CN111321111A - Method for promoting skin injury repair by using adipose-derived stem cell extracellular vesicles - Google Patents
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Abstract
The invention discloses a method for promoting skin injury repair by using adipose-derived stem cell extracellular vesicles, which comprises a cell extraction and identification step, an adipose-derived stem cell extracellular vesicle extraction step, an adipose-derived stem cell extracellular vesicle identification step, a skin photoinjury nude mouse model construction and treatment step, an adipose-derived stem cell extracellular vesicle treatment effect evaluation step for skin photoinjury, an in vitro skin fibroblast photoinjury model establishment step, a cell viability detection step, an β -galactosidase staining step, a mouse macrophage polarization induction and treatment experiment step, a skin fibroblast endogenous active oxygen detection step and a cell cycle detection step.
Description
Technical Field
The invention relates to a method for promoting skin injury repair by using adipose-derived stem cell extracellular vesicles.
Background
With the development of economic society, skin aging resistance is more and more concerned by the public. Aging of the skin not only manifests itself in a morphologically old state, but also results in a series of pathological conditions, such as: dry skin, reduced skin barrier function, skin tumor development, etc. Therefore, how to effectively prevent skin aging has become a hot issue of concern for plastic prosthetists and dermatologists. Etiologically, skin aging is determined by endogenous (genetic) and exogenous (environmental) factors. Intrinsic aging, which is natural aging, is degenerative change directly related to age, and extrinsic aging, which is aging after the skin of the body is exposed to exposure factors, in which irradiation of ultraviolet rays is the main exposure factor, causes aging by causing damage to the skin.
At present, exogenous skin aging is mainly prevented and treated by oral or external medicines, the mechanism is complex, generally, the skin aging is delayed mainly by antagonizing oxidative stress, regulating inflammatory response and inhibiting the expression of aging-related genes and pathways, but the application of the medicine is limited by the problems of low bioavailability, more adverse reactions of partial medicines, limited curative effect of partial medicines on resisting skin aging and the like. In recent years, a number of studies have shown that the use of stem cells can ameliorate photodamage to the skin. Among them, the application of Adipose-derived stem cells is receiving wide attention, mainly because a large amount of Adipose tissues can be extracted by liposuction, and the Adipose tissues are rich in a large amount of Adipose Derived Stem Cells (ADSCs), so that the Adipose-derived stem cells have a wide source and are proved to have many biological effects: such as promoting blood vessel production, promoting collagen synthesis, regulating inflammatory reaction, inhibiting scar hyperplasia, and improving skin photodamage. However, the application of adipose-derived stem cells has the problems of the risk of culture contamination in vitro, the risk of tumorigenesis in transplants, the insufficient supply area for autografting, the immunological rejection of allograft, ethics and the like, and the clinical application of adipose-derived stem cells is limited by the problems. In addition, recent studies have shown that: stem cells act primarily through some of their paracrine factors, of which the extracellular vesicles of the cells play an important role.
Compared to cell therapy, Extracellular Vesicles (EVs) of cells are cell-free components, and their application has many advantages: more stable, convenient to store, avoids tumorigenic risks, no/low immunological rejection in xenogenic applications, and the like. Previous reports have shown that: the use of extracellular vesicles of adipose stem cells can prevent photodamage of skin Fibroblasts (fibroplasts), but the exact mechanism and the effect of their use in animals is still unclear.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a method for promoting skin injury repair by using adipose-derived stem cell extracellular vesicles, improves skin photoinjury by extracting the adipose-derived stem cell extracellular vesicles to repair skin injury, discloses a mechanism for preventing skin fibroblast photoinjury by using the adipose-derived stem cell extracellular vesicles, and verifies the treatment effect of the adipose-derived stem cell extracellular vesicles in an animal body.
