CN117106712B - Stem cell exosome for skin regeneration, application and product thereof - Google Patents
Stem cell exosome for skin regeneration, application and product thereof Download PDFInfo
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Classifications
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/16—Emollients or protectives, e.g. against radiation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0629—Keratinocytes; Whole skin
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- C12N2501/90—Polysaccharides
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1352—Mesenchymal stem cells
- C12N2502/1388—Mesenchymal stem cells from other natural sources
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Abstract
The invention provides a stem cell exosome for skin regeneration, and application and a product thereof, belonging to the technical field of biology. The stem cell exosome provided by the invention is extracted from umbilical cord mesenchymal stem cell culture medium pretreated by bighead atractylodes rhizome polysaccharide. The experimental result shows that compared with untreated exosomes, the exosomes provided by the invention can remarkably improve the proliferation capability of normal and UV damaged skin cells, thereby effectively promoting the rapid repair of skin. Secondly, the invention further discloses application of the gel containing the exosome in alleviating skin photodamage.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a stem cell exosome for skin regeneration, and application and a product thereof.
Background
The skin is the organ with the largest area of the human body, can isolate the invasion of the outside to the environment in the human body, prevents harmful microorganisms, ultraviolet rays, chemicals and the like from entering the body, and plays a role in defending. Meanwhile, the skin plays an important role in the injury repair process, contains abundant epidermal stem cell reserves, can participate in wound repair through proliferation, differentiation and migration, and is coordinated with epidermal cells and fibroblasts to reconstruct the epidermal and dermal structures at the injury part.
In recent years, mesenchymal stem cells (Mesenchymal stromalcells, MSCs) have been attracting attention in the repair and regeneration of skin wounds. MSCs are actively involved in the skin wound repair process, not only differentiating into fibroblasts involved in matrix synthesis, but also releasing various factors involved in tissue regeneration, such as anti-scarring, anti-apoptotic and angiogenic factors. However, many studies have shown that MSCs do not participate in the therapeutic process themselves, but rather act primarily in dependence on paracrine signals in the body, releasing certain bioactive molecules, proliferating and differentiating into the desired damaged tissue cell types, thereby achieving wound repair. Therefore, MSCs-derived exosome therapy is applied as a new method for repair and regeneration of skin wounds.
Exosomes are an important extracellular vesicle, and in recent years, there has been a great deal of attention to the role in intercellular communication and biological therapy. Exosomes were originally discovered in 1983 and were initially considered to be only one way for cells to expel metabolic waste. Later studies have found that exosomes can be produced and released from almost all cells including dendritic cells, epithelial cells, tumor cells, immune cells, stem cells, etc., and can also be extracted from body fluids such as plasma, saliva, urine and breast milk. The existing research shows that the exosomes can effectively realize the repair of skin injury and promote the repair of skin, but the exosomes which are not subjected to specific stimulation or pretreatment have lower bioactive factors, and have very limited recovery effect on the skin structure and function. Therefore, how to more effectively exert the effect of exosomes is a problem that needs to be solved at present.
Disclosure of Invention
The invention aims at providing a stem cell exosome for skin regeneration, application thereof and a prepared gel product.
In order to achieve the above purpose, the present invention provides the following technical solutions:
in a first aspect, the present invention provides a stem cell exosome for skin regeneration, the stem cell exosome being obtained from a culture medium of umbilical cord mesenchymal stem cells pretreated with bighead atractylodes rhizome polysaccharide;
the stem cell exosomes are used for rapid skin regeneration after photodamage.
Preferably, the preparation method of the stem cell exosome comprises the following steps:
(1) Resuscitates umbilical cord mesenchymal stem cells, places the umbilical cord mesenchymal stem cells in a culture dish, and cultures the umbilical cord mesenchymal stem cells to a logarithmic phase by using alpha-MEM complete culture solution;
(2) Pretreating umbilical cord mesenchymal stem cells for 6h by using 10mg/mL bighead atractylodes rhizome polysaccharide;
(3) Removing the culture medium of umbilical cord mesenchymal stem cells treated by using bighead atractylodes rhizome polysaccharide, adding a new serum-free alpha-MEM culture medium for continuous culture for 48 hours, and collecting the culture medium;
(4) Extracting exosomes by using an exosome extraction kit to obtain stem cell exosomes for rapid skin regeneration.
