Method for preparing muscle stem cell exosome for delaying extraocular muscle degeneration, product obtained thereby, and use thereof

Abstract

Provided is a method for loading a muscle stem cell exosome with a Notch activator and a senolytic drug, fisetin, that is capable of targeting and killing senescent cells, and further with lutein that has an eye protection effect, such that senescent muscle cells can be eliminated while the regeneration of muscle stem cells is promoted. By means of high-activity stem cell separation and culture technology, combined with exosome large-scale preparation and separation technology, simple and low-cost exosome drug-loading technology, senescent cell targeted clearance technology, etc., the present invention effectively helps to ameliorate the problems of extraocular muscle aging and atrophy, and can be widely applied to the damage repair of various muscle tissues. The present invention provides a new option for ameliorating the symptom of degeneration of extraocular muscle function, and has outstanding advantages in terms of safety, biocompatibility, and effectiveness.

Description

A method for preparing muscle stem cell exosomes for delaying extraocular muscle degeneration, the resulting product, and its applications. Technical Field

[0001] This invention belongs to the field of stem cell preparations, specifically relating to a method for preparing muscle stem cell exosomes for alleviating extraocular muscle degeneration, the resulting product, and its applications. Background Technology

[0002] Eye movements play a crucial role in an animal's ability to better perceive its surroundings. The extraocular muscles that control eye movement often atrophy and degenerate in aging individuals or due to certain diseases. This is related to eye misalignment, paralytic strabismus, myopia, presbyopia, and other eye function impairments, and may also lead to damage to the balanced, coordinated, and unidirectional movement of the extraocular muscles in both eyes. Muscle stem cells and their exosomes can effectively repair muscle tissue damage with minimal side effects. Muscle stem cell exosomes contain various active ingredients that promote muscle regeneration. Activation of the Notch signaling pathway is essential for muscle stem cells to maintain long-term self-renewal capacity, but its expression is significantly reduced in aging muscle tissue. The presence of senescent cells in muscle tissue can produce a zombie effect, impairing the function of normal muscle stem cells and hindering muscle regeneration.

[0003] In recent years, the rapid development of various cell-related drugs with significant efficacy in the pharmaceutical industry has attracted widespread attention. However, medical research and applications related to muscle stem cells are still in their early stages. Due to aging, tissue damage, or disease, there is a lack of safe and effective treatments for extraocular muscle atrophy and degeneration. Therefore, there is a significant demand for this type of technology in both clinical treatment and daily eye health maintenance. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for preparing muscle stem cell exosomes for delaying the aging and atrophy of extraocular muscles. By loading muscle stem cell exosomes with Notch activator and fisetin, a senolytic drug that can target and kill senescent cells, and further loading them with lutein, which has eye-protecting effects, it can effectively improve the atrophy and aging of extraocular muscles and maintain eye health.

[0005] The present invention also provides muscle stem cell exosomes prepared using the above-described preparation method.

[0006] Another object of the present invention is to provide the use of the above-mentioned muscle stem cell exosomes in the preparation of a drug for delaying the aging and atrophy of extraocular muscles.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0008] This invention provides a method for preparing muscle stem cell exosomes for delaying aging and atrophy of extraocular muscles, comprising the following steps:

[0009] (1) Isolation and culture of highly active muscle stem cells: The extracted muscle stem cells (hMPC) were screened and isolated by flow cytometry for cells with high ALDH1 and Notch3 expression (ALDH1+ / Notch3+ hMPC), and then cultured and expanded using a medium containing Jagged1 peptide and laccasein.

[0010] (2) Efficiently obtain muscle stem cell exosomes loaded with Jagged1 peptide, laccasein and lutein: Muscle stem cells with high expression of ALDH / Notch3 were cultured, and serum-free culture medium containing Jagged1 peptide, laccasein and lutein was added. After continuous culture, the supernatant of the culture medium was taken and centrifuged at low temperature to obtain exosomes loaded with Jagged1 peptide, laccasein and lutein.

[0011] Furthermore, in step (1), cells expressing the highest levels of ALDH1 and Notch3 (20-40%) are screened and isolated.

[0012] Furthermore, in step (1), the concentration of Jagged1 polypeptide in the culture medium is 10~30 mg / L, and the concentration of lacquer flavonoid is 5~25 mg / L; the composition of the culture medium is: low sugar DMEM culture medium containing 5-15% FBS.

[0013] Furthermore, in step (1), the culture and amplification conditions are 5% CO2, 37 o The cell culture was carried out in a cell culture incubator of type C.