The technical scheme for realizing the purpose is as follows: a method for promoting skin injury repair by using adipose-derived stem cell extracellular vesicles comprises the following steps:
s1, the cell extraction and identification step comprises the following procedures:
(1) extraction and identification of adipose-derived stem cells: digesting the waste granular fat after liposuction by using a collagenase digestion method, extracting adipose-derived stem cells, carrying out passage after the lipolysis, and identifying the extracted adipose-derived stem cells by flow cytometry and three-way induction respectively;
(2) extraction of skin fibroblasts: soaking the waste foreskin with Dispase for 24 hours, removing the epidermis, digesting the residual dermis by a collagenase digestion method, extracting skin fibroblasts, carrying out passage after the skin fibroblasts grow full, and substituting for 3-5 in vitro for the research of the photoaging of the skin fibroblasts by extracellular vesicles;
s2, extracting the fat stem cell extracellular vesicles: after the third-generation adipose-derived stem cells are full, starving the adipose-derived stem cells for 48 hours, collecting cell supernatants, extracting adipose-derived stem cell Extracellular Vesicles (EVs) by using an ultrafiltration method, suspending the extracted extracellular vesicles in PBS, and storing at minus 80 ℃;
s3, identification of the fat stem cell extracellular vesicles: observing the morphology and the size of the extracellular vesicles through a transmission electron microscope, calculating the average diameter of the extracellular vesicles through Nanoparticle Tracking Analysis (NTA), and detecting markers CD81, CD63, CD9 and TSG101 of the extracellular vesicles through Western Blot;
s4, construction and treatment steps of a skin photodamage nude mouse model: taking 40 female BALB/c nude mice of 5 weeks old, and conventionally breeding the mice in an SPF environment for 1 week to adapt to the environment; starting at week 6, nude mice were randomly divided into 4 groups:
(1) control group: UVB irradiation is not carried out, and subcutaneous injection treatment is not carried out;
(2) UVB group: carrying out UVB irradiation and subcutaneous injection of PBS on a nude mouse;
(3) UVB +150ug/week group: nude mice were subjected to UVB irradiation + subcutaneous injection of 150ug/week Extracellular Vesicles (EVs);
(4) UVB +300ug/week group: nude mice were subjected to UVB irradiation + subcutaneous injection of 300ug/week Extracellular Vesicles (EVs);
the process for establishing the skin photodamage nude mouse model is as follows: erecting a UVB lamp tube above a mouse cage to enable a nude mouse to be under direct irradiation of UVB and to move freely, placing an energy detector under the UVB lamp, wherein the irradiation energy of the first week is determined by the Minimum Erythema Dose (MED), irradiating for 5 days every week, and increasing the irradiation dose of the Minimum Erythema Dose (MED) every subsequent week for 8 weeks;
s5, an evaluation step of the effect of the adipose-derived stem cell extracellular vesicles on treatment of skin photodamage, comprising the following steps:
(1) carrying out skin multifunctional imaging on the injection part of each group of nude mice to detect the general wrinkle condition, reflecting the thickness of the skin through a polarized light mode, evaluating the roughness of the skin through 3D roughness, and comprehensively scoring;
(2) drawing materials of injection parts of each group of nude mice, performing HE staining and masson staining respectively, calculating and counting the thickness of epidermis and the thickness of dermis, and performing PCNA and CD68 staining to know the proliferation condition and macrophage infiltration condition of cells in the skin;
s6, establishing an in vitro skin fibroblast photodamage model: inoculating skin fibroblast in 96-well plate or 6-well plate, adding or not adding extracellular vesicles with different concentrations according to groups, culturing for 24 hr, removing cell supernatant, washing twice with PBS, covering cells with a thin layer of PBS, placing ultraviolet lamp on the cells, and adjusting energy density of the ultraviolet lamp to 100mJ/cm2After irradiation, removing the covered PBS, replacing fresh culture solution, continuing to culture and carrying out subsequent experiments;
s7, cell viability detection step: skin fibroblasts are inoculated in a 96-well plate, extracellular vesicles with different concentrations are added or not added according to groups, cells are irradiated by ultraviolet rays, after the cells are well irradiated, a fresh culture solution is replaced, the cells are continuously cultured for 72 hours, the OD value of the cells under 450nm is measured by a CCK-8 kit according to the steps of a specification, and the cell activity is calculated by the following formula:
cell viability (OD value of cells in treatment group/OD value in control group) × 100%
Determining the concentration of extracellular vesicles used in subsequent cell experiments by two dimensions of the non-illuminated cells and the illuminated cells;
s8, β -galactosidase staining step, namely inoculating skin fibroblasts into a 6-well plate, staining each group of cells by using a β -galactosidase kit according to the instruction steps 72 hours after the cells are illuminated, observing the cells under an inverted microscope and taking pictures after staining is finished, randomly taking 5 visual fields for each well, and calculating the average number of staining positive cells, wherein the calculation formula is as follows:
the positive rate (number of staining positive cells/total number of cells) × 100%;
s9 mouse macrophage polarization induction and treatment experiment step to study the role of extracellular vesicles in regulating macrophages, 5 × 105The cell line of the/well raw 264.7 is inoculated in a 6-well plate, after the cells adhere to the wall, the culture solution is removed, the culture solution of a common culture system, the culture solution plus 1mg/mL LPS plus 20ng/mL IFN-gamma, the culture solution plus 1mg/mLLPS plus 20 ng/mLIFN-gamma and extracellular vesicles with different concentrations are respectively added, after incubation for 24 hours, the flow cytometry, qPCR and Western Blot are respectively used for detecting giant vesiclesPolarization of phagocytes;
s10, detecting endogenous active oxygen of skin fibroblasts: inoculating skin fibroblasts in a 6-well plate, incubating the skin fibroblasts with extracellular vesicles of different concentrations for 24 hours, removing a culture solution, incubating a fluorescent reagent DCFH2-DA (10 μ M) and the cells at 37 ℃ for 20 minutes according to a kit specification, removing the reagent, washing the cells for 3 times by serum-free DMEM, performing flow analysis on the digested cells, calculating average fluorescence intensity, and observing and photographing the undigested cells under a fluorescence microscope;
s11, cell cycle detection step: skin fibroblasts were seeded in 6-well plates, incubated with extracellular vesicles of different concentrations for 24 hours, then the cells were irradiated with ultraviolet light and detected 24 hours after irradiation with a cell cycle kit according to the instructions.
The method for promoting skin injury repair by using adipose-derived stem cell extracellular vesicles, wherein the seeding density of the 96-well plate is 2000 cells/well, and the seeding density of the 6-well plate is 1 × 105Individual cells/well.
In the method for promoting skin injury repair by using adipose-derived stem cell extracellular vesicles, in steps S6-S7 and S9-S11, the extracellular vesicles with different concentrations include extracellular vesicles with a concentration of 100ug/mL and extracellular vesicles with a concentration of 200 ug/mL.
The research method of the function and mechanism of the adipose-derived stem cell extracellular vesicle for promoting skin injury repair disclosed by the invention discloses a mechanism of preventing skin fibroblast photodamage by using the adipose-derived stem cell extracellular vesicle, and verifies the treatment effect of the adipose-derived stem cell extracellular vesicle in an animal body.
Drawings
FIG. 1 is a diagram of the experimental process of the research method of the action and mechanism of the adipose-derived stem cell extracellular vesicles in promoting skin injury repair;
FIG. 2 is an identification chart of EVs;
FIG. 3 is a graph showing a general assessment of the improvement in photodamage to skin in animal models following treatment with EVs injections;
FIG. 4 is a graph showing the results of measurements of epidermal thickness and dermal thickness in an animal model after EVs injection therapy;
FIG. 5 is a graph of the results of measurements of cell proliferation and inflammatory infiltration in animal models following EVs injection therapy;
FIG. 6 is a graph showing the results of EVs promoting skin fibroblast proliferation and reducing β -galactosidase staining;
FIG. 7 is a graph showing the results of the mouse macrophage polarization induction and treatment experiment;
FIG. 8 is a graph showing the results of measurement of endogenous reactive oxygen species and antioxidant enzymes in skin fibroblasts;
FIG. 9 is a graph showing the results of the cell cycle arrest assay of skin fibroblasts.