Preferably, the preparation method of the bighead atractylodes rhizome polysaccharide comprises the following steps:
(1) Pulverizing the dried Atractylodis rhizoma into powder, and sieving with a sieve to obtain Atractylodis rhizoma powder;
(2) Adding petroleum ether 10 times of the weight of the mixture, stirring and soaking the mixture for 48 hours at room temperature, and drying the mixture to obtain defatted white atractylodes rhizome powder;
(3) Adding 10 times of distilled water, heating at 60deg.C, ultrasonic extracting for 40min with ultrasonic power of 100W to obtain Atractylodis rhizoma extractive solution;
(4) Placing the Atractylodis rhizoma extractive solution in a flask of rotary evaporator, concentrating at 60deg.C under reduced pressure to 1/5 of the original volume;
(5) Adding 4 times of absolute ethyl alcohol, and centrifuging at 4 ℃ for 12h at 8000rpm to obtain a white atractylodes rhizome polysaccharide precipitate;
(6) Re-dissolving the obtained polysaccharide precipitate into 1% polysaccharide precipitate, adding chloroform-n-butanol mixed solution, oscillating for 10min, collecting water phase, adding chloroform-n-butanol mixed solution again, repeating for 6 times, and removing protein by Sevage method to obtain polysaccharide solution;
(7) Filtering Atractylodis rhizoma polysaccharide solution with 25kDa ultrafiltration membrane, and filtering the permeate with 10kDa ultrafiltration membrane;
(8) And (3) carrying out alcohol precipitation on the obtained permeate liquid, and then carrying out freeze drying to obtain the bighead atractylodes rhizome polysaccharide.
Preferably, the stem cell exosomes achieve rapid repair of skin following photodamage by increasing the proliferative capacity of keratinocytes following photodamage.
In a second aspect, the invention provides an application of a stem cell exosome for skin regeneration, wherein the stem cell exosome is prepared by the preparation method of the stem cell exosome;
the application comprises the application of the stem cell exosome in preparing a pharmaceutical preparation or a cosmetic product for rapidly repairing photodamaged skin.
Preferably, the use further comprises the use of the stem cell exosomes in the preparation of a medium for promoting the proliferative capacity of skin keratinocytes.
Preferably, the pharmaceutical formulation is a liquid pharmaceutical formulation;
preferably, the cosmetic article is a gel cosmetic article.
In a third aspect, the present invention provides a product of stem cell exosomes for skin regeneration, the product being a gel prepared by using the stem cell exosomes as core active ingredients, the stem cell exosomes being prepared by the above-described preparation method of stem cell exosomes.
Preferably, the method for preparing the gel per 100mL comprises the following steps:
(1) Placing 1.5g of gelatin powder in a container, slowly adding 60mL PBS buffer solution at 37 ℃ and slowly stirring to dissolve gelatin during the adding process;
(2) Heating in water bath at 75deg.C for 15min to dissolve gelatin completely, taking out from water bath, standing, and cooling to 37-40deg.C to obtain gelatin solution;
(3) Weighing 0.5g of chitosan powder, adding 30mL of PBS buffer solution, and stirring for dissolution to obtain chitosan solution;
(4) Fully mixing gelatin solution and chitosan solution, adding 10mL of 100 mug/mL of stem cell exosomes, gently stirring until the mixture is uniformly dispersed, and then fixing the volume to 100mL to obtain gel solution;
(5) Rapidly adding the gel solution into a preheated container, and placing in a 37 ℃ incubator for 30min to obtain the gel for reducing photodamage.