[0014] Furthermore, in step (2), the muscle stem cells are ALDH1 / Notch3 highly expressed cells in the 4th to 6th generation of the logarithmic growth phase.

[0015] Furthermore, in step (2), the concentration of Jagged1 polypeptide in the serum-free culture medium is 10~30 mg / L, the concentration of lacquinone is 5~20 mg / L, and the concentration of lutein is 5~15 mg / L.

[0016] Furthermore, in step (2), the continuous culture period is 2-4 days, with 5% CO2 and 37... o In cell culture incubator C.

[0017] The present invention also provides a muscle stem cell exosome prepared using the above-described preparation method.

[0018] This invention also provides the application of the above-mentioned muscle stem cell exosomes in the preparation of drugs that delay the aging and atrophy of extraocular muscles.

[0019] The muscle stem cells (hMPC) used in this invention are extracted from the human body.

[0020] In the preparation method provided by this invention, after cell culture for 48-72 hours, the final concentration of exosomes in the cell culture medium can reach 10-20 μg / ml; the exosomes contain 0.02-0.05 mg / mg Jagged1 peptide, 0.01-0.04 mg / mg lacquer, and 0.01-0.03 mg / mg lutein.

[0021] The exosomes prepared by this invention can help produce exosome-based drugs for clinical treatment or eye drop products for daily eye care.

[0022] Treatment of extraocular muscles: Dissolve exosomes in physiological saline at a certain ratio and instill them into the eye socket as eye drops.

[0023] This invention utilizes the safe and benign effects of highly active muscle stem cell exosomes on muscle tissue, and combines Notch activator, senolytics drugs and lutein to enhance the positive efficacy of muscle stem cell exosomes on damaged extraocular muscle tissue.

[0024] The beneficial effects of this invention are as follows:

[0025] (1) This invention provides a method for preparing muscle stem cell exosomes for delaying the aging, atrophy, and functional degeneration of extraocular muscles. The obtained exosomes can help improve the health of extraocular muscles. When used, the exosomes are quantitatively suspended and dissolved in physiological saline and dripped into the orbit. They can interact with extraocular muscle cells and be absorbed by the cells, thereby exerting the effects of the components carried by the exosomes.

[0026] (2) This invention is the first to load muscle stem cell exosomes with Notch activator and fisetin, a senolytic drug that can target and kill senescent cells, and then load lutein, which has eye protection effects, to clear senescent muscle cells and promote the regeneration of muscle stem cells.

[0027] (3) The muscle stem cell exosomes of the present invention have biologically active factors and anti-aging drugs unique to stem cells, which can be used to prepare biomedical products such as synthetic drugs or cell repair drugs that promote healthy muscle growth and repair after injury.

[0028] (4) This invention utilizes highly active stem cell isolation and culture technology, combined with large-scale exosome preparation and separation technology, simple and low-cost exosome drug delivery technology, and senescent cell targeted clearance technology to effectively help improve the aging and atrophy of extraocular muscles. It can be widely applied to the repair of various muscle tissue injuries, such as helping to improve ptosis caused by levator palpebrae superioris muscle dysfunction, loss of facial expression or deepening of nasolabial folds caused by facial muscle atrophy, and the repair of various muscle tissue injuries, providing a new technological option for more effective and safe eye health maintenance.

[0029] (5) This invention provides a new option for improving symptoms of extraocular muscle degeneration, and has significant advantages in terms of safety, biocompatibility, and effectiveness. In the future, it can be combined with technologies such as 3D cell culture to further improve the activity of muscle stem cells and the yield of exosomes, and can also be combined with technologies such as cell membrane disruption to prepare exosomes to further reduce costs and better serve medical-grade applications. Attached Figure Description

[0030] Figure 1 is a schematic diagram of the technical route for preparing muscle stem cell exosomes according to the present invention;

[0031] Figure 2 shows the experimental results of the extraocular muscles of aging mice;

[0032] Figure 3 shows a comparison of the content of muscle stem cells in the extraocular muscles of aging mice. Detailed Implementation

[0033] The technical solution of the present invention will be further explained and described below with reference to specific embodiments.

[0034] Figure 1 shows a schematic diagram of the technical route for preparing muscle stem cell exosomes provided by the present invention.

[0035] Unless otherwise specified, all raw materials used in this invention are commercially available.

[0036] The Jagged1 peptide used in this invention was purchased from Abcam.