Detailed Description
In order that those skilled in the art will better understand the technical solution of the present invention, the following detailed description is given with reference to the accompanying drawings:
referring to fig. 1, according to an embodiment of the present invention, a method for promoting skin injury repair by using adipose-derived stem cell extracellular vesicles includes the following steps:
s1, the cell extraction and identification step comprises the following procedures:
(1) extraction and identification of adipose-derived stem cells: digesting the waste granular fat after liposuction by using a collagenase digestion method, extracting adipose-derived stem cells, carrying out passage after the lipolysis, and identifying the extracted adipose-derived stem cells by flow cytometry and three-way induction respectively;
(2) extraction of skin fibroblasts: soaking the waste foreskin with Dispase for 24 hours, removing the epidermis, digesting the residual dermis by a collagenase digestion method, extracting skin fibroblasts, carrying out passage after the skin fibroblasts grow full, and substituting for 3-5 in vitro for the research of the photoaging of the skin fibroblasts by extracellular vesicles;
s2, extracting the fat stem cell extracellular vesicles: after the third-generation adipose-derived stem cells are full, starving the adipose-derived stem cells for 48 hours, collecting cell supernatants, extracting the extracellular vesicles of the adipose-derived stem cells by an ultrafiltration method, and suspending the extracted extracellular vesicles in PBS (phosphate buffer solution) for preservation at minus 80 ℃;
s3, identification of the fat stem cell extracellular vesicles: observing the shape and size of the extracellular vesicles through a transmission electron microscope, calculating the average diameter of the extracellular vesicles through nanoparticle tracking analysis, and detecting markers CD81, CD63, CD9 and TSG101 of the extracellular vesicles through Western Blot;
referring to fig. 2, fig. 2A shows the extracellular vesicles of the adipose-derived stem cells observed under a transmission electron microscope, with a scale of 200 um. 2B is a Nanoparticle Tracking Analysis (NTA) assay, showing that the average diameter of the vesicles is 127 + -3 nm in size. 2C is a vesicle-associated protein marker detected by Western Blot, and the result shows that the vesicles express CD81, CD63, CD9 and TSG 101.
S4, construction and treatment steps of a skin photodamage nude mouse model: taking 40 female BALB/c nude mice of 5 weeks old, and conventionally breeding the mice in an SPF environment for 1 week to adapt to the environment; starting at week 6, nude mice were randomly divided into 4 groups:
(1) control group (control): UVB irradiation is not carried out, and subcutaneous injection treatment is not carried out;
(2) UVB group: carrying out UVB irradiation and subcutaneous injection of PBS on a nude mouse;
(3) UVB +150ug/week group: UVB irradiation + subcutaneous injection of 150ug/week extracellular vesicles was performed on nude mice;
(4) UVB +300ug/week group: UVB irradiation + subcutaneous injection of 300ug/week extracellular vesicles was performed on nude mice;
the process for establishing the skin photodamage nude mouse model is as follows: erecting a UVB lamp tube above a mouse cage to enable a nude mouse to be under direct irradiation of UVB and to move freely, placing an energy detector under the UVB lamp, wherein the irradiation energy of the first week is determined by the minimum erythema dose, irradiating for 5 days every week, and increasing the irradiation dose of the minimum erythema dose every subsequent week for 8 weeks;
s5, an evaluation step of the effect of the adipose-derived stem cell extracellular vesicles on treatment of skin photodamage, comprising the following steps:
(1) carrying out skin multifunctional imaging on the injection part of each group of nude mice to detect the general wrinkle condition, reflecting the thickness of the skin through a polarized light mode, evaluating the roughness of the skin through 3D roughness, and comprehensively scoring;
referring to fig. 3, 3A shows that the skin damage and wrinkles of each group of nude mice are observed under the skin detector, 3B shows that the UVB group is observed to be transparent to the blood vessels of the control group under the polarized light mode of the skin detector, which indicates that the skin is thinned, and UVB +150ug/week group and UVB +300ug/week have no obvious blood vessel transparent condition, which indicates that EVs increases the skin thickness. And 3C shows the roughness of each group of skin by the 3D roughness reconstruction of the skin detector. 3D is the clinical wrinkle score showing that after UV irradiation, the UVB group scores higher and the degree of aging after photodamage is high, while the UVB +150ug/week group and UVB +300ug/week reduce the degree of aging after photodamage.
Experimental results show that the aging condition of the skin after photodamage is obviously improved after 2 months of EVs injection treatment.
(2) Drawing materials of injection parts of each group of nude mice, performing HE staining and masson staining respectively, calculating and counting the thickness of epidermis and the thickness of dermis, and performing PCNA and CD68 staining to know the proliferation condition and macrophage infiltration condition of cells in the skin;
referring to fig. 4, 4A shows HE and masson staining showing skin thickness for each group, with 150um scale. 4B shows the skin thickness statistics for each group, with significant thickening of the UVB group skin after UV irradiation, and dose-dependent improvement in the UVB +150ug/week and UVB +300ug/week groups. 4C shows a statistic of dermis thickness for each group, with significant thinning of the UVB group dermis after UV irradiation, while the dermis thickness was improved for the UVB +150ug/mL and UVB +300ug groups, but there was no significant difference between the UVB +150ug/week and UVB +300 ug/week.