The invention has the beneficial effects that:
the invention provides an exosome generated after pretreatment by using bighead atractylodes rhizome polysaccharide, and experimental results show that the exosome can improve the proliferation activity of normal skin cells HaCaT by 81.8%, which is obviously higher than the proliferation promoting effect of untreated exosome a on skin cells;
secondly, the exosomes can significantly increase the proliferation capacity of skin cells damaged by ultraviolet light. Compared with untreated exosomes, the exosomes provided by the invention can improve the activity of the HaCaT cells after ultraviolet injury from 0.548 to 0.904, and has better repair promoting effect;
secondly, the invention provides the gel for reducing the ultraviolet damage prepared by using the exosome, and the oxidative damage caused by ultraviolet is obviously reduced after the gel is smeared.
Drawings
FIG. 1 is a scanning electron microscope image of an exosome prepared according to the present invention;
FIG. 2 is a graph showing the proliferation promoting effect of exosomes prepared according to the present invention on skin keratinocytes;
FIG. 3 is a graph showing the results of measuring the cell proliferation ability of the exosomes prepared according to the present invention after photodamage to skin keratinocytes;
FIG. 4 is a graph showing the effect of the gel prepared according to the present invention on MDA level change in mouse skin tissue.
Detailed Description
Example 1: (1) Pulverizing dried 50g of Atractylodis rhizoma into powder, and sieving with 50 mesh sieve to obtain Atractylodis rhizoma powder;
(2) Adding 500mL of petroleum ether, stirring and soaking for 48 hours at room temperature, and drying to obtain defatted white atractylodes rhizome powder;
(3) Adding 500mL distilled water, heating at 60deg.C, ultrasonic extracting for 40min with ultrasonic power of 100W to obtain Atractylodis rhizoma extractive solution;
(4) Placing the bighead atractylodes rhizome extract into a flask of a rotary evaporator, and concentrating under reduced pressure to 100mL at 60 ℃;
(5) Adding 400mL of absolute ethyl alcohol, and centrifuging at the temperature of 4 ℃ for 12h at the speed of 8000rpm to obtain a white atractylodes rhizome polysaccharide precipitate;
(6) Re-dissolving the obtained polysaccharide precipitate into 1% polysaccharide precipitate, adding mixed solution of chloroform-n-butanol (4:1), oscillating for reacting for 10min, collecting water phase, adding mixed solution of chloroform-n-butanol (4:1) again, repeating for 6 times, and removing protein by Sevage method to obtain polysaccharide solution;
(7) Filtering Atractylodis rhizoma polysaccharide solution with 25kDa ultrafiltration membrane, and filtering the permeate with 10kDa ultrafiltration membrane;
(8) And respectively freeze-drying the obtained trapped fluid and permeate after alcohol precipitation to obtain bighead atractylodes rhizome polysaccharide I and bighead atractylodes rhizome polysaccharide II.
Example 2: (1) Resuscitates umbilical cord mesenchymal stem cells stored by the company, places the umbilical cord mesenchymal stem cells in a culture dish, and cultures the umbilical cord mesenchymal stem cells to a logarithmic phase by using alpha-MEM complete culture solution (10% FBS);
(2) Pretreating umbilical cord mesenchymal stem cells for 6h by using 10mg/mL bighead atractylodes rhizome polysaccharide I (experimental group I) and 10mg/mL bighead atractylodes rhizome polysaccharide II (experimental group II), wherein the control group is not treated;
(3) Removing the culture medium of umbilical cord mesenchymal stem cells treated by the control group and the bighead atractylodes rhizome polysaccharide, adding a new serum-free alpha-MEM culture medium for continuous culture for 48 hours, and collecting the culture medium;
(4) Referring to the specification of a Kaiyi biological exosome extraction kit, placing the collected culture medium into a centrifuge tube for 3000g, centrifuging for 10min, and placing 10mL of supernatant into a sterile centrifuge tube;
(5) 2500 mu L of exosome separating reagent added into the kit, and after being gently mixed, standing for 2 hours at the temperature of 4 ℃;
(6) The supernatant was discarded after centrifugation at 12000g for 20min at 4℃and the exosome pellet obtained in control, experimental group I and experimental group II was resuspended in PBS to 100. Mu.g/mL for use, designated exosome a, exosome b and exosome c, respectively.