[0037] Example 1

[0038] (1) Method for isolating and culturing highly active muscle stem cells: Human muscle stem cells (hMPCs) were extracted, and cells with high ALDH1 and Notch3 expression (ALDH1+ / Notch3+ hMPCs) were screened and isolated using flow cytometry, resulting in approximately 40% of the cells with the highest ALDH1 and Notch3 expression. These cells were then cultured and expanded using a medium supplemented with Jagged1 peptide (20 mg / L) and lacquer yellow pigment (10 mg / L) (low-glucose DMEM medium containing 15% FBS), at 5% CO2, 37°C. oThe cell culture was carried out in a cell culture incubator of type C;

[0039] (2) Efficiently obtain muscle stem cell exosomes loaded with Jagged1 peptide and laccasein: ALDH / Notch3-overexpressing muscle stem cells in the logarithmic growth phase (passage 4) were cultured (~0.6x10⁻¹²). 4 cells / cm 2 (Surface of culture flask) , Add serum-free culture medium (95% OriCell basal medium + 5% serum-free stem cell culture additive) containing Jagged1 peptide (20 mg / L), laccasein (10 mg / L), and then lutein (10 mg / L) (0.2 mL / cm²). 2 (Surface of culture flask), after continuous culture for 3 days, take the supernatant of the culture medium and centrifuge at low temperature and ultra-high speed (4). o Exosomes loaded with Jagged1 peptide, laccasein, and lutein were obtained by cell culture at 100K rmp for 4 h. After 72 h of cell culture, the final concentration of exosomes in the cell culture medium reached approximately 10 μg / ml; the exosomes contained approximately 0.03 mg / mg Jagged1 peptide and approximately 0.02 mg / mg laccasein.

[0040] Effect Example

[0041] The method for treating the extraocular muscles of 18-month-old male mice with exosomes prepared in Example 1 was as follows: the exosomes were suspended and dissolved in physiological saline at a certain ratio (4 μg / mL), and then instilled into the orbit of the mice as eye drops. This treatment was repeated every 2 days. After 10 days, the eyeballs and extraocular muscles were collected for immunofluorescence staining. The control group was treated with physiological saline.

[0042] As shown in Figure 1, after 10 days of using the exosomes prepared according to the present invention, the muscle fibers of the extraocular muscles have a larger diameter (utrophin+) and more capillary structures (CD31+) in the muscles.

[0043] As shown in Figures 2 and 3, after 10 days of using the exosomes prepared according to the present invention, there were more muscle stem cells (Pax7+) in the extraocular muscles.

Claims

1. A method for preparing muscle stem cell exosomes for delaying the atrophic aging of the extraocular muscles, characterized by, The method comprises the following steps: (1) Isolation and culture of high-activity muscle stem cells: the extracted muscle stem cells (hMPC) are screened and separated by flow cytometry to obtain cells with high expression of ALDH1 and Notch3 (ALDH1+ / Notch3+ hMPC), and then cultured and expanded by using a culture medium containing a Jagged1 polypeptide and fisetin; (2) Efficiently obtain muscle stem cell exosomes loaded with Jagged1 polypeptide, fisetin and lutein: the muscle stem cells with high expression of ALDH / Notch3 are cultured, and a serum-free culture solution containing Jagged1 polypeptide, fisetin and lutein is added, and after continuous culture, the supernatant of the culture solution is obtained by low-temperature high-speed centrifugation to obtain exosomes loaded with Jagged1 polypeptide, fisetin and lutein.

2. The production method according to claim 1, characterized by, In step (1), 20-40% of the cells with the highest expression of ALDH1 and Notch3 are screened and separated.

3. The production method according to claim 1 or 2, characterized by, In step (1), the concentration of Jagged1 polypeptide in the culture medium is 10-30 mg / L, and the concentration of fisetin is 5-25 mg / L; the composition of the culture medium is low-sugar DMEM culture medium containing 5-15% FBS.

4. The production method according to claim 1 or 3, characterized by, In step (1), the culture expansion condition is 5% CO2, 37 o in a cell incubator of C.

5. The method of any one of claims 1-4, wherein, In step (2), the muscle stem cells are ALDH1 / Notch3 high-expression cells in the logarithmic growth phase and in the 4th to 6th generations.

6. The production method according to claim 1 or 5, characterized by, In step (2), the concentration of Jagged1 polypeptide in the serum-free culture solution is 10-30 mg / L, the concentration of fisetin is 5-20 mg / L, and the concentration of lutein is 5-15 mg / L.

7. The production method according to claim 1 or 6, characterized by, In step (2), the number of days of the continuous culture is 2-4 days, 5% CO2, 37 o C in a cell incubator.

8. A muscle stem cell exosome prepared by the preparation method of claims 1-7.

9. Use of the muscle stem cell exosome of claim 8 in the preparation of a drug for delaying the aging and atrophy of extraocular muscles.

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