Amniotic mesenchymal stem cell exosome as well as preparation method and application thereof

Abstract

The invention discloses an amniotic mesenchymal stem cell exosome and a preparation method and application thereof.The preparation method comprises the following steps that amniotic mesenchymal stem cells are inoculated into a complete culture medium to be cultured, and after the cells adhere to the wall, epidermal growth factors and polypeptide are added into the complete culture medium to continue to be cultured; replacing the complete culture medium, continuously culturing until the cell fusion degree is not lower than 85%, replacing the complete culture medium again, culturing, and collecting supernate; and extracting the exosome in the supernate by adopting an ultracentrifugation method to obtain the amniotic mesenchymal stem cell exosome. According to the preparation method of the amniotic mesenchymal stem cell exosome, the epidermal growth factor and the polypeptide are added into the culture medium, so that secretion of the cell exosome can be stimulated, meanwhile, carrying substances of the exosome are improved, the functions of promoting cell proliferation, migration and the like of the exosome are improved, and then healing of skin injury can be better promoted; the wound healing time is obviously shortened, and the pain of a patient is relieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to amniotic mesenchymal stem cell exosomes and a preparation method and application thereof. Background Art

[0002] The amnion is a thin membrane composed of a single layer of epithelial cells connected to each other. The single layer of epithelial cells has the function of secreting amniotic fluid. As the embryo grows and develops, the amnion and the chorion are closely attached to form the fetal membrane. As the amniotic cavity gradually expands, the fetal membrane becomes a translucent film with no blood vessels and is tough (the fetal membrane is also the cyst wall of the amniotic cavity). The liquid filled in the amniotic cavity is amniotic fluid. The amnion only covers the fetal surface of the placenta and does not penetrate into the placental tissue. As the pregnancy progresses, the amniotic cavity gradually expands and occupies the entire uterine cavity. The amnion is the innermost layer of the fetal membrane. It is a translucent film that is connected to the amniotic layer covering the placenta and umbilical cord.

[0003] Amniotic mesenchymal stem cells are a multifunctional stem cell with the ability to self-renew and regenerate. They can differentiate into various types of cells under certain conditions and have a wide range of medical application potential. The main effects of amniotic mesenchymal stem cells include: Tissue repair and regeneration: Amniotic mesenchymal stem cells can differentiate into a variety of cell types, including osteocytes, chondrocytes, and adipocytes, thereby promoting tissue repair and regeneration. This plays an important role in fracture healing, cartilage repair, and skin regeneration. Immunomodulation: Amniotic mesenchymal stem cells have immunomodulatory functions, which can inhibit inflammatory responses and regulate the activity of immune cells. This helps to reduce the occurrence and development of autoimmune diseases and reduce rejection reactions in situations such as organ transplantation. Angiogenesis: These stem cells have the ability to promote the formation of new blood vessels, which can improve blood circulation and promote tissue repair. This is very important in the treatment of cardiovascular diseases, ischemic diseases, and vascular damage. Anti-tumor effects: Recent studies have shown that amniotic mesenchymal stem cells may have anti-tumor effects, which can inhibit tumor growth and spread and improve the efficacy of chemotherapy and radiotherapy.

[0004] Exosomes refer to small membrane vesicles (30-150nm) containing complex RNA and proteins. Nowadays, they specifically refer to discoid vesicles with a diameter of 40-100nm. In 1983, exosomes were first discovered in sheep reticulocytes, and Johnstone named them "exosomes" in 1987. A variety of cells can secrete exosomes under normal and pathological conditions. They are mainly derived from multivesicular bodies formed by the invagination of intracellular lysosomal microparticles, and are released into the extracellular matrix after the multivesicular outer membrane fuses with the cell membrane.

[0005] As the largest organ of the human body, the skin has the functions of barrier, protection, regulation of body temperature and sensation. Skin defects caused by factors such as inflammation, ulcers, scalds, burns, etc. can be life-threatening in severe cases. Skin damage is caused by infection by external pathogenic microorganisms or lesions in the body. At present, skin damage is also the focus and difficulty of clinical treatment during the treatment process. Chronic wounds have a long treatment cycle and high costs, which affects the patient's quality of life and mental health, and also puts tremendous pressure on the medical system. In recent years, although there have been great advances in wound treatment, the therapeutic effects of various treatment measures are still difficult to be satisfactory.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide an amniotic mesenchymal stem cell exosome and a preparation method and application thereof. The amniotic mesenchymal stem cell exosome of the present invention can better promote the healing of skin injuries, significantly shorten the wound healing time, and relieve the patient's pain.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] The first aspect of the present invention provides a method for preparing amniotic mesenchymal stem cell exosomes, the preparation method comprising the following steps:

[0010] (a) inoculating amniotic membrane mesenchymal stem cells into a complete medium for culturing, and after the cells adhere to the wall, adding epidermal growth factor and a polypeptide to the complete medium for further culturing, wherein the polypeptide is YSRNEYGTF;

[0011] (b) replacing the complete medium and continuing to culture until the cell confluence is not less than 85%, replacing the complete medium again for culturing, and collecting the supernatant;

[0012] (c) extracting exosomes in the supernatant by ultracentrifugation to obtain the amnion mesenchymal stem cell exosomes.

[0013] Preferably, in step (a), the amniotic membrane mesenchymal stem cells are P3 to P6 cells.

[0014] Preferably, in step (a), the culture temperature is 37° C. and the carbon dioxide concentration is 5%.

[0015] Preferably, in step (a), epidermal growth factor and polypeptide are added to the complete culture medium and the culture is continued for 15 to 40 hours.

[0016] Preferably, in step (a), the final concentration of epidermal growth factor added is 5-10 ng / mL, and the final concentration of polypeptide added is 2-4 ng / mL.

[0017] Preferably, in step (a), the amniotic membrane mesenchymal stem cells are seeded at a concentration of (2-6)×10 5 Pieces / mL.

[0018] Preferably, in step (b), the complete culture medium is replaced again for 40 to 60 hours of culturing.

[0019] A second aspect of the present invention provides amniotic mesenchymal stem cell exosomes prepared by the above-mentioned method for preparing amniotic mesenchymal stem cell exosomes.

[0020] A third aspect of the present invention provides an application of amniotic membrane mesenchymal stem cell exosomes prepared by the method for preparing amniotic membrane mesenchymal stem cell exosomes in preparing skin damage products.

[0021] Compared with the prior art, the beneficial effects of the present invention include at least:

[0022] The preparation method of amniotic mesenchymal stem cell exosomes of the present invention can stimulate the secretion of exosomes by adding epidermal growth factor and polypeptide to the culture medium, and at the same time improve the carrying substances of the exosomes, thereby enhancing the functions of the exosomes in promoting cell proliferation and migration, thereby better promoting the healing of skin injuries, significantly shortening the wound healing time, and alleviating the pain of patients. DETAILED DESCRIPTION

[0023] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0024] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0025] The present invention provides a method for preparing amniotic mesenchymal stem cell exosomes, the method comprising the following steps:

[0026] (a) inoculating amniotic membrane mesenchymal stem cells into a complete medium for culturing, and after the cells adhere to the wall, adding epidermal growth factor and a polypeptide to the complete medium for further culturing, wherein the polypeptide is YSRNEYGTF;

[0027] (b) replacing the complete medium and continuing to culture until the cell confluence is not less than 85%, replacing the complete medium again for culturing, and collecting the supernatant;

[0028] (c) extracting exosomes in the supernatant by ultracentrifugation to obtain the amnion mesenchymal stem cell exosomes.

[0029] The preparation method of amniotic mesenchymal stem cell exosomes of the present invention can stimulate the secretion of exosomes by adding epidermal growth factor and polypeptide to the culture medium, and at the same time improve the carrying substances of the exosomes, thereby enhancing the functions of the exosomes in promoting cell proliferation and migration, thereby better promoting the healing of skin injuries, significantly shortening the wound healing time, and alleviating the pain of patients.

[0030] In some embodiments, in step (a), the amniotic membrane mesenchymal stem cells are P3 to P6 cells.

[0031] In some embodiments, in step (a), the culture temperature is 37° C. and the carbon dioxide concentration is 5%.

[0032] In some embodiments, in step (a), epidermal growth factor and polypeptide are added to the complete culture medium and the culture is continued for 15 to 40 hours.

[0033] In some embodiments, in step (a), the final concentration of epidermal growth factor added is 5-10 ng / mL, and the final concentration of polypeptide added is 2-4 ng / mL.

[0034] In some embodiments, in step (a), the amniotic membrane mesenchymal stem cells are seeded at a concentration of (2-6)×10 5 Pieces / mL.

[0035] In some embodiments, in step (b), the complete culture medium is replaced again for 40 to 60 hours of culturing.

[0036] Another embodiment of the present invention provides amniotic mesenchymal stem cell exosomes prepared by the above-mentioned method for preparing amniotic mesenchymal stem cell exosomes.

[0037] Yet another embodiment of the present invention provides a use of the amniotic membrane mesenchymal stem cell exosomes prepared by the method for preparing the amniotic membrane mesenchymal stem cell exosomes in preparing a skin damage product.

[0038] The technical solution of the present invention is further described in detail below through specific embodiments.

[0039] Example 1

[0040] This embodiment is a method for preparing amniotic mesenchymal stem cell exosomes, and the preparation method comprises the following steps:

[0041] (a) The amniotic membrane mesenchymal stem cells were inoculated into a complete medium for culture, and after the cells adhered to the wall, epidermal growth factor and polypeptide were added to the complete medium for further culture for 15 hours, wherein the polypeptide was YSRNEYGTF; the amniotic membrane mesenchymal stem cells were P3 cells; the culture temperature was 37°C, the carbon dioxide concentration was 5%; the final concentration of epidermal growth factor was 10 ng / mL, and the final concentration of polypeptide was 2 ng / mL; the inoculation concentration of the amniotic membrane mesenchymal stem cells was 4×10 5 Pieces / mL

[0042] (b) replacing the complete medium and continuing to culture until the cell confluence is not less than 85%, replacing the complete medium again and culturing for 60 hours, and collecting the supernatant;

[0043] (c) extracting exosomes in the supernatant by ultracentrifugation to obtain the amnion mesenchymal stem cell exosomes.

[0044] Example 2

[0045] This embodiment is a method for preparing amniotic mesenchymal stem cell exosomes, and the preparation method comprises the following steps:

[0046] (a) The amniotic membrane mesenchymal stem cells were inoculated into a complete medium for culture, and after the cells adhered to the wall, epidermal growth factor and polypeptide were added to the complete medium for further culture for 40 hours, wherein the polypeptide was YSRNEYGTF; the amniotic membrane mesenchymal stem cells were P3 cells; the culture temperature was 37°C, the carbon dioxide concentration was 5%; the final concentration of epidermal growth factor was 5 ng / mL, and the final concentration of polypeptide was 4 ng / mL; the inoculation concentration of the amniotic membrane mesenchymal stem cells was 4×10 5 Pieces / mL

[0047] (b) replacing the complete medium and continuing to culture until the cell confluence is not less than 85%, replacing the complete medium again and culturing for 40 hours, and collecting the supernatant;

[0048] (c) extracting exosomes in the supernatant by ultracentrifugation to obtain the amnion mesenchymal stem cell exosomes.

[0049] Example 3

[0050] This embodiment is a method for preparing amniotic mesenchymal stem cell exosomes, and the preparation method comprises the following steps:

[0051] (a) The amniotic membrane mesenchymal stem cells were inoculated into a complete medium for culture, and after the cells adhered to the wall, epidermal growth factor and polypeptide were added to the complete medium for further culture for 32 hours, wherein the polypeptide was YSRNEYGTF; the amniotic membrane mesenchymal stem cells were P3 cells; the culture temperature was 37°C, the carbon dioxide concentration was 5%; the final concentration of epidermal growth factor was 8 ng / mL, and the final concentration of polypeptide was 3 ng / mL; the inoculation concentration of the amniotic membrane mesenchymal stem cells was 4×10 5 Pieces / mL

[0052] (b) replacing the complete medium and continuing to culture until the cell confluence is not less than 85%, replacing the complete medium again and culturing for 50 hours, and collecting the supernatant;

[0053] (c) extracting exosomes in the supernatant by ultracentrifugation to obtain the amnion mesenchymal stem cell exosomes.

[0054] Comparative Example 1

[0055] This comparative example is a method for preparing amniotic mesenchymal stem cell exosomes, and the preparation method comprises the following steps:

[0056] (a) The amniotic mesenchymal stem cells were inoculated into complete culture medium for culture. After the cells adhered to the wall, the culture was continued for 32 hours. The amniotic mesenchymal stem cells were P3 cells. The culture temperature was 37°C and the carbon dioxide concentration was 5%. The inoculation concentration of the amniotic mesenchymal stem cells was 4×10 5 Pieces / mL

[0057] (b) replacing the complete medium and continuing to culture until the cell confluence is not less than 85%, replacing the complete medium again and culturing for 50 hours, and collecting the supernatant;

[0058] (c) extracting exosomes in the supernatant by ultracentrifugation to obtain the amnion mesenchymal stem cell exosomes.

[0059] Comparative Example 2

[0060] This comparative example is a method for preparing amniotic mesenchymal stem cell exosomes, and the preparation method comprises the following steps:

[0061] (a) The amniotic membrane mesenchymal stem cells were inoculated into a complete medium for culture. After the cells adhered to the wall, epidermal growth factor was added to the complete medium and cultured for 32 hours. The amniotic membrane mesenchymal stem cells were P3 cells. The culture temperature was 37°C and the carbon dioxide concentration was 5%. The final concentration of epidermal growth factor was 11 ng / mL. The inoculation concentration of the amniotic membrane mesenchymal stem cells was 4×10 5 Pieces / mL

[0062] (b) replacing the complete medium and continuing to culture until the cell confluence is not less than 85%, replacing the complete medium again and culturing for 50 hours, and collecting the supernatant;

[0063] (c) extracting exosomes in the supernatant by ultracentrifugation to obtain the amnion mesenchymal stem cell exosomes.

[0064] Comparative Example 3

[0065] This comparative example is a method for preparing amniotic mesenchymal stem cell exosomes, and the preparation method comprises the following steps:

[0066] (a) The amniotic membrane mesenchymal stem cells were inoculated into a complete medium for culture, and after the cells adhered to the wall, a polypeptide was added to the complete medium for further culture for 32 hours, wherein the polypeptide was YSRNEYGTF; the amniotic membrane mesenchymal stem cells were P3 cells; the culture temperature was 37°C, the carbon dioxide concentration was 5%; the final concentration of the polypeptide was 11 ng / mL; the inoculation concentration of the amniotic membrane mesenchymal stem cells was 4×10 5 Pieces / mL

[0067] (b) replacing the complete medium and continuing to culture until the cell confluence is not less than 85%, replacing the complete medium again and culturing for 50 hours, and collecting the supernatant;

[0068] (c) extracting exosomes in the supernatant by ultracentrifugation to obtain the amnion mesenchymal stem cell exosomes.

[0069] Experimental example

[0070] The amniotic mesenchymal stem cell exosomes prepared in Example 3 and Comparative Examples 1 to 3 were obtained respectively, and resuspended in PBS to obtain an exosome solution with a concentration of 0.15 mg / mL;

[0071] The experiment was conducted on BALA / c mice, which were randomly divided into 5 groups. Group 1 was the control and smeared with PBS solution. Groups 2 to 5 were smeared with the exosome solutions of Example 3 and Comparative Examples 1 to 3, respectively. The experimental process was as follows: the back of the mouse was depilated, and then a 0.8×0.8 cm skin defect was made under anesthesia; then, the corresponding exosome solution or PBS solution was applied when the skin defect was made, 6 hours, and 12 hours later; the application amount was 0.1 ml; then, the healing of the skin defect was observed after one week (7 days), and the area of ​​the healed skin defect of each group of mice was analyzed by Image J software, and the average healing rate (i.e., healing area / initial skin defect area×100%) was calculated, and the calculation results are shown in Table 1;

[0072] Table 1

[0073] Group Healing rate 1 group 43.26％ 2 groups 71.85％ 3 groups 52.16％ 4 groups 58.37％ 5 groups 59.28％

[0074] From Table 1, we can see that:

[0075] Compared with the exosomes in the comparative example, the exosomes prepared in the embodiment of the present invention can better promote wound healing and significantly shorten the healing time.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A method for preparing amniotic mesenchymal stem cell exosomes, characterized in that: The preparation method comprises the following steps: (a) inoculating amniotic membrane mesenchymal stem cells into a complete medium for culturing, and after the cells adhere to the wall, adding epidermal growth factor and a polypeptide to the complete medium for further culturing, wherein the polypeptide is YSRNEYGTF; (b) replacing the complete medium and continuing to culture until the cell confluence is not less than 85%, replacing the complete medium again for culturing, and collecting the supernatant; (c) extracting exosomes in the supernatant by ultracentrifugation to obtain the amnion mesenchymal stem cell exosomes.

2. The method for preparing amniotic mesenchymal stem cell exosomes according to claim 1, characterized in that: In the step (a), the amniotic membrane mesenchymal stem cells are P3 to P6 cells.

3. The method for preparing amniotic mesenchymal stem cell exosomes according to claim 1, characterized in that: In the step (a), the culture temperature is 37° C. and the carbon dioxide concentration is 5%.

4. The method for preparing amniotic mesenchymal stem cell exosomes according to claim 1, characterized in that: In the step (a), epidermal growth factor and polypeptide are added to the complete culture medium and the culture is continued for 15 to 40 hours.

5. The method for preparing amniotic mesenchymal stem cell exosomes according to claim 1, characterized in that: In the step (a), the final concentration of epidermal growth factor added is 5-10 ng / mL, and the final concentration of polypeptide added is 2-4 ng / mL.

6. The method for preparing amniotic mesenchymal stem cell exosomes according to claim 1, characterized in that: In the step (a), the amniotic membrane mesenchymal stem cells are seeded at a concentration of (2-6)×10 5 Pieces / mL.

7. The method for preparing amniotic mesenchymal stem cell exosomes according to claim 1, characterized in that: In the step (b), the complete culture medium is replaced again for 40 to 60 hours of culture.

8. Amniotic membrane mesenchymal stem cell exosomes prepared by the method for preparing amniotic membrane mesenchymal stem cell exosomes according to any one of claims 1 to 7.

9. Use of the amniotic membrane mesenchymal stem cell exosomes prepared by the method for preparing amniotic membrane mesenchymal stem cell exosomes according to any one of claims 1 to 7 in preparing skin damage products.