Example 3: exosome detection by transmission electron microscope
(1) Dropwise adding 10 mu L of exosomes a, b and c obtained in the example 2 onto a copper mesh respectively, standing for 3min, and sucking off the floating liquid by using dust-free paper;
(2) Dropwise adding 10 mu L of phosphotungstic acid on a copper wire, standing for 1min, and sucking off floating liquid by using dust-free paper;
(3) After drying at room temperature for 5min, imaging was performed using a transmission electron microscope, and the results obtained are shown in fig. 1.
From FIG. 1, a discoid exosome can be observed, demonstrating the success of the exosome extraction of the present invention.
Example 4: exosomes produced after treatment of bighead atractylodes rhizome polysaccharide increase proliferation capacity of keratinocytes (HaCaT)
(1) Keratinocytes in the logarithmic growth phase were digested and prepared into cell suspensions, and 100 μl of the cell suspensions were inoculated into 96-well cell culture plates in an amount of 1w cells per well;
(2) Placing the cell culture plate in a cell culture box for culturing until the cells are completely adhered;
(3) Fresh DMEM medium (10% FBS) was removed from the original medium, 100. Mu.L of DMEM medium was added to the control group, 10. Mu.L of exosomes a and 90. Mu.L of DMEM medium were added to the experimental group a, 10. Mu.L of exosomes b and 90. Mu.L of DMEM medium were added to the experimental group b, and 10. Mu.L of exosomes c and 90. Mu.L of DMEM medium were added to the experimental group c;
(4) After further culturing for 48 hours, the absorbance of the cells was measured by adding CCK-8, and the cell activity was calculated.
As can be seen from fig. 2, compared with the control group, the cell activity of the experimental group a is increased by 22.3%, the cell activity of the experimental group b is increased by 27.3%, and the cell activity of the experimental group c is increased by 81.8%, so that the proliferation capacity of keratinocytes can be effectively promoted after exosomes are added;
meanwhile, it can be seen that the exosomes b produced after treatment with atractylenolide I have slightly higher promotion of proliferation capacity of keratinocytes than exosomes a produced without treatment with atractylenolide I, but the difference is not statistically significant;
however, the promotion effect of exosomes c produced after treatment with atractylenolide ii was significantly higher than that of untreated exosomes a, indicating that the proliferation promotion effect of exosomes produced by stem cells on keratinocytes could be significantly improved by treatment with atractylenolide ii.
Example 5: exosomes produced after treatment of bighead atractylodes rhizome polysaccharide increase proliferation capacity of keratinocytes (HaCaT) after photodamage
(1) Keratinocytes in the logarithmic growth phase were digested and prepared into cell suspensions, and 100 μl of the cell suspensions were inoculated into 96-well cell culture plates in an amount of 1w cells per well;
(2) Placing the cell culture plate in a cell culture box for culturing until the cells are completely adhered;
(3) Control group was not treated, test group a, test group b and test group c were treated at 60mJ/cm 2 Is irradiated to cells;
(4) After the irradiation, the culture medium is replaced, the control group and the experimental group are replaced by new DMEM culture medium, the experimental group b is replaced by DMEM culture medium containing 10 mug/mL exosome a prepared in advance, and the experimental group c is replaced by DMEM culture medium containing 10 mug/mL exosome c;
(5) After further culturing for 48 hours, the absorbance of the cells was measured by adding CCK-8, and the cell activity was calculated.