The experimental results show that: EVs reduce the increase in epidermal thickness and increase the thickness of the dermis.
Referring to FIG. 5, 5A shows cell proliferation (PCNA, scale 50um) and macrophage infiltration (CD68, scale 100um) in skin tissue. 5B shows an epidermal proliferation index which is significantly reduced in the UVB group upon UV irradiation, whereas the UVB +150ug/week and UVB +300ug/week groups show a dose-dependent improvement. 5C shows dermal proliferation index: there was no significant difference between the groups. 5D shows the statistics of CD68 staining positive cells, and the number of CD68+ cells infiltrated by the UVB group is obviously increased through ultraviolet irradiation, while the number of CD68+ cells of the UVB +150ug/week group and the UVB +300ug/week group is obviously reduced, but the UVB +150ug/week group and the UVB +300ug/week group have no obvious difference.
The experimental results show that: EVs increase the proliferative capacity of epidermal cells, decrease macrophage infiltration, increase epidermal thickness in UVB group but decrease cell proliferation index, suggesting that epidermal thickening is caused by hyperkeratosis due to decreased regenerative capacity of epidermis.
S6, establishing an in vitro skin fibroblast photodamage model: inoculating skin fibroblast in 96-well plate or 6-well plate, adding or not adding extracellular vesicles with different concentrations according to groups, culturing for 24 hr, removing cell supernatant, washing twice with PBS, covering cells with a thin layer of PBS, placing ultraviolet lamp on the cells, and adjusting energy density of the ultraviolet lamp to 100mJ/cm2After irradiation, removing the covered PBS, replacing fresh culture solution, continuing to culture and carrying out subsequent experiments;
s7, cell viability detection step: skin fibroblasts are inoculated in a 96-well plate, extracellular vesicles with different concentrations are added or not added according to groups, cells are irradiated by ultraviolet rays, after the cells are well irradiated, a fresh culture solution is replaced, the cells are continuously cultured for 72 hours, the OD value of the cells under 450nm is measured by a CCK-8 kit according to the steps of a specification, and the cell activity is calculated by the following formula:
cell viability (OD value of cells in treatment group/OD value in control group) × 100%
Determining the concentration of extracellular vesicles used in subsequent cell experiments by two dimensions of the non-illuminated cells and the illuminated cells;
referring to fig. 6, fig. 6A shows the effect of vesicles with different concentrations on the proliferation activity of skin fibroblasts in the absence of uv irradiation, and the results show that: the concentration of more than 100ug/mL can obviously promote the proliferation activity of cells. 6B is the promoting effect of the vesicles with different concentrations on the proliferation activity of skin fibroblasts under the condition of ultraviolet irradiation. The results show that: UVB irradiation obviously weakens the proliferation activity of cells, the proliferation activity can be obviously restored at the concentration of more than 100ug/mL, and the two results are combined, so that the subsequent cell experiment adopts 100ug/mL vesicles with low concentration and 200ug/mL vesicles with high concentration.
The experimental results show that: EVs with a concentration of 100ug/mL or more can promote the proliferation of skin fibroblasts both without and with ultraviolet irradiation.
S8, β -galactosidase staining step, namely inoculating skin fibroblasts into a 6-well plate, staining each group of cells by using a β -galactosidase kit according to the instruction steps 72 hours after the cells are illuminated, observing the cells under an inverted microscope and taking pictures after staining is finished, randomly taking 5 visual fields for each well, and calculating the average number of staining positive cells, wherein the calculation formula is as follows:
the positive rate (number of staining positive cells/total number of cells) × 100%;
referring to fig. 6,6C is a graph of β -galactosidase staining results (scale 100um) for each group, 6D is a statistic of the 6C graph, showing that upon UVB irradiation, β -galactosidase staining was significantly enhanced, while adipose stem cell extracellular vesicles showed dose-dependent reduction in intensity of β -galactosidase staining.
Experimental results show that EVs reduce the expression of β -galactosidase after ultraviolet irradiation in vitro.
