Application of mesenchymal stem cell-derived exosome in resisting UVB induced skin photoaging

Abstract

The invention discloses an application of mesenchymal stem cell-derived exosomes in resisting UVB (ultraviolet B) induced skin photoaging, which comprises the following steps: S1, determining a system boundary, namely determining which parts belong to a system range and which parts do not belong to the system range, so as to conveniently formulate a corresponding strategy in the system range subsequently; s2, determining the role of an attacker: according to the attack mode, means and angle of the attacker, preliminarily determining the type of the attacker, such as hackers, internal employees and the like; s3, constructing an attack tree: generally, determining a final target of an attacker, dividing the final target into sub-targets and constructing a tree diagram of the attack tree; wherein the final target is a tree root, the sub-target is a branch, and the means for completing the sub-target is a leaf; s4, analyzing an attack tree: identifying heavy vulnerabilities and weaknesses of the system, and formulating a corresponding defense strategy; and S5, actively setting the trapping vulnerabilities: actively setting the trapping vulnerabilities on the final target and the sub-target so as to obtain the information of an attacker or capture the attacker when the attacker attacks the trapping vulnerabilities.

Description

Technical Field

[0001] The present invention relates to the technical field of exosomes derived from mesenchymal stem cells, and specifically to application of exosomes derived from mesenchymal stem cells in resisting UVB-induced skin photoaging. Background Art

[0002] Ultraviolet radiation (UV), especially the UVB part (280-315nm), poses a significant long-term threat to the skin and is an important cause of skin photoaging. Photoaging not only seriously affects the appearance and functional state of the skin, such as loss of elasticity, increased wrinkles, pigmentation, etc., but is also closely related to the occurrence and development of skin tumors, posing a major risk to individual health. This chronic skin damage process, marked by the degradation of the extracellular matrix and a significant decrease in collagen content, not only damages the skin barrier function, but also profoundly affects the individual's mental health, exacerbating the dual burden of physiology and psychology.

[0003] Mesenchymal stem cells (MSCs) are known to play a role in the treatment of various tissue injuries and immune diseases. We have also demonstrated that MSCs have therapeutic effects in a variety of ophthalmic disease models, including retinal ischemia-reperfusion, diabetic retinopathy, uveoretinitis, retinal degeneration, and retinal injury. MSCs can be isolated from several adult tissues (including bone marrow, adipose tissue, and umbilical cord blood) and expanded in vitro. Exosomes are a natural mechanism for transporting genetic information (including nucleic acids, proteins, and lipids) between cells, which can promote cell-to-cell communication. Extracellular vesicles derived from mesenchymal stem cells, exosomes, have attracted widespread attention from researchers due to their low immunogenicity, excellent biocompatibility, and strong delivery capabilities.

[0004] However, there are no reports on the application of mesenchymal stem cell-derived extracellular vesicles (EVs) in the prevention of UVB-induced skin photoaging. Therefore, the research and development of exosomes that can be used to prevent UVB-induced skin photoaging has important scientific significance and clinical translation value. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above-mentioned and / or existing problems in the application of exosomes derived from mesenchymal stem cells in resisting UVB-induced skin photoaging, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to provide the application of exosomes derived from mesenchymal stem cells in resisting UVB-induced skin photoaging. The exosomes reduce the damage of UVB to the skin through the antioxidant pathway. The mechanism of the exosomes in resisting UVB-induced skin photoaging includes: the exosomes derived from mesenchymal stem cells can upregulate the expression of collagen in HDF, downregulate the expression of MMP-1, and promote the migration and proliferation of HDF cells under ultraviolet irradiation; the exosomes derived from mesenchymal stem cells can also promote the recovery of wrinkles in photoaged skin and increase collagen deposition. In this process, miR-125b-5p in the exosomes derived from mesenchymal stem cells plays a role by targeting the TGF-β1 / Smad signaling pathway. This fills the technical gap in the current exosomes extracted from mesenchymal stem cells in resisting UVB-induced skin photoaging.

[0008] To solve the above technical problems, according to one aspect of the present invention, the present invention provides the following technical solutions:

[0009] The application of exosomes derived from mesenchymal stem cells in anti-UVB-induced skin photoaging includes:

[0010] Step 1: Isolation and culture of mesenchymal stem cells: Take fresh umbilical cords from sterile newborns, rinse them repeatedly with phosphate buffer, and cut them into tissue blocks with a diameter of about 1-2 mm; digest them with type 2 collagenase and trypsin in turn, centrifuge the supernatant, take the cell pellet and put it into a culture bottle, and culture it in DMEM / F12 medium containing 10% fetal bovine serum, 5% CO2, and 37°C saturated humidity; remove non-adherent cells, and digest them with 0.25% trypsin after 80% fusion of adherent cells for subculture;