As can be seen from fig. 3, the cell activity of the experimental group a was 0.548±0.059, the cell activity of the experimental group b was 0.676±0.048, and the cell activity of the experimental group c was 0.904±0.046;
the cell activity of experimental group a was significantly reduced compared to the control group, indicating that UVB irradiation produced significant photodamage to keratinocytes;
compared with the experimental group a, the cell activity of the experimental group b is obviously improved, which indicates that the proliferation capacity of keratinocytes after photodamage can be effectively improved by adding exosomes;
compared with the experimental group a, the cell activity of the experimental group c is obviously improved, and compared with the experimental group b, the exosome c generated after treatment by using the bighead atractylodes rhizome polysaccharide II can obviously realize rapid regeneration and repair of skin after photodamage.
Example 6: preparation of gel 1 for reduction of photodamage
(1) Placing 1.5g of gelatin powder in a beaker, slowly adding 60mL of PBS buffer at 37 ℃ and slowly stirring by using a glass rod during the adding process to dissolve gelatin;
(2) Heating in water bath at 75deg.C for 15min to dissolve gelatin completely, taking out from water bath, standing, and cooling to 37-40deg.C to obtain gelatin solution;
(3) Weighing 0.5g of chitosan powder, adding 40mL of PBS buffer solution, and stirring for dissolution to obtain chitosan solution;
(4) Fully mixing gelatin solution and chitosan solution, and then fixing the volume to 100mL to obtain gel solution;
(5) The gel solution was rapidly added to a preheated 24-well plate and placed in a 37 ℃ incubator for 30min to give gel 1.
Example 7: preparation of gel 2 for reduction of photodamage
(1) Placing 1.5g of gelatin powder in a beaker, slowly adding 60mL of PBS buffer at 37 ℃ and slowly stirring by using a glass rod during the adding process to dissolve gelatin;
(2) Heating in water bath at 75deg.C for 15min to dissolve gelatin completely, taking out from water bath, standing, and cooling to 37-40deg.C to obtain gelatin solution;
(3) Weighing 0.5g of chitosan powder, adding 30mL of PBS buffer solution, and stirring for dissolution to obtain chitosan solution;
(4) Fully mixing gelatin solution and chitosan solution, adding 10mL of exosome a with the concentration of 100 mug/mL, gently stirring until the exosome a is uniformly dispersed, and fixing the volume to 100mL to obtain gel solution;
(5) The gel solution was rapidly added to a preheated 24-well plate and placed in a 37 ℃ incubator for 30min to give gel 2.
Example 8: preparation of gel 3 for reduction of photodamage
(1) Placing 1.5g of gelatin powder in a beaker, slowly adding 60mL of PBS buffer at 37 ℃ and slowly stirring by using a glass rod during the adding process to dissolve gelatin;
(2) Heating in water bath at 75deg.C for 15min to dissolve gelatin completely, taking out from water bath, standing, and cooling to 37-40deg.C to obtain gelatin solution;
(3) Weighing 0.5g of chitosan powder, adding 30mL of PBS buffer solution, and stirring for dissolution to obtain chitosan solution;
(4) Fully mixing gelatin solution and chitosan solution, adding 10mL of exosome c with the concentration of 100 mug/mL, gently stirring until the exosome c is uniformly dispersed, and then fixing the volume to 100mL to obtain gel solution;
(5) The gel solution was rapidly added to a preheated 24-well plate and placed in a 37 ℃ incubator for 30min to give gel 3.
