Umbilical cord mesenchymal stem cell exosomes and preparation method and application thereof

Abstract

The present invention provides an umbilical cord mesenchymal stem cell exosome and a preparation method and application thereof, belonging to the technical field of wound repair. The present invention cultures the umbilical cord mesenchymal stem cells three times, and by adjusting the culture medium components and growth conditions of each culture, promotes the proliferation of umbilical cord mesenchymal stem cells and makes them express in a specific direction, which can increase the expression of cytokines such as EGF, HGF, IGF-1, PDGF, KGF and HB-EGF, TGF-β1, FGF-2, VEGF-A, etc. The umbilical cord mesenchymal stem cell exosomes of the present invention can effectively repair the wound, improve its wound healing rate, and shorten the number of days of healing. The umbilical cord mesenchymal stem cell exosomes prepared by the present invention have good application prospects in the fields of skin regeneration and wound repair.

Description

Technical Field

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

[0002] Human mesenchymal stem cells (MSC) are a type of stem cells that have a fibroblast-like morphology (spindle-shaped and fusiform) after adherent culture, can self-renew in vitro, and have the ability to differentiate into osteoblasts, adipocytes, and chondrocytes. Mesenchymal stem cells can not only differentiate into a variety of human cells, including adipocytes, osteoblasts, chondrocytes, muscle cells, tendon cells, etc.; at the same time, they can also be induced to differentiate into hepatocytes, pancreatic β cells, nerve cells, etc. Human mesenchymal stem cells can be isolated from a variety of human tissues (such as bone marrow, umbilical cord, placenta, fat, umbilical cord blood, etc.), and can also be obtained through differentiation or transdifferentiation; mesenchymal stem cells from different sources differ in gene expression and differentiation ability. In terms of application, it has been proven that mesenchymal stem cells are very safe and are the most widely used stem cells in clinical practice.

[0003] The topical use of mesenchymal stem cells to treat refractory wounds and skin injuries can greatly reduce their uncontrollability and tumorigenicity in vivo, but mesenchymal stem cells have a certain survival time in vitro, and cells will undergo apoptosis after 24 hours, affecting the therapeutic effect. Moreover, the skin surface environment is extremely unstable, and it is difficult for cells to survive for a long time. Dead cells form new pollutants, affecting the therapeutic effect. The exosomes of mesenchymal stem cells exist in the culture medium collected after the culture of mesenchymal stem cells, which contain a variety of cytokines, such as stem cell factor (SCF), fibroblast growth factor (FGF), endothelial growth factor (VEGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor 1-like growth factor (IGF), granulocyte colony stimulating factor (G-CSF), interleukin (IL), anti-inflammatory factors, transforming growth factor, etc. The exosomes of MSC have strong repair, regeneration and protection effects in the cardiovascular system, traumatic brain injury, musculoskeletal injury, liver injury, kidney injury, etc. However, in the field of wound repair or skin regeneration, the functional proteins contained therein are required to promote cell proliferation, regeneration and differentiation. Since the functional proteins in exosomes are often determined by the cells that are secreted by them, it is necessary to regulate the differential expression of the functional protein components in exosomes to meet the needs of wound repair or skin regeneration. Summary of the invention

[0004] The present invention obtains exosomes that are adapted to the needs of wound repair or skin regeneration by inducing and culturing human umbilical cord mesenchymal stem cells three times and adjusting the formula of the culture medium and the culture conditions.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing umbilical cord mesenchymal stem cell exosomes, comprising the following steps:

[0007] S1. Umbilical cord mesenchymal stem cells were cultured at 2-8×10 5 The cells were inoculated into the first culture medium at a density of 100 cells / mL and cultured for 23 to 25 hours to obtain the first generation of cells;

[0008] The first culture medium is: stem cell basal culture medium + 3-6 μM 1-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazine + 1.5-2.5 μM dexamethasone + 40-60 μg / mL ascorbic acid + 8-12 mM sodium β-glycerophosphate;

[0009] S2. The first generation cells were cultured at 2 to 8 × 10 5 The cells were inoculated at a density of 100 cells / mL in the second culture medium and cultured for 70 to 75 hours, with the medium replaced every 23 to 25 hours to obtain the second generation of cells;

[0010] The second culture medium is: stem cell basal culture medium + 150-200 μg / mL pioglitazone hydrochloride + 5-10 μg / mL naringin + 60-80 μg / mL ascorbic acid.