[0011] Step 2: Collection of mesenchymal stem cell culture medium: Take 3-5 generations of mesenchymal stem cells and culture them serum-free for 48 hours; collect the culture supernatant; filter through a 0.22 μm sterile membrane to obtain the mesenchymal stem cell conditioned medium;

[0012] Step 3: Isolation and purification of exosomes: collect the filtered mesenchymal stem cell conditioned medium, centrifuge at 4°C, 1000g for 10 min, collect the supernatant; centrifuge the collected supernatant at 4°C, 2000g for 20 min, collect the supernatant; centrifuge the collected supernatant at 4°C, 10000g for 30 min, collect the supernatant; centrifuge the collected supernatant at 110000g for 70 min, discard the supernatant, and resuspend the precipitate with phosphate buffer; centrifuge again at 110000g for 70 min, discard the supernatant, resuspend the precipitate with a small amount of phosphate buffer, filter and sterilize with 0.22μm filter membrane to obtain exosomes derived from mesenchymal stem cells;

[0013] Step 4: Identification of exosomes: Scanning electron microscopy to observe the general morphology of exosomes: Take a uniformly suspended exosome solution, dehydrate, dry, stick, and conduct the sample, observe the particle morphology under a scanning electron microscope, take electron microscope photos, randomly select 20 exosomes, and record their diameters according to the ruler. Under scanning electron microscopy, it can be seen that the diameter of the exosomes is about 50-100nm, with a complete lipid membrane, a low electron density component in the center of the body, and a round or oval membrane vesicle structure; take exosome samples and use high performance liquid chromatography and mass spectrometry to analyze the proteins contained in the exosomes;

[0014] Step 5: Prepare a UVB-induced mouse control group: 6-8 week-old healthy female mice (weight 15-18 g) were randomly divided into an irradiation group and a treatment group in equal numbers. The backs of the mice in the irradiation group and the treatment group were shaved, and then the backs were irradiated with UVB 430 mJ / cm2 for 2 hours;

[0015] Step 6: Observation of the therapeutic effect of exosomes: After injecting 5 μl (0.5 mg / ml) of exosomes into the treatment group, photos were taken to observe the back conditions of the irradiation group and the treatment group. After a certain period of time, the back skin tissues of the mice in the treatment group and the irradiation group were obtained, and then the tissues were fixed, dehydrated, paraffin-embedded, sliced ​​and stained with HE. The skin thickness and necrosis of the two groups were compared to analyze the application effect of exosomes derived from mesenchymal stem cells in anti-UVB-induced skin photoaging.

[0016] As a preferred solution for the application of the exosomes derived from mesenchymal stem cells in the present invention in resisting UVB-induced skin photoaging, human placental mesenchymal stem cells and human adipose mesenchymal stem cells may also be used in step 1.

[0017] As a preferred embodiment of the application of the mesenchymal stem cell-derived exosomes in the prevention of UVB-induced skin photoaging, in step 6, a treatment group can be added, and exosomes with different concentrations can be injected into the added treatment group to compare the application of the exosome concentrations in the prevention of UVB-induced skin photoaging.

[0018] Compared with the prior art, the present invention has the following beneficial effects: the exosomes can reduce the damage of UVB to the skin through the anti-oxidation pathway, and the mechanism of the exosomes against UVB-induced skin photoaging includes: the exosomes derived from mesenchymal stem cells can upregulate the expression of collagen in HDF, downregulate the expression of MMP-1, and promote the migration and proliferation of HDF cells under ultraviolet irradiation; the exosomes derived from mesenchymal stem cells can also promote the recovery of wrinkles in photoaged skin and increase collagen deposition. In this process, miR-125b-5p in the exosomes derived from mesenchymal stem cells plays a role by targeting the TGF-β1 / Smad signaling pathway. This fills the technical gap in the current exosomes extracted from mesenchymal stem cells in resisting UVB-induced skin photoaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail below in combination with the accompanying drawings and detailed embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0020] Figure 1 A diagram of a method for using exosomes derived from mesenchymal stem cells in the present invention to resist UVB-induced skin photoaging. DETAILED DESCRIPTION

[0021] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0022] Secondly, the present invention is described in detail with reference to schematic diagrams. When describing the embodiments of the present invention in detail, for the sake of convenience, the cross-sectional diagrams showing the device structure will not be partially enlarged according to the general scale, and the schematic diagrams are only examples, which should not limit the scope of protection of the present invention. In addition, in actual production, the three-dimensional dimensions of length, width and depth should be included.