Example 9: exosomes produced after treatment of bighead atractylodes rhizome polysaccharide reduce photodamage of ultraviolet rays to skin
(1) Selecting 20 BALB/c mice which are healthy and have no specific pathogen, and carrying out experiments after the female BALB/c mice are kept for 1 week under the same conditions, wherein the body mass (20+/-2) g and the age of the mice are 6-8 weeks;
(2) After 3% pentobarbital sodium is injected into the abdominal cavity for anesthesia, long hairs on the back of the mouse are cut off by using surgical scissors, and the hairs are removed by using a dehairing knife;
(3) Dipping 6% Na with medicinal cotton 2 S solution is smeared on the back of the mouse for dehairing, and the dehairing range is 3cm multiplied by 3 cm;
(4) The mice are randomly divided into 4 groups, a control group is not treated, an experimental group a is coated with gel 1, an experimental group b is coated with gel 2, and an experimental group c is coated with gel 3;
(5) After 30min, 300 mJ/cm was used 2 Is irradiated for 3 days;
(6) After the irradiation is finished, killing all mice by cervical dislocation, and taking down the skin at the dehairing position by using scissors;
(7) Rinsing the obtained skin in pre-cooled physiological saline, removing blood, wiping the skin with filter paper, measuring the weight with a balance, and placing the skin into a beaker;
(8) Adding physiological saline with the weight of 9 times, shearing the obtained skin tissue into small tissue blocks, using a low-temperature grinding instrument, fully grinding, and using a low-temperature centrifuge for centrifugation at 2000 rpm for 15min;
(9) After taking the supernatant, the MDA levels in the skin tissue of each group of mice were determined according to the MDA kit instructions.
As can be seen from fig. 4, the MDA level of the control group was 21.17±2.29, the MDA level of the experimental group a was 37.34±2.17, the MDA level of the experimental group b was 30.73 ±2.19, and the MDA level of the experimental group c was 25.41 ±2.16;
it can be seen that the level of oxidative damage marker MDA in the skin tissue of the mice coated with gel 1 is significantly increased after the ultraviolet irradiation treatment compared with the control group; the skin tissue of the mice coated with the gel 2 is reduced to a certain extent compared with the experimental group a, but the effect is general;
compared with experimental group a, the skin tissue of the mice coated with the gel 3 is obviously reduced, which proves that the photo-damage gel 3 for photo-damage prepared by the invention can effectively reduce the photo-damage of the skin caused by ultraviolet rays.
Claims (3)
1. A stem cell exosome for skin regeneration capability, characterized in that the stem cell exosome is an exosome obtained from a culture medium of umbilical cord mesenchymal stem cells pretreated with bighead atractylodes rhizome polysaccharide;
the stem cell exosomes are used for rapid regeneration of skin after photodamage;
the preparation method of the stem cell exosome comprises the following steps:
(1) Resuscitates umbilical cord mesenchymal stem cells, places the umbilical cord mesenchymal stem cells in a culture dish, and cultures the umbilical cord mesenchymal stem cells to a logarithmic phase by using alpha-MEM complete culture solution;
(2) Pretreating umbilical cord mesenchymal stem cells for 6h by using 10mg/mL bighead atractylodes rhizome polysaccharide;
(3) Removing the culture medium of umbilical cord mesenchymal stem cells treated by using bighead atractylodes rhizome polysaccharide, adding a new serum-free alpha-MEM culture medium for continuous culture for 48 hours, and collecting the culture medium;
(4) Extracting exosomes by using an exosome extraction kit to obtain stem cell exosomes for rapid skin regeneration;
the preparation method of the bighead atractylodes rhizome polysaccharide comprises the following steps:
(1) Pulverizing the dried Atractylodis rhizoma into powder, and sieving with a sieve to obtain Atractylodis rhizoma powder;
(2) Adding petroleum ether 10 times of the weight of the mixture, stirring and soaking the mixture for 48 hours at room temperature, and drying the mixture to obtain defatted white atractylodes rhizome powder;
(3) Adding 10 times of distilled water, heating at 60deg.C, ultrasonic extracting for 40min with ultrasonic power of 100W to obtain Atractylodis rhizoma extractive solution;
(4) Placing the Atractylodis rhizoma extractive solution in a flask of rotary evaporator, concentrating at 60deg.C under reduced pressure to 1/5 of the original volume;
(5) Adding 4 times of absolute ethyl alcohol, and centrifuging at 4 ℃ for 12h at 8000rpm to obtain a white atractylodes rhizome polysaccharide precipitate;
(6) Re-dissolving the obtained polysaccharide precipitate into 1% polysaccharide precipitate, adding chloroform-n-butanol mixed solution, oscillating for 10min, collecting water phase, adding chloroform-n-butanol mixed solution again, repeating for 6 times, and removing protein by Sevage method to obtain polysaccharide solution;
(7) Filtering Atractylodis rhizoma polysaccharide solution with 25kDa ultrafiltration membrane, and filtering the permeate with 10kDa ultrafiltration membrane;
(8) Precipitating the obtained permeate with ethanol, and freeze-drying to obtain Atractylodis rhizoma polysaccharide;
the bighead atractylodes rhizome polysaccharide realizes the rapid repair of the skin after photodamage by improving the proliferation capacity of keratinocytes after photodamage of stem cell exosomes.