[0011] S3. The second generation cells were cultured at 2 to 8 × 10 5 The density of cells / mL was inoculated into the third culture medium, cultured for 45-50 hours, and the medium was changed every 23-25 ​​hours;

[0012] The third culture medium is: stem cell basal culture medium + 0.5-2 μg / mL transforming growth factor-β1 + 1-5 μg / mL stromal cell-derived factor-1α + 4-6 μg / mL insulin-like growth factor 1.

[0013] S4. Collect the culture medium in step S3, and after differential centrifugation, collect the precipitate, which is the umbilical cord mesenchymal stem cell-containing exosomes.

[0014] Preferably, the culture conditions in S1 are 36-38° C., 2-5% CO 2 ; and the culture conditions in S2 are 36-38° C., 2-5% O 2 .

[0015] Preferably, the stem cell basal culture medium includes one of DMEM / F12 complete culture medium, IMDM culture medium and RPMI-1640 complete culture medium.

[0016] Preferably, the culture conditions in S3 are 36-38° C., 2-5% CO 2 , and pulse ultrasonic treatment is performed at the 16th to 18th hour of culture, and the pulse treatment time is 10 to 20 minutes.

[0017] Preferably, the frequency of the pulsed ultrasonic treatment is 0.5-2 MHz, the duty cycle is 20-30%, and the intensity is 40-60 W / cm 2 .

[0018] Preferably, the differential centrifugation is: 400-500g for 10-15min, 1800-2200g for 10-15min, and 9000-11000g for 25-35min.

[0019] The present invention also provides umbilical cord mesenchymal stem cell exosomes prepared by the preparation method.

[0020] The present invention provides the use of the umbilical cord mesenchymal stem cell exosomes in preparing a product for repairing skin wounds.

[0021] The present invention also provides a medicine for skin wound repair, wherein the active ingredients of the medicine include the umbilical cord mesenchymal stem cell exosomes.

[0022] Preferably, the dosage form of the drug includes injection, spray or ointment.

[0023] By adopting the above technical scheme, the present invention has the following beneficial effects: the present invention cultures umbilical cord mesenchymal stem cells three times, and by adjusting the culture medium components and growth conditions of each culture, the umbilical cord mesenchymal stem cells are promoted to proliferate and express in a specific direction, and the expression of cytokines such as EGF, HGF, IGF-1, PDGF, KGF, HB-EGF, TGF-β1, FGF-2, and VEGF-A can be increased. The umbilical cord mesenchymal stem cell exosomes of the present invention can effectively repair the wound surface, improve its wound healing rate, and shorten the number of days of healing. The exosomes prepared by the present invention have good application prospects in the fields of skin regeneration and wound repair. DETAILED DESCRIPTION

[0024] The present invention provides a method for preparing umbilical cord mesenchymal stem cell exosomes, comprising the following steps:

[0025] S1. Umbilical cord mesenchymal stem cells were cultured at 2-8×10 5The cells were inoculated into the first culture medium at a density of 100 cells / mL and cultured for 23 to 25 hours to obtain the first generation of cells;

[0026] The first culture medium is: stem cell basal culture medium + 3-6 μM 1-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazine + 1.5-2.5 μM dexamethasone + 40-60 μg / mL ascorbic acid + 8-12 mM sodium β-glycerophosphate;

[0027] S2. The first generation cells were cultured at 2 to 8 × 10 5 The cells were inoculated at a density of 100 cells / mL in the second culture medium and cultured for 70 to 75 hours, with the medium replaced every 23 to 25 hours to obtain the second generation of cells;

[0028] The second culture medium is: stem cell basal culture medium + 150-200 μg / mL pioglitazone hydrochloride + 5-10 μg / mL naringin + 60-80 μg / mL ascorbic acid.