S9 mouse macrophage polarization induction and treatment experiment step to study the role of extracellular vesicles in regulating macrophages, 5 × 105The cell line of the/well raw 264.7 is inoculated in a 6-well plate, after the cells adhere to the wall, the culture solution is removed, the culture solution of a common culture system, the culture solution plus 1mg/mL LPS plus 20ng/mL IFN-gamma, the culture solution plus 1mg/mLLPS plus 20 ng/mLIFN-gamma plus extracellular vesicles with different concentrations are respectively added, after incubation for 24 hours, the polarization condition of the macrophages is respectively detected by flow cytometry, qPCR and Western Blot;
referring to FIG. 7, in which 7A is flow cytometry showing that M0 macrophages are induced to M1 type macrophages (CD86+) by LPS + IFN- γ induction, whereas the LPS + IFN- γ +100ug/mL and LPS + IFN- γ +200ug/mL groups showed dose-dependent improvement. The surface markers of the M2-type macrophages of each group were negative in expression, so that subsequent experiments were mainly performed to examine the effects of EVs on modulating M1-type macrophages (M1-type macrophages are pro-inflammatory cells, and the decrease is beneficial for inhibiting inflammation). Since M2-type macrophages are negative, subsequent experiments mainly discuss the effect of EVs on M1-type macrophages.
Statistics for 7B and 7C on CD68+ cells and CD206+ cells of 7A, respectively.
qPCR in 7D and 7E showed a clear increase in macrophage secretion of inflammatory factors (TNF- α and IL- β) induced by LPS + IFN- γ, whereas the LPS + IFN- γ +100ug/mL and LPS + IFN- γ +200ug/mL groups showed dose-dependent improvement.
In 7F and 7G, NF-. kappa.B 1 pathway was activated by LPS + IFN-. gamma.induction, whereas the addition of EVs (LPS + IFN-. gamma. +100ug/mL and LPS + IFN-. gamma. +200 ug/mL) showed a dose-dependent improvement, indicating that inflammation stimulated activation of NF-. kappa.B 1 pathway, while EVs suppressed inflammation by inhibiting its activation.
The experimental results show that: EVs reduce the inflammatory response of macrophages in vitro.
S10, detecting endogenous active oxygen of skin fibroblasts: inoculating skin fibroblasts in a 6-well plate, incubating the skin fibroblasts with extracellular vesicles of different concentrations for 24 hours, removing a culture solution, incubating a fluorescent reagent DCFH2-DA (10 μ M) and the cells at 37 ℃ for 20 minutes according to a kit specification, removing the reagent, washing the cells for 3 times by serum-free DMEM, performing flow analysis on the digested cells, calculating average fluorescence intensity, and observing and photographing the undigested cells under a fluorescence microscope;
referring to FIG. 8, FIG. 8A is a graph showing the results of detection of endogenous reactive oxygen species (scale: 250 um). 8B is the statistics of the detection results of endogenous active oxygen in the 8A picture, and the results show that: after UV irradiation, there was a significant increase in endogenous Reactive Oxygen Species (ROS) production in the UVB group, whereas dose-dependent reduction in reactive oxygen species production was observed in the test groups (UVB +100ug/mL and UVB +200 ug/mL) to which EVs was added. The qPCR at 8C and Western Blot at 8D showed a significant decrease in endogenous antioxidant enzyme production (SOD1 and CAT) in the UVB group, a significant increase in SOD1 in the UVB +100ug/mL group, and a significant increase in SOD1 and CAT production in the UVB +200ug/mL group, after uv irradiation.
The experimental results show that: EVs reduce the production of endogenous reactive oxygen species by skin fibroblasts and increase the production of endogenous antioxidant enzymes in vitro.
S11, cell cycle detection step: skin fibroblasts were seeded in 6-well plates, incubated with extracellular vesicles of different concentrations for 24 hours, then the cells were irradiated with ultraviolet light, and 24 hours after irradiation were detected with a cell cycle kit according to the instructions.
Referring to fig. 9, fig. 9A shows flow cytometry: the S phase and G2 phase of UVB group are obviously increased after ultraviolet irradiation, which indicates that (S + G2 phase) cell retardation occurs, and UVB +100ug/mL group and UVB +200ug/mL group are dose-dependent and improve cell cycle retardation. 9B is a statistic of the 9A graph. 9C is the detection of cell cycle arrest related proteins P53 and P21, the trend and flow results are the same, and after ultraviolet irradiation, the expression of P53 and P21 of UVB group is obviously increased, which indicates that cell arrest occurs, while the dose-dependent improvement of cell cycle arrest (the expression of P53 and P21 is obviously reduced) is performed on UVB +100ug/mL group and UVB +200ug/mL group.
