A mesenchymal stem cell exosome and its preparation method and application

Abstract

The present invention provides a method for preparing mesenchymal stem cell exosomes, which is convenient to obtain materials and has low cost. It can quickly and efficiently prepare a large amount of high-purity mesenchymal stem cell-derived exosomes, which can effectively reduce costs and increase yields compared to the prior art, and is suitable for large-scale application; the exosomes can be used to promote the growth and repair of nerve cells, and can induce neural progenitor cells to differentiate into various nerve cell forms such as neurons and peripheral nerves; drugs can enter the cerebrospinal fluid through the exosomes, alleviate brain nerve degeneration, can pass through the blood-brain barrier, and can be used as a natural carrier for drugs for treating central nervous system diseases; it can regulate the growth of epidermal cells, promote the secretion of inflammatory cytokines in the microenvironment, stimulate angiogenesis and collagen deposition at skin defects, and regulate the proliferation and differentiation of skin fibroblasts, thereby accelerating skin tissue regeneration and repair; it can effectively promote hair follicle regeneration, thereby achieving a hair growth effect.

Description

Technical Field

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

[0002] Exosomes refer to small membrane vesicles with a diameter of 30-150nm, containing complex RNA and proteins, specifically disc-shaped vesicles with a diameter of 40-100nm. In 1983, exosomes were first discovered in sheep reticulocytes, and Johnstone named them "exosomes" in 1987. All cultured cell types can secrete exosomes, and exosomes exist naturally in body fluids, including blood, saliva, urine, cerebrospinal fluid, and milk. 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.

[0003] Current studies have shown that specifically secreted exosomes can participate in intercellular communication and have the potential to be used as carriers to transport specific substances to specific sites. Currently, exosomes are mainly extracted by ultracentrifugation, kit method or magnetic bead method. However, ultracentrifugation requires a centrifugal force of up to 100,000 g, the process is time-consuming, and the recovery rate is unstable. In addition, ultra-high speeds can easily damage exosome vesicles, thereby affecting the yield; the kits are diverse in type, quality, and highly targeted, making it difficult to meet different sample and extraction requirements; the magnetic bead method is often costly and complex to operate, and the extracted exosomes have problems such as low specificity and poor integrity, which limits the application of exosomes. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a mesenchymal stem cell exosome and a preparation method and application thereof.

[0005] The specific technical solutions of the present invention are as follows:

[0006] In one aspect, the present invention provides a method for preparing mesenchymal stem cell exosomes, comprising the following steps:

[0007] Add 0.65M potassium glycerophosphate solution or 0.075M potassium chloride solution to an equal volume of cultured mature mesenchymal stem cells, place them at 37°C for hypotonic treatment for 20 minutes, then wash with physiological saline, and place them at 37°C for hypotonic treatment for 40 minutes to destroy the cell membrane and further expose the exosomes; centrifuge the hypotonic treated cells at 4°C at a speed of 300-2000g for 20 minutes, transfer the supernatant to a new centrifuge tube, and filter the supernatant using 0.45μm and 0.22μm filter membranes to obtain a solution containing the exosomes.

[0008] The method is simple to operate and easy to use, and the mesenchymal stem cells obtained have a high yield and good purity, making it suitable for subsequent research.

[0009] Another aspect of the present invention provides mesenchymal stem cell exosomes prepared by the above method, wherein the exosomes contain specific antibodies CD9, CD63, CD81 and PLAP.

[0010] The exosomes contain exosome-specific markers CD9, CD63, CD81 and PLAP, and the content is relatively high, with a total amount of 100-2000 μg / ml, among which CD9 can reach 300-1000 ng / ml, CD63 can reach 20-200 ng / ml, CD81 can reach 30-400 ng / ml, and PLAP can reach 20-200 ng / ml, indicating that the yield of exosomes obtained using the above method is relatively high.

[0011] The present invention also provides the use of the above-mentioned mesenchymal stem cell exosomes in the preparation of a preparation for promoting the differentiation, growth and replication of nerve cells.

[0012] The exosomes can promote the differentiation of neurogenic cells into neural progenitor cells, neurons, peripheral nerves and other neural structure formation, and can detect cell markers at each neural differentiation stage.

[0013] The present invention also provides the use of the above-mentioned mesenchymal stem cell exosomes in the preparation of a drug carrier.

[0014] The natural structural morphology of exosomes provides a basis for the uptake and transportation of small molecule chemical drugs (such as paclitaxel, curcumin, etc.). Since the drugs can be wrapped in exosomes without being absorbed by the blood and can reach the medication site directly, the toxic side effects of the drugs are reduced and the utilization rate of the drugs is improved. Compared with traditional chemical drug delivery systems, they have outstanding advantages.