[0029] S3. The second generation cells were cultured at 2 to 8 × 10 5 The density of cells / mL was inoculated into the third culture medium, cultured for 45-50 hours, and the medium was changed every 23-25 ​​hours;

[0030] The third culture medium is: stem cell basal culture medium + 0.5-2 μg / mL transforming growth factor-β1 + 1-5 μg / mL stromal cell-derived factor-1α + 4-6 μg / mL insulin-like growth factor 1.

[0031] S4. Collect the culture medium in S3, perform differential centrifugation, and collect the precipitate, which is the umbilical cord mesenchymal stem cell-containing exosomes.

[0032] The present invention firstly cultures the umbilical cord mesenchymal stem cells. The present invention has no special limitation on the culture of the umbilical cord mesenchymal stem cells. The culture can be carried out by conventional methods and conventional culture conditions of those skilled in the art. The culture medium used in the culture is preferably α-MEM culture medium. After the cells have grown to 80-90% of the culture bottle, the culture medium in the culture solution is discarded. After washing with PBS, the umbilical cord mesenchymal stem cells are inoculated in the first culture medium for the first culture to obtain the first generation of cells. The inoculation density is 2-8×10 5 pcs / mL, more preferably 4 to 6×10 5 / mL, more preferably 5×10 5 / mL; the culture temperature is 36-38°C, more preferably 36.5-37.5°C, more preferably 37°C; the CO2 content in the culture environment is 2-5%, more preferably 3-4%, more preferably 3.5%; the culture time is 23-25h, more preferably 23.5-24.5h, more preferably 24h; the first culture medium is stem cell basal medium + 3-6μM 1-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazine + 1.5-2.5μM dexamethasone + 40-60μg / mL ascorbic acid + 8-12mM sodium β-glycerophosphate;

[0033] In the first culture medium, the concentration of 1-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazine is 3 to 6 μM, more preferably 4 to 5 μM, and more preferably 4.5 μM;

[0034] The concentration of dexamethasone is 1.5 to 2.5 μM, more preferably 1.8 to 2.2 μM, and more preferably 2.0 μM;

[0035] The concentration of ascorbic acid is 40 to 60 μg / mL, more preferably 45 to 55 μg / mL, and more preferably 50 μg / mL;

[0036] The concentration of the sodium β-glycerophosphate is 8 to 12 mM, more preferably 9 to 11 mM, and even more preferably 10 mM.

[0037] 1-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazine, dexamethasone, ascorbic acid and sodium beta-glycerophosphate in the first culture medium act together to make the umbilical cord mesenchymal stem cells grow and express in a specific direction.

[0038] In the present invention, the first generation cells are washed with PBS and then inoculated in a second culture medium for a second culture to obtain second generation cells. The inoculation density is 2 to 8×10 5 pcs / mL, more preferably 4 to 6×10 5 / mL, more preferably 5×10 5 / mL; the culture temperature is 36-38°C, more preferably 36.5-37.5°C, more preferably 37°C; the O2 content in the culture environment is 2-5% (v / v), more preferably 3-4%, more preferably 3.5%; the CO2 content is 2-5% (v / v), more preferably 3-4%, more preferably 3.5%, and the remaining gas is N2. The culture time is 70-75h, more preferably 71-74h, more preferably 72h; the liquid is changed every 23-25h, more preferably every 23.5-24.5h, more preferably every 24h. The second culture medium is stem cell basal medium + 150-200μg / mL pioglitazone hydrochloride + 5-10μg / mL naringin + 60-80μg / mL ascorbic acid;

[0039] In the second culture medium, the concentration of pioglitazone hydrochloride is 150-200 μg / mL, more preferably 170-190 μg / mL, and more preferably 180 μg / mL;

[0040] The concentration of naringin is 5 to 10 μg / mL, more preferably 7 to 9 μg / mL, and more preferably 8 μg / mL;

[0041] The concentration of ascorbic acid is 60 to 80 μg / mL, more preferably 65 to 75 μg / mL, and even more preferably 70 μg / mL.