2. The use of a stem cell exosome according to claim 1, wherein the use is the use of the stem cell exosome in the preparation of a pharmaceutical formulation for rapid repair of photodamaged skin.
3. A product of stem cell exosomes for skin regeneration, characterized in that the product is a gel prepared using the stem cell exosomes of claim 1 as core active ingredients;
the preparation method of the gel comprises the following steps of:
(1) Placing 1.5g of gelatin powder in a container, slowly adding 60mL PBS buffer solution at 37 ℃ and slowly stirring to dissolve gelatin during the adding process;
(2) Heating in water bath at 75deg.C for 15min to dissolve gelatin completely, taking out from water bath, standing, and cooling to 37-40deg.C to obtain gelatin solution;
(3) Weighing 0.5g of chitosan powder, adding 30mL of PBS buffer solution, and stirring for dissolution to obtain chitosan solution;
(4) Fully mixing gelatin solution and chitosan solution, adding 10mL of 100 mug/mL of stem cell exosomes, gently stirring until the mixture is uniformly dispersed, and then fixing the volume to 100mL to obtain gel solution;
(5) Rapidly adding the gel solution into a preheated container, and placing in a 37 ℃ incubator for 30min to obtain the gel for reducing photodamage.
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| CN108251363A (en) * | 2017-12-31 | 2018-07-06 | 南华生物医药股份有限公司 | A kind of polysaccharide composition for promoting human umbilical cord mesenchymal stem cells proliferation and its application |
| CN108392494A (en) * | 2018-05-15 | 2018-08-14 | 江苏大学 | Applications of the exosome of people's umbilical cord mesenchymal stem cells secretion in preparing the drug for repairing skin ultraviolet injury |
| CN110548001A (en) * | 2019-09-06 | 2019-12-10 | 沈阳细胞治疗工程技术研发中心有限公司 | Repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes |
| CN111826345A (en) * | 2019-04-17 | 2020-10-27 | 深圳国科靶点药物有限公司 | Human umbilical cord mesenchymal stem cell source exosome preparation |
| CN116829700A (en) * | 2020-12-04 | 2023-09-29 | 株式会社普里莫里斯治疗 | Method for preparing high-concentration stem cell exosome with enhanced anti-inflammatory and regeneration functions by using lipopolysaccharide and lipoteichoic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108251363A (en) * | 2017-12-31 | 2018-07-06 | 南华生物医药股份有限公司 | A kind of polysaccharide composition for promoting human umbilical cord mesenchymal stem cells proliferation and its application |
| CN108392494A (en) * | 2018-05-15 | 2018-08-14 | 江苏大学 | Applications of the exosome of people's umbilical cord mesenchymal stem cells secretion in preparing the drug for repairing skin ultraviolet injury |
| CN111826345A (en) * | 2019-04-17 | 2020-10-27 | 深圳国科靶点药物有限公司 | Human umbilical cord mesenchymal stem cell source exosome preparation |
| CN110548001A (en) * | 2019-09-06 | 2019-12-10 | 沈阳细胞治疗工程技术研发中心有限公司 | Repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes |
| CN116829700A (en) * | 2020-12-04 | 2023-09-29 | 株式会社普里莫里斯治疗 | Method for preparing high-concentration stem cell exosome with enhanced anti-inflammatory and regeneration functions by using lipopolysaccharide and lipoteichoic acid |
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