[0015] Furthermore, the preparation method of the drug carrier is as follows:

[0016] 10% bovine serum is added to the mesenchymal stem cell culture medium for culturing, and the use of bovine serum is stopped 3 days before the end of the culture. After the end of the culture, extraction and purification are performed to obtain the drug carrier.

[0017] The present invention also provides the use of the above-mentioned mesenchymal stem cell exosomes in the preparation of a preparation for promoting skin tissue repair and regeneration.

[0018] The exosomes provided by the present invention contain components such as proteins and RNA of mesenchymal stem cells, which can regulate inflammatory responses and promote the regeneration of skin-related cells. The preparations prepared with the exosomes can repair and accelerate cell growth, promote angiogenesis, and promote skin tissue repair and regeneration, thereby playing a good therapeutic role on skin damage, ulceration, etc.

[0019] Furthermore, the preparation comprises the hydrogel and the exosomes in a volume ratio of 1:1.

[0020] The present invention also provides the use of the above-mentioned mesenchymal stem cell exosomes in the preparation of a preparation for promoting hair follicle regeneration.

[0021] The exosomes provided by the present invention contain components such as proteins and RNA of mesenchymal stem cells, which can regulate inflammatory responses and promote the regeneration of hair follicle-related cells. The preparation prepared with the exosomes can accelerate the growth of hair follicle cells, promote angiogenesis, and effectively promote the regeneration of hair follicles, thereby having a good hair growth effect.

[0022] Furthermore, the preparation comprises the exosomes and physiological saline, wherein the content of the exosomes is 0.1-10.4%.

[0023] The beneficial effects of the present invention are as follows: the present invention provides a method for preparing mesenchymal stem cell exosomes, which is easy to obtain and has low cost. It can quickly and efficiently prepare a large amount of high-purity exosomes derived from mesenchymal stem cells. Compared with the existing technology, it can effectively reduce costs and increase yields, and is suitable for large-scale applications; the exosomes can be used to promote the growth and repair of nerve cells, and can induce neural progenitor cells to differentiate into various nerve cell forms such as neurons and peripheral nerves; drugs can enter the cerebrospinal fluid through exosomes, alleviate brain nerve degeneration, can pass through the blood-brain barrier, and can be used as a natural carrier for drugs for treating central nervous system diseases; it can regulate the growth of epidermal cells, promote the secretion of inflammatory cytokines in the microenvironment, stimulate angiogenesis and collagen deposition in skin defects, and regulate the proliferation and differentiation of skin fibroblasts, thereby accelerating skin tissue regeneration and repair; it can effectively promote hair follicle regeneration, thereby achieving a hair growth effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The quantitative determination results of exosome immunoenzyme-linked reaction in different parts in Experimental Example 1, where A is CD19, CD63, CD81, and B is PLAP;

[0025] Figure 2 This is the tracking result of the immunofluorescence reaction of the exosome antibody in Experimental Example 1;

[0026] Figure 3 This is the morphology of exosomes under electron microscope in Experimental Example 2;

[0027] Figure 4 The cell surface changes on the 6th day of culture after adding exosomes to the culture medium in Experimental Example 3;

[0028] Figure 5 This is the immunofluorescence morphology of neural cell differentiation on the 12th day of culture in Experimental Example 3;

[0029] Figure 6 The pathological healing of the skin wound of the rat in Experimental Example 4;

[0030] Figure 7 This is the skin hair growth of nude mice in Experimental Example 5;

[0031] Figure 8 This is the detection of exosomes labeling alveoli in vitro in Experimental Example 6. DETAILED DESCRIPTION

[0032] Example 1

[0033] Placental tissue was used to prepare mesenchymal stem cell exosomes. The specific method is as follows:

[0034] S1: In the GMP preparation workshop, the placental tissue was thoroughly washed with physiological saline containing 1% double antibody, the washed tissue was placed in a sterile culture dish, cut into 1 cm small pieces, fixed with serrated forceps, the blood vessels in the tissue were peeled off, and cut into pieces of about 1 mm 3 Small pieces were washed with saline;

[0035] S2: Add the blood vessel fragments to 20 ml serum-free culture medium for culture. Place them in an incubator at 37°C and 5% CO2 for the first 4 days. Change the medium starting from the 5th day and every 3 days thereafter. After 7 to 15 days, spindle cells can be observed to grow, and mesenchymal stem cells can be obtained. When 7 to 8 clones are obtained, they can be passaged and frozen.