[0042] In addition, the second culture of the present invention is carried out under hypoxic conditions. Compared with normal oxygen conditions, hypoxic conditions promote the proliferation of mesenchymal stem cells. The oxygen content of the present invention is preferably adjusted using a purification incubator, and under 2-5% hypoxic conditions, pioglitazone hydrochloride, naringin and ascorbic acid produce a synergistic effect, so that the first generation cells grow and express in a specific direction, thereby obtaining the second generation cells.

[0043] After obtaining the second generation cells, the present invention inoculates them into a third culture medium for third culture to obtain the second generation cells. The inoculation density is 2 to 8×10 5 pcs / mL, more preferably 4 to 6×10 5 / mL, more preferably 5×10 5 / mL; the culture temperature is 36-38°C, more preferably 36.5-37.5°C, more preferably 37°C; the CO2 content in the culture environment is 2-5%, more preferably 3-4%, more preferably 3.5%; the culture time is 45-50h, more preferably 47-49h, more preferably 48h; the liquid is replaced every 23-25h, more preferably every 23.5-24.5h, more preferably every 24h. Pulse ultrasonic treatment is performed at the 16th to 18th hour of the third culture, more preferably at the 16.5th to 17.5th hour, more preferably at the 17th hour, and the frequency of the pulse ultrasonic treatment is 0.5-2MHz, more preferably 1-1.5MHz, more preferably 1.2MHz; the duty cycle is 20-30%, more preferably 23-27%, more preferably 25%; the intensity is 40-60W / cm 2 , more preferably 45 to 55 W / cm 2 , more preferably 50W / cm 2 The pulse ultrasonic treatment time is 10 to 20 minutes, more preferably 12 to 18 minutes, and more preferably 15 minutes. The third culture medium is: stem cell basal medium + 0.5 to 2 μg / mL transforming growth factor-β1 + 1 to 5 μg / mL stromal cell derived factor-1α + 4 to 6 μg / mL insulin-like growth factor 1;

[0044] In the third culture medium, the concentration of transforming growth factor-β1 is 0.5 to 2 μg / mL, more preferably 1 to 1.5 μg / mL, and more preferably 1.3 μg / mL;

[0045] The concentration of the stromal cell-derived factor-1α is 1 to 5 μg / mL, more preferably 2 to 4 μg / mL, and more preferably 3 μg / mL;

[0046] The concentration of the insulin-like growth factor 1 is 4 to 6 μg / mL, more preferably 4.5 to 5.5 μg / mL, and even more preferably 5 μg / mL.

[0047] During the third culture, transforming growth factor-β1, stromal cell-derived factor-1α and insulin-like growth factor 1 in the third culture medium combined with low-intensity pulsed ultrasound can maximize the activation of the biological response of the second-generation cells, improve cell proliferation, activity and functionality, so that the exosomes secreted by them can accelerate biological processes such as cell proliferation, angiogenesis and matrix synthesis in skin wounds, and ultimately promote wound healing.

[0048] The basal culture medium of the first culture medium, the second culture medium and the third culture medium of the present invention comprises one of DMEM / F12 complete culture medium, IMDM culture medium and RPMI-1640 complete culture medium.

[0049] After the third culture is completed, the culture fluid is collected for differential centrifugation, and the precipitate is collected to obtain the exosomes containing umbilical cord mesenchymal stem cells. The differential centrifugation includes a first centrifugation, a second centrifugation and a third centrifugation, wherein the centrifugal force of the first centrifugation is 400-500 g, more preferably 420-480 g, more preferably 450 g, and the time of the first centrifugation is 10-15 min, more preferably 11-14 min, more preferably 12 min; the centrifugal force of the second centrifugation is 1800-2200 g, more preferably 1900-2100 g, more preferably 2000 g, and the time of the second centrifugation is 10-15 min, more preferably 11-14 min, more preferably 12 min; the centrifugal force of the third centrifugation is 9000-11000 g, more preferably 9500-10500 g, more preferably 10000 g, and the time of the third centrifugation is 25-35 min, more preferably 28-32 min, more preferably 30 min. After the third centrifugation, the centrifugation is repeated at a centrifugal force of 9000-11000 g, preferably 9500-10500 g, and more preferably 10000 g, and the time of the repeated centrifugation is 60-80 min, more preferably 65-75 min, and more preferably 70 min. The obtained precipitate is the exosomes of umbilical cord mesenchymal stem cells.