The experimental results show that: EVs improve the cycle arrest of skin fibroblasts in vitro.
At present, iPS and EVs derived from skin fibroblasts are proved to be capable of improving the photodamage of skin, but iPS cells need to be generated by transferring tumorigenic genes, the operation process is complex, certain tumorigenic risks exist in application, and the sources of the fibroblasts are limited. The adipose-derived stem cells can extract a large amount of adipose through liposuction, and the operation is safe and has wide sources. Therefore, the skin photodamage is improved by extracting the vesicles of the adipose-derived stem cells, and clinical transformation is expected to be rapidly carried out in the future. The invention provides application data of adipose-derived stem cell extracellular vesicles in improving skin photodamage in animal models and discloses a possible mechanism of improving photodamage at an in vitro cell level.
In conclusion, the method for promoting skin injury repair by using the adipose-derived extracellular vesicles disclosed by the invention reveals a mechanism for preventing skin fibroblast photodamage by using the adipose-derived extracellular vesicles, and verifies the therapeutic effect of the adipose-derived extracellular vesicles in animals.
It should be understood by those skilled in the art that the above embodiments are only for illustrating the present invention and are not to be used as a limitation of the present invention, and that changes and modifications to the above described embodiments are within the scope of the claims of the present invention as long as they are within the spirit and scope of the present invention.
Claims (3)
1. A method for promoting skin injury repair by using adipose-derived stem cell extracellular vesicles, which is characterized by comprising the following steps:
s1, the cell extraction and identification step comprises the following procedures:
(1) extraction and identification of adipose-derived stem cells: digesting the waste granular fat after liposuction by using a collagenase digestion method, extracting adipose-derived stem cells, carrying out passage after the lipolysis, and identifying the extracted adipose-derived stem cells by flow cytometry and three-way induction respectively;
(2) extraction of skin fibroblasts: soaking the waste foreskin with Dispase for 24 hours, removing the epidermis, digesting the residual dermis by a collagenase digestion method, extracting skin fibroblasts, carrying out passage after the skin fibroblasts grow full, and replacing with 3 rd to 5 th to research on the skin fibroblast photodamage caused by extracellular vesicles in vitro;
s2, extracting the fat stem cell extracellular vesicles: after the third-generation adipose-derived stem cells are full, starving the adipose-derived stem cells for 48 hours, collecting cell supernatants, extracting the extracellular vesicles of the adipose-derived stem cells by an ultrafiltration method, and suspending the extracted extracellular vesicles in PBS (phosphate buffer solution) for preservation at minus 80 ℃;
s3, identification of the fat stem cell extracellular vesicles: observing the shape and size of the extracellular vesicles through a transmission electron microscope, calculating the average diameter of the extracellular vesicles through nanoparticle tracking analysis, and detecting markers CD81, CD63, CD9 and TSG101 of the extracellular vesicles through Western Blot;
s4, construction and treatment steps of a skin photodamage nude mouse model: taking 40 female BALB/c nude mice of 5 weeks old, and conventionally breeding the mice in an SPF environment for 1 week to adapt to the environment; starting at week 6, nude mice were randomly divided into 4 groups:
(1) control group: UVB irradiation is not carried out, and subcutaneous injection treatment is not carried out;
(2) UVB group: carrying out UVB irradiation and subcutaneous injection of PBS on a nude mouse;
(3) UVB +150ug/week group: UVB irradiation + subcutaneous injection of 150ug/week extracellular vesicles was performed on nude mice;
(4) UVB +300ug/week group: UVB irradiation + subcutaneous injection of 300ug/week extracellular vesicles was performed on nude mice;
the process for establishing the skin photodamage nude mouse model is as follows: erecting a UVB lamp tube above a mouse cage to enable a nude mouse to be under direct irradiation of UVB and to move freely, placing an energy detector under the UVB lamp, wherein the irradiation energy of the first week is determined by the minimum erythema dose, irradiating for 5 days every week, and increasing the irradiation dose of the minimum erythema dose every subsequent week for 8 weeks;
s5, the evaluation step of the effect of the adipose-derived stem cell extracellular vesicles on photoaging comprises the following steps:
(1) carrying out skin multifunctional imaging on the injection part of each group of nude mice to detect the general wrinkle condition, reflecting the thickness of the skin through a polarized light mode, evaluating the roughness of the skin through 3D roughness, and comprehensively scoring;
(2) drawing materials of injection parts of each group of nude mice, performing HE staining and masson staining respectively, calculating and counting the thickness of epidermis and the thickness of dermis, and performing PCNA and CD68 staining to know the proliferation condition and macrophage infiltration condition of cells in the skin;
s6, establishing an in vitro skin fibroblast photodamage model: inoculating skin fibroblast in 96-well plate or 6-well plate, adding or not adding extracellular vesicles with different concentrations according to groups, culturing for 24 hr, removing cell supernatant, washing twice with PBS, covering cells with a thin layer of PBS, placing ultraviolet lamp on the cells, and adjusting energy density of the ultraviolet lamp to 100mJ/cm2After irradiation, removing the covered PBS, replacing fresh culture solution, continuing to culture and carrying out subsequent experiments;
s7, cell viability detection step: skin fibroblasts are inoculated in a 96-well plate, extracellular vesicles with different concentrations are added or not added according to groups, cells are irradiated by ultraviolet rays, after the cells are well irradiated, a fresh culture solution is replaced, the cells are continuously cultured for 72 hours, the OD value of the cells under 450nm is measured by a CCK-8 kit according to the steps of a specification, and the cell activity is calculated by the following formula:
cell viability (OD value of cells in treatment group/OD value in control group) × 100%
Determining the concentration of extracellular vesicles used in subsequent cell experiments by two dimensions of the non-illuminated cells and the illuminated cells;
s8, β -galactosidase staining step, namely inoculating skin fibroblasts into a 6-well plate, staining each group of cells by using a β -galactosidase kit according to the instruction steps 72 hours after the cells are illuminated, observing the cells under an inverted microscope and taking pictures after staining is finished, randomly taking 5 visual fields for each well, and calculating the average number of staining positive cells, wherein the calculation formula is as follows:
the positive rate (number of staining positive cells/total number of cells) × 100%;
s9 mouse macrophage polarization induction and treatment experiment step to study the role of extracellular vesicles in regulating macrophages, 5 × 105The cell line of the/well raw 264.7 is inoculated in a 6-well plate, after the cells adhere to the wall, the culture solution is removed, the culture solution of a common culture System, the culture solution plus 1mg/mL LPS plus 20ng/mL IFN-gamma, the culture solution plus 1mg/mLLPS plus 20 ng/mLIFN-gamma and extracellular vesicles with different concentrations are respectively added, after incubation for 24 hours, the macrophage polarization condition is respectively detected by flow cytometry, Real-time Quantitative PCR detection (PCR) and Western Blot;
s10, detecting endogenous active oxygen of skin fibroblasts: inoculating skin fibroblasts in a 6-well plate, incubating the skin fibroblasts with extracellular vesicles of different concentrations for 24 hours, removing a culture solution, incubating a fluorescent reagent DCFH2-DA (10 μ M) and the cells at 37 ℃ for 20 minutes according to a kit specification, removing the reagent, washing the cells for 3 times by serum-free DMEM, performing flow analysis on the digested cells, calculating average fluorescence intensity, and observing and photographing the undigested cells under a fluorescence microscope;
s11, cell cycle detection step: skin fibroblasts were seeded in 6-well plates, incubated with extracellular vesicles of different concentrations for 24 hours, then the cells were irradiated with ultraviolet light and detected 24 hours after irradiation with a cell cycle kit according to the instructions.
2. The method for promoting skin injury repair by using adipose-derived stem cell extracellular vesicles as claimed in claim 1, wherein the 96-well plate is seeded at a density of 2000 cells/well, and the 6-well plate is seeded at a density of 1 × 105Individual cells/well.
3. The method for promoting skin injury repair by using adipose-derived stem cell extracellular vesicles, according to claim 1, wherein the different concentrations of extracellular vesicles include 100ug/mL extracellular vesicles and 200ug/mL extracellular vesicles in steps S6-S7 and S9-S11.
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