[0036] S3: MSCs were cultured in non-additive medium for 48-72 h 3 days before culture to remove exogenous exosomes;

[0037] S4: The above mesenchymal stem cells were washed with PBS, and an equal volume of 0.65 M potassium glycerophosphate solution or 0.075 M potassium chloride solution was added, and the mixture was placed at 37°C for hypotonic treatment for 20 min. After that, the mixture was washed with normal saline, and then placed at 37°C for hypotonic treatment for 40 min to destroy the cell membrane and expose the exosomes.

[0038] S5: Centrifuge the hypotonic cell fluid at 300-2000 g for 20 min at 4° C., transfer the supernatant to a new centrifuge tube, and filter the supernatant using 0.45 μm and 0.22 μm filter membranes to obtain a solution containing the exosomes.

[0039] Example 2

[0040] Umbilical cord tissue was used to prepare mesenchymal stem cell exosomes, and the specific method was the same as that in Example 1.

[0041] Example 3

[0042] The amniotic membrane tissue was used to prepare mesenchymal stem cell exosomes, and the specific method was the same as that in Example 1.

[0043] Example 4

[0044] Umbilical cord blood was used to prepare mesenchymal stem cell exosomes. The specific method is as follows:

[0045] S1: Separate the whole blood cells from the umbilical cord blood and pre-treat the plasma; coat the cell culture dish with anti-CD34 antibody and anti-CD44 antibody, add the pre-treated whole blood cells from the umbilical cord blood, and separate and culture the nucleated cells;

[0046] S2: Add the separated nucleated cells to 20 ml serum-free medium for culture. Culture them in a 37°C, 5% CO2 incubator for the first 4 days. Change the medium starting from the 5th day and every 3 days thereafter. After 7 to 15 days, spindle cells can be observed to grow, and mesenchymal stem cells are obtained. When 7 to 8 clones are obtained, they can be passaged and frozen.

[0047] S3~S5 are the same as in Example 1.

[0048] Experimental Example 1

[0049] Comparison of exosome production

[0050] The exosomes produced by Examples 1 to 4 and the amniotic cell lysate were tested for CD9, CD63, CD81, and PLAP markers by enzyme-linked immunosorbent assay. The results are as follows: Figure 1 and Figure 2 As shown, the above four markers were detected in each group of samples, with a total amount of 100-2000 μg / ml, among which CD9 could reach 300-1000 ng / ml, CD63 could reach 20-200 ng / ml, CD81 could reach 30-400 ng / ml, and PLAP could reach 20-200 ng / ml, indicating that the yield of exosomes obtained by the above method was high. And the PLAP content in the placenta tissue lysate was the highest, indicating that the exosomes prepared by mesenchymal stem cells derived from placenta tissue had the highest yield and the strongest preparation capacity.

[0051] The above products were observed under an electron microscope, and cup-shaped bodies with a diameter of less than or equal to 100 nm were clearly visible, confirming the production of exosomes.

[0052] Experimental Example 2

[0053] Exosome expression and localization

[0054] Using three surface antigens marked by CD9, CD63, and CD81, exosomes were identified and traced by fluorescent immunoreaction. Figure 3 As shown, exosomal CD63-FITC can be accurately located and tracked.

[0055] Experimental Example 3

[0056] Application of mesenchymal stem cell exosomes in promoting neural cell differentiation

[0057] 10%, 20%, 30% and 50% of the extract prepared in Example 1 were added to the MEM culture medium, respectively, and filtered with a 0.22 μm filter membrane to remove cell debris and particulate impurities, as intervention groups 2 to 5. In addition, the neural cell culture medium added with serum was added to a six-well plate as a control group; amniotic membrane mesenchymal stem cells were inoculated in a six-well plate at a quantity of 1×105 cells / well for culture, and the growth of the cells was observed.

[0058] The experimental results on the 6th day of culture are as follows Figure 4 As shown, adding the extract of Example 1 to the culture medium can effectively support cell passage and promote the differentiation and growth of neuronal cells, and the effect is positively correlated with the amount of exosomes added. It can be seen that exosomes can promote the differentiation of neural cells into various neural structures such as neural progenitor cells, neuronal cells and peripheral nerves, and can detect markers of various neural differentiation stages.

[0059] The experimental results on the 12th day of culture are as follows Figure 5 As shown, the cells were stained with Anti-Nestin FITC antibody on the surface. The cell morphology and immunostaining results showed that the exosomes provided by the present invention have the ability to promote the differentiation of nerve cells; the number of differentiated neural stem cells in the intervention group was more than 10% higher than that in the control group, and the mass ratio of immunostaining of neuronal cells was 1-8%. It can be seen that the exosomes provided by the present application have a prominent effect of promoting the differentiation of nerve cells and promoting nerve growth.