[0023] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0024] The present invention provides the application of exosomes derived from mesenchymal stem cells in resisting UVB-induced skin photoaging. The exosomes reduce the damage of UVB to the skin through the antioxidant pathway. The mechanism of the exosomes in resisting UVB-induced skin photoaging includes: the exosomes derived from mesenchymal stem cells can upregulate the expression of collagen in HDF, downregulate the expression of MMP-1, and promote the migration and proliferation of HDF cells under ultraviolet irradiation; the exosomes derived from mesenchymal stem cells can also promote the recovery of wrinkles in photoaged skin and increase collagen deposition. In this process, miR-125b-5p in the exosomes derived from mesenchymal stem cells plays a role by targeting the TGF-β1 / Smad signaling pathway. This fills the technical gap in the current exosomes extracted from mesenchymal stem cells in resisting UVB-induced skin photoaging.

[0025] Figure 1 The figure shows the overall structure of an embodiment of the application of exosomes derived from mesenchymal stem cells in the present invention in the prevention of UVB-induced skin photoaging. The main part of the application of exosomes derived from mesenchymal stem cells in the present embodiment in the prevention of UVB-induced skin photoaging includes:

[0026] Step 1: Isolation and culture of mesenchymal stem cells: Take fresh umbilical cords from sterile newborns, rinse them repeatedly with phosphate buffer, and cut them into tissue blocks with a diameter of about 1-2 mm; digest them with type 2 collagenase and trypsin in turn, centrifuge the supernatant, take the cell pellet and put it into a culture bottle, and culture it in DMEM / F12 medium containing 10% fetal bovine serum, 5% CO2, and 37°C saturated humidity; remove non-adherent cells, and digest them with 0.25% trypsin after 80% fusion of adherent cells for subculture;

[0027] Step 2: Collection of mesenchymal stem cell culture medium: Take 3-5 generations of mesenchymal stem cells and culture them serum-free for 48 hours; collect the culture supernatant; filter through a 0.22 μm sterile membrane to obtain the mesenchymal stem cell conditioned medium;

[0028] Step 3: Isolation and purification of exosomes: collect the filtered mesenchymal stem cell conditioned medium, centrifuge at 4°C, 1000g for 10 min, collect the supernatant; centrifuge the collected supernatant at 4°C, 2000g for 20 min, collect the supernatant; centrifuge the collected supernatant at 4°C, 10000g for 30 min, collect the supernatant; centrifuge the collected supernatant at 110000g for 70 min, discard the supernatant, and resuspend the precipitate with phosphate buffer; centrifuge again at 110000g for 70 min, discard the supernatant, resuspend the precipitate with a small amount of phosphate buffer, filter and sterilize with 0.22μm filter membrane to obtain exosomes derived from mesenchymal stem cells;

[0029] Step 4: Identification of exosomes: Scanning electron microscopy to observe the general morphology of exosomes: Take a uniformly suspended exosome solution, dehydrate, dry, stick, and conduct the sample, observe the particle morphology under a scanning electron microscope, take electron microscope photos, randomly select 20 exosomes, and record their diameters according to the ruler. Under scanning electron microscopy, it can be seen that the diameter of the exosomes is about 50-100nm, with a complete lipid membrane, a low electron density component in the center of the body, and a round or oval membrane vesicle structure; take exosome samples and use high performance liquid chromatography and mass spectrometry to analyze the proteins contained in the exosomes;

[0030] Step 5: Prepare a UVB-induced mouse control group: 6-8 week-old healthy female mice (weight 15-18 g) were randomly divided into an irradiation group and a treatment group in equal numbers. The backs of the mice in the irradiation group and the treatment group were shaved, and then the backs were irradiated with UVB 430 mJ / cm2 for 2 hours;

[0031] Step 6: Observation of the efficacy of exosomes: After the treatment group was injected with 5 μl (0.5 mg / ml) of exosomes, photos were taken to observe the back conditions of the irradiation group and the treatment group. After a certain period of time, the back skin tissues of the mice in the treatment group and the irradiation group were obtained, and then the tissues were fixed, dehydrated, paraffin-embedded, sliced ​​and HE-stained. The skin thickness and necrosis of the two groups were compared to analyze the application effect of exosomes derived from mesenchymal stem cells in anti-UVB-induced skin photoaging;

[0032] Among them; human placental mesenchymal stem cells and human adipose mesenchymal stem cells can also be selected in step 1; in step 6, a treatment group can be added, and exosomes with different concentrations can be injected into the added treatment group to compare the application of exosome concentrations in resisting UVB-induced skin photoaging.

[0033] Although the present invention has been described above with reference to the embodiments, various modifications may be made thereto and parts thereof may be replaced by equivalents without departing from the scope of the present invention. In particular, as long as there is no structural conflict, the various features in the embodiments disclosed in the present invention may be used in combination with each other in any manner, and the fact that these combinations are not exhaustively described in this specification is only for the sake of omitting space and saving resources. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. Application of exosomes derived from mesenchymal stem cells in preventing UVB-induced skin photoaging, characterized in that: include: Step 1: Isolation and culture of mesenchymal stem cells: Take fresh umbilical cords from sterile newborns, rinse them repeatedly with phosphate buffer, and cