100 μg / ml), use an electroporator at 300 V (4 electric shocks) to perform electroporation of exosomes, then place in a 37°C water bath for 30 minutes to load more SR12343. The final content of SR12343 is approximately 2.8 μg SR12343 / 1 mg exosome.

[0031] Effect Example 2 Use of drug-loaded muscle fiber exosomes on an animal model: Suspend the drug-loaded muscle fiber exosomes prepared in Example 2 in physiological saline. The animal model is an 8-year-old dog, and it is intravenously injected at an equivalent of 7.5 mg / kg SR12343. Inject once a week for 6 times. The effects are as Figure 4 。

[0032] From Figure 4 it can be seen that the hind limb muscle mass of the senescent dogs treated with exosomes (6 weeks, once a week) increased significantly.

[0033] Comparative Example 1 Steps 1-2 are the same as in Example 1; 3. Obtain muscle fiber cell exosomes (without SR12343): Add serum-free culture medium (2x10 5 cells / 10 ml culture medium) for culture, and collect the culture medium after 60 hours to isolate exosomes 4. Load SR12343 into muscle fiber exosomes: For the muscle fiber cell exosome physiological saline solution (100 μg exosome / mL), add SR12343 (55 μg / ml), use an electroporator at 200 V (4 electric shocks) to perform electroporation of exosomes, then place in a 37°C water bath for 20 minutes to load SR12343 to obtain drug-loaded muscle fiber exosomes. The final content of SR12343 is approximately 2.1 μg SR12343 / 1 mg exosome.

Claims

1. A preparation method of drug-loaded muscle fiber exosomes for improving sarcopenia in pet dogs, characterized in that, It includes the following steps: (1) Isolate and culture highly active muscle stem cells: Isolate muscle stem cells (MPCs) from the hindlimb skeletal muscle of pets, screen and isolate cells with high ALDH1 expression (ALDH1+MPC) to obtain highly active muscle stem cells; Cultivate and expand the highly active muscle stem cells to obtain primary muscle cells; (2) When the cultured primary muscle stem cells (MPCs) are cultured and expanded to the 4th - 6th generation, add DMEM basal medium containing horse serum to differentiate into muscle fiber cells; (3) Add serum-free medium containing SR12343 drug to the differentiated muscle fiber cells for culture. After the culture is completed, collect the culture medium and isolate exosomes to obtain muscle fiber cell exosomes loaded with SR12343; (4) Add the muscle fiber cell exosome solution loaded with SR12343, add SR12343), then perform electroporation on the exosomes, and then perform water bath treatment to obtain drug-loaded muscle fiber exosomes.

2. The preparation method according to claim 1, characterized in that, In step (1), the cells with high ALDH1 expression have an expression level of 40 - 50%.

3. The preparation method according to claim 1 or 2, characterized in that, In step (1), the culture and amplification are to culture highly active muscle stem cells in a cell culture medium at 37 o °C for 4-5 days; the cell culture medium is DMEM + 20% fetal bovine serum.

4. The preparation method according to claim 1, wherein In step (2), the DMEM basal medium contains 4 - 6% horse serum; the differentiation time is 5 days.

5. The preparation method according to claim 1 or 4, characterized in that, In step (3), the final concentration of the SR12343 drug in the culture medium is 5 - 20 μg / ml; the culture time is 48 - 72 h.

6. The preparation method according to claim 1, characterized in that, In step (4), the concentration of the muscle fiber cell exosome solution loaded with SR12343 is 100 - 200 μg / ml in physiological saline; the concentration of SR12343 in the system is 50 - 150 μg / ml.

7. The preparation method according to claim 1 or 6, characterized in that, In step (4), the electroporation is to perform 3 - 5 electric shocks using an electroporator at 200 - 400 V; the water bath treatment is to treat in a 37°C water bath for 20 - 40 min.

8. A drug-loaded muscle fiber exosome prepared by using the preparation method according to any one of claims 1 - 7.

9. Use of a drug-loaded muscle fiber exosome according to claim 8 in the preparation of a drug for improving and treating sarcopenia in pet dogs.

Images