[0050] The present invention also provides umbilical cord mesenchymal stem cell exosomes prepared by the preparation method.

[0051] The present invention also provides the use of the exosomes in preparing a product for repairing skin wounds.

[0052] The present invention also provides a medicine for skin wound repair, wherein the active ingredients of the medicine include the umbilical cord mesenchymal stem cell exosomes.

[0053] Preferably, the dosage form of the drug includes injection, spray or ointment.

[0054] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0055] The human umbilical cord mesenchymal stem cells (HUMSC) of the present invention were purchased from ATCC.

[0056] The DMEM / F12 complete medium, IMDM medium and RPMI-1640 complete medium of the present invention were purchased from Wuhan Pronocell Life Science Co., Ltd., and other reagents can be purchased through commercial channels unless otherwise specified.

[0057] Example 1

[0058] Human umbilical cord mesenchymal stem cells (HUMSC) were purchased from ATCC. The cryopreserved tubes of human umbilical cord mesenchymal stem cells were taken out of dry ice and quickly thawed in a 37°C water bath. The cell suspension was then transferred to a centrifuge tube containing α-MEM medium and centrifuged at 400g for 5 min. The centrifuge tube was removed, the supernatant was discarded, α-MEM medium was added to resuspend the cells, and then the cell suspension was transferred to a 100mL cell culture flask containing α-MEM medium and cultured in a 37°C incubator; the medium was changed after 24 hours. When the cells filled 90% of the culture flask, the medium in the culture medium was discarded, PBS was washed once, and the umbilical cord mesenchymal stem cells were cultured at 2×10 5 The cells were inoculated at a density of 100 / mL in the first culture medium and cultured at 36°C, 2% CO2 for 23 hours to obtain the first generation of cells. The first culture medium was: DMEM / F12 complete medium + 3 μM 1-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazine + 1.5 μM dexamethasone + 40 μg / mL ascorbic acid + 8 mM sodium β-glycerophosphate.

[0059] The first generation cells were washed once with PBS and then cultured at 2×10 5 The cells were inoculated at a density of 100 μg / mL in the second culture medium, cultured at 36°C, 2% O2, 5% CO2 for 70 hours, and the medium was changed every 23 hours to obtain the second generation of cells. The second culture medium was: DMEM / F12 complete culture medium + 150 μg / mL pioglitazone hydrochloride + 5 μg / mL naringin + 60 μg / mL ascorbic acid.

[0060] The second generation cells were washed once with PBS and then cultured at 2×10 5 The cells were inoculated into the third culture medium at a density of 100 / mL and cultured at 36°C and 2% CO2 for 45 h, with the medium changed every 23 h. Pulse ultrasound treatment was performed at the 16th hour of culture, with a frequency of 0.5 MHz, a duty cycle of 20%, and an intensity of 40 W / cm 2 The pulse ultrasonic treatment time was 10 min. The third culture medium was: DMEM / F12 complete culture medium + 0.5 μg / mL transforming growth factor-β1 + 1 μg / mL stromal cell derived factor-1α + 4 μg / mL insulin-like growth factor 1.

[0061] The second-generation cell culture medium was centrifuged at 400g for 10 min, 1800g for 10 min, 9000g for 25 min, and then centrifuged again at 9500g for 60 min. The precipitate obtained was the human umbilical cord mesenchymal stem cell exosomes.

[0062] Example 2

[0063] Human umbilical cord mesenchymal stem cells (HUMSC) were purchased from ATCC. The cryopreserved tubes of human umbilical cord mesenchymal stem cells were taken out of dry ice and quickly thawed in a 37°C water bath. The cell suspension was then transferred to a centrifuge tube containing α-MEM medium and centrifuged at 400g for 5 min. The centrifuge tube was removed, the supernatant was discarded, α-MEM medium was added to resuspend the cells, and then the cell suspension was transferred to a 100mL cell culture flask containing α-MEM medium and cultured in a 37°C incubator; the medium was changed after 24 hours. When the cells filled 90% of the culture flask, the medium in the culture medium was discarded, PBS was washed once, and the umbilical cord mesenchymal stem cells were cultured at 5×10 5 The cells were inoculated at a density of 100 / mL in the first culture medium and cultured at 37°C, 3% CO2 for 24 hours to obtain the first generation of cells. The first culture medium was: IMDM medium + 5 μM 1-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazine + 2 μM dexamethasone + 50 μg / mL ascorbic acid + 10 mM sodium β-glycerophosphate.

[0064] The first generation cells were washed once with PBS and then 5×10 5 The cells were inoculated at a density of 100 μg / mL in the second culture medium, cultured at 37°C, 3% O2, 5% CO2 for 72 hours, and the medium was changed every 24 hours to obtain the second generation of cells. The second culture medium was: IMDM medium + 180 μg / mL pioglitazone hydrochloride + 8 μg / mL naringin + 70 μg / mL ascorbic acid.

[0065] The second generation cells were washed once with PBS and then 5×10 5 The cells were inoculated into the third culture medium at a density of 100 / mL and cultured at 37°C and 3% CO2 for 48 hours, with the medium changed every 24 hours. Pulse ultrasound treatment was performed at the 17th hour of culture, with a frequency of 1 MHz, a duty cycle of 25%, and an intensity of 50 W / cm 2 The pulse ultrasonic treatment time was 15 min. The third culture medium was: IMDM culture medium + 1 μg / mL transforming growth factor-β1 + 3 μg / mL stromal cell derived factor-1α + 5 μg / mL insulin-like growth factor-1.

[0066] The second-generation cell culture medium was centrifuged at 450g for 12 min, 2000g for 12 min, 10000g for 30 min, and then centrifuged again at 10000g for 70 min. The precipitate obtained was the human umbilical cord mesenchymal stem cell exosomes.

[0067] Example 3

[0068] Human umbilical cord mesenchymal stem cells (HUMSC) were purchased from ATCC. The cryopreserved tubes of human umbilical cord mesenchymal stem cells were taken out of dry ice and quickly thawed in a 37°C water bath. The cell suspension was then transferred to a centrifuge tube containing α-MEM medium and centrifuged at 400g for 5 min. The centrifuge tube was removed, the supernatant was discarded, α-MEM medium was added to resuspend the cells, and then the cell suspension was transferred to a 100mL cell culture flask containing α-MEM medium and cultured in a 37°C incubator; the medium was changed after 24 hours. When the cells filled 90% of the culture flask, the medium in the culture medium was discarded, PBS was washed once, and the umbilical cord mesenchymal stem cells were cultured at 8×10 5 The cells were inoculated at a density of 100 / mL in the first culture medium and cultured at 38°C, 5% CO2 for 25 hours to obtain the first generation of cells. The first culture medium was: RPMI-1640 complete medium + 6 μM 1-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazine + 2.5 μM dexamethasone + 60 μg / mL ascorbic acid + 12 mM sodium β-glycerophosphate.

[0069] The first generation cells were washed once with PBS and then 8×10 5 The cells were inoculated at a density of 100 μg / mL in the second culture medium, cultured at 38°C, 5% O2, 5% CO2 for 75 hours, and the medium was changed every 25 hours to obtain the second generation of cells. The second culture medium was: RPMI-1640 complete culture medium + 200 μg / mL pioglitazone hydrochloride + 10 μg / mL naringin + 80 μg / mL ascorbic acid.

[0070] The second generation cells were washed once with PBS and then 8×10 5 The cells were inoculated into the third culture medium at a density of 100 / mL and cultured at 38°C, 5% O2, 5% CO2 for 50 h, with the medium changed every 25 h. Pulse ultrasound treatment was performed at the 18th hour of culture, with a frequency of 2 MHz, a duty cycle of 30%, and an intensity of 60 W / cm 2 The pulse ultrasonic treatment time is 20 minutes. The third culture medium is: RPMI-1640 complete culture medium + 2μg / mL transforming growth factor-β1 + 5μg / mL stromal cell derived factor-1α + 6μg / mL insulin-like growth factor 1.

[0071] The second-generation cell culture medium was centrifuged at 500 g for 15 min, 2200 g for 15 min, 11000 g for 35 min, and then centrifuged again at 10500 g for 80 min. The precipitate obtained was the human umbilical cord mesenchymal stem cell exosomes.

[0072] Comparative Example 1

[0073] The difference from Example 1 is that 1-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazine is not added to the first culture medium, and other culture conditions and culture processes are the same as those in Example 1.

[0074] Comparative Example 2

[0075] The difference from Example 1 is that naringin is not added to the second culture medium, and other culture conditions and culture processes are the same as those in Example 1.

[0076] Comparative Example 3

[0077] The difference from Example 1 is that during the culture in the second culture medium, the 2% O2 environment is replaced by a 21% (v / v) O2 environment (normoxic conditions), and the other culture conditions and culture process are the same as those in Example 1.

[0078] Comparative Example 4

[0079] The difference from Example 1 is that stromal cell-derived factor-1α is not added to the third culture medium. Other culture conditions and culture processes are the same as those in Example 1.

[0080] Comparative Example 5

[0081] The difference from Example 1 is that during the culture in the third culture medium, pulse ultrasonic treatment is performed. Other culture conditions and culture processes are the same as those in Example 1.

[0082] Experimental Example 1

[0083] The human umbilical cord mesenchymal stem cell exosomes obtained in Examples 1 to 3 and Comparative Examples 1 to 5 were tested by ELISA to determine the concentrations of EGF, HGF, IGF-1, PDGF, KGF and HB-EGF, TGF-β1, FGF-2, and VEGF-A cytokines. The ELISA kit was purchased from Shanghai Hengyuan Biotechnology Co., Ltd., and the test was performed according to the instructions. The first culture medium, the second culture medium, and the third culture medium of the control group were DMEM / F12 basal culture medium, and hypoxic culture and pulsed ultrasonic treatment were not performed during the culture process. The measured data were normalized to 1 with the concentration in the control culture medium. The data of the embodiment group were displayed as relative multiples. Three groups were repeated in each group, and the average was taken. The results are shown in Table 1.

[0084] Table 1 Relative expression of cytokines in exosomes of human umbilical cord mesenchymal stem cells in different groups

[0085]

[0086]

[0087] EGF, HGF, IGF-1, PDGF, KGF, HB-EGF, TGF-β1, FGF-2, VEGF-A are respectively: epidermal growth factor, hepatocyte growth factor, insulin growth factor-1, platelet-derived growth factor, keratinocyte growth factor, heparin-binding epidermal growth factor, human transforming growth factor-β1, fibroblast growth factor-2, vascular endothelial growth factor-A. The above cytokines are all involved in skin wound repair or skin regeneration. As can be seen from Table 1, the relative expression of cytokines in human umbilical cord mesenchymal stem cell exosomes in Examples 1 to 3 of the present invention increased to varying degrees compared with the control group, while the expression of the above cytokines in Comparative Examples 1 to 5 was increased, but not as significant as that in Examples 1 to 3. It shows that the culture medium and culture conditions of the first culture, the second culture, and the third culture have a positive regulatory effect on the expression of EGF, HGF, IGF-1, PDGF, KGF, HB-EGF, TGF-β1, FGF-2, and VEGF-A.

[0088] Experimental Example 2 Animal Experiment

[0089] Experimental animals: SPF grade SD rats (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.), weighing 280-300g, female.

[0090] Reagent preparation: Doxorubicin hydrochloride was prepared into a 2 mg / mL solution with normal saline, and the solution was prepared immediately before use. The exosomes obtained in Example 1 of the present invention were dissolved in normal saline to obtain an exosome solution with a concentration of 50 μg / mL.

[0091] A rat model of refractory burn wounds was established: the rats were anesthetized by intraperitoneal injection of 0.5 mL of 3% sodium pentobarbital, and the backs of the rats were depilated; a wound was made on the midline of the back of the rats; the wound was exposed to 95°C for 30 s using a scald mold (aluminum block) with a diameter of 2 cm, and 60 μL of doxorubicin hydrochloride (2 mg / mL) was injected subcutaneously 0.2 cm from the edges of the wound; the scab on the back of the rats was removed on the 28th day after the operation to obtain a rat model of refractory burn wounds.