[0060] Experimental Example 4

[0061] Application of mesenchymal stem cell exosomes in promoting skin tissue repair and regeneration

[0062] S1: Six healthy SD male rats aged 6 to 8 weeks were randomly divided into two groups. After anesthesia, the backs were shaved and six 1.5 cm × 1.5 cm full-thickness skin defects were made on both sides of the back with the spine as the center.

[0063] S2: preparing transplant materials, wherein group A: mixing the fulin honey hydrogel with the exosomes prepared in Example 1 at a volume ratio of 1:1, spreading it on the bottom of a 24-well plate, placing it in a 37°C incubator for 30 minutes, and taking it out for use after the mixture solidifies; group B: spreading the fulin honey hydrogel on the bottom of a 24-well plate, placing it in a 37°C incubator for 30 minutes, and taking it out for use after solidification;

[0064] S3: Equal amounts of transplant materials from the two groups were transplanted onto the wound surface. Ten days later, the rats were killed for dissection and the skin healing was observed using HE staining.

[0065] The experimental results are as follows Figure 6 As shown, the skin wounds of rats in group A healed well, and abundant new blood vessels were observed in pathology (indicated by arrows in Figure A), while the skin wounds of rats in group B did not heal, no new blood vessels were observed, and a large number of inflammatory cells were present (Figure B). It can be seen that the preparation of exosomes combined with hydrogels provided in this application can effectively promote the repair of skin defects.

[0066] Experimental Example 5

[0067] Application of mesenchymal stem cell exosomes in promoting hair follicle regeneration / treating hair loss

[0068] Six healthy nude mice aged 6 to 8 weeks were randomly divided into two groups. The intervention group was sprayed with 30% exosome spray (including exosomes and normal saline, of which the content of exosomes was 10%) on the body surface once a day, 0.5 ml each time, and the control group was sprayed with an equal amount of normal saline spray daily. The skin and hair conditions were observed after continuous spraying for 30 days.

[0069] The experimental results are as follows Figure 7 As shown, the hair on the body surface and mouth and nose of the nude mice in the intervention group increased significantly (Figures A and B), while there was no change in the nude mice in the control group (Figure C). It can be seen that the exosomes provided in the present application can effectively promote hair growth.

[0070] Experimental Example 6

[0071] Application of mesenchymal stem cell exosomes as drug carriers

[0072] The exosomes obtained in Example 1 were fluorescently stained using Hoechst 3325, and the stained exosomes were diluted 1000 times with PBS and sprayed into the mouth and nose of healthy nude mice (using the spray in Example 5), and then dissected, frozen and fixed 3 days later.

[0073] The experimental results are as follows Figure 8 As shown, the frozen sections were observed under a fluorescence microscope, showing that the alveoli were fluorescently labeled, indicating that the exosomes were able to reach the lungs. It can be seen that the exosomes provided in the present application can be used as carriers to carry drugs to specific sites for release.

[0074] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing mesenchymal stem cell exosomes, characterized in that: The steps include: Adding an equal volume of 0.65M potassium glycerophosphate solution or 0.075M potassium chloride solution to the cultured mature mesenchymal stem cells, placing them under hypotonic treatment at 37°C for 20 minutes, then washing with physiological saline, and then placing them under hypotonic treatment at 37°C for 40 minutes; centrifuging the hypotonic cell fluid at 4°C at a speed of 300-2000g for 20 minutes, transferring the supernatant to a new centrifuge tube, and filtering the supernatant using 0.45μm and 0.22μm filter membranes in turn to obtain a solution containing the exosomes; The exosomes are mesenchymal stem cell exosomes prepared from placental tissue.

2. The mesenchymal stem cell exosomes prepared according to the method provided in claim 1, characterized in that: The exosomes contain markers CD9, CD63, CD81 and PLAP.

3. Use of the mesenchymal stem cell exosomes according to claim 2 in the preparation of a drug carrier.

4. The use according to claim 3, characterized in that The preparation method of the drug carrier is as follows: 10% bovine serum is added to the mesenchymal stem cell culture medium for culturing, and the use of bovine serum is stopped 3 days before the end of the culture. After the end of the culture, extraction and purification are performed to obtain the drug carrier.

5. Use of the mesenchymal stem cell exosomes according to claim 2 in the preparation of a preparation for promoting skin tissue repair and regeneration.

6. The use according to claim 5, characterized in that The preparation includes the hydrogel and the exosomes in a volume ratio of 1:

1.

7. Use of the mesenchymal stem cell exosomes according to claim 2 in the preparation of a preparation for promoting hair follicle regeneration.

8. The use according to claim 7, characterized in that The preparation comprises the exosomes and normal saline, wherein the content of the exosomes is 0.1-10.4%.

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