[0092] The model rats were randomly divided into 9 groups, 3 in each group, and the solutions containing growth factors prepared by the compositions of Examples 1 to 3 and Comparative Examples 1 to 5 were injected into the rats at 5 mm from the edge of the wound, 300 μL each time, and the control group was injected with an equal amount of normal saline. After the operation, the rats were covered with sterile gauze and bandaged with an elastic bandage of appropriate tightness, and the above operation was repeated every 4 days. At 0, 4, 8, 12, and 16 days of drug treatment, digital cameras were used to take pictures, record the wound conditions of the rats, and digital image analysis software was used to analyze the wound area and calculate the healing rate. Wound healing rate = (original wound area-unhealed wound area) / original wound area. The wound healing rates of the rats in each group at 4, 8, 12, and 16 days after drug treatment are shown in Table 2.

[0093] Table 2 Wound healing rates of rats in each group at 4, 8, 12 and 16 days

[0094]

[0095] Table 2 shows the wound repair of rats after each group of experiments. As shown in Table 2, after the exosome solution provided in Examples 1 to 3 was injected into the wound of rats, the wound healing rate of rats was significantly higher than that of Comparative Examples 1 to 5 and the control group, indicating that the exosomes provided in Examples 1 to 3 can effectively repair the wounds of model rats, suggesting that the exosomes provided in Examples 1 to 3 have better wound repair function.

[0096] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing umbilical cord mesenchymal stem cell exosomes, characterized in that: The following steps are involved: S1. The umbilical cord mesenchymal stem cells were cultured at 2-8×10 5 The cells were inoculated into the first culture medium at a density of 100 cells / mL and cultured for 23 to 25 hours to obtain the first generation of cells; The first culture medium is: stem cell basal culture medium + 3-6 μM 1-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazine + 1.5-2.5 μM dexamethasone + 40-60 μg / mL ascorbic acid + 8-12 mM sodium β-glycerophosphate; S2. The first generation cells were cultured at 2 to 8 × 10 5 The cells were inoculated into the second culture medium at a density of 100 cells / mL and cultured for 70 to 75 hours, with the medium replaced every 23 to 25 hours to obtain the second generation of cells; The second culture medium is: stem cell basal culture medium + 150-200 μg / mL pioglitazone hydrochloride + 5-10 μg / mL naringin + 60-80 μg / mL ascorbic acid; S3. The second generation cells were cultured at 2 to 8 × 10 5 The density of cells / mL was inoculated into the third culture medium, cultured for 45-50 hours, and the medium was changed every 23-25 ​​hours; The third culture medium is: stem cell basal culture medium + 0.5-2 μg / mL transforming growth factor-β1 + 1-5 μg / mL stromal cell derived factor-1α + 40-60 μg / mL insulin-like growth factor 1; S4. collecting the culture medium in S3, and collecting the precipitate after differential centrifugation, which is the umbilical cord mesenchymal stem cell exosomes; The culture conditions of S1 are 36-38° C., 2-5% CO2; the culture conditions of S2 are 36-38° C., 2-5% O2; The culture conditions in S3 are 36-38°C, 2-5% CO2, and pulse ultrasonic treatment is performed at the 16th to 18th hour of culture, and the pulse treatment time is 10-20 minutes; The pulse ultrasonic treatment has a frequency of 0.5 to 2 MHz, a duty cycle of 20 to 30%, and an intensity of 40 to 60 W / cm 2 .

2. The preparation method according to claim 1, characterized in that: The stem cell basal culture medium includes one of DMEM / F12 complete culture medium, IMDM culture medium and RPMI-1640 complete culture medium.

3. The preparation method according to claim 1, characterized in that: The differential centrifugation is as follows: 400-500g centrifugation for 10-15min, 1800-2200g centrifugation for 10-15min, and 9000-11000g centrifugation for 25-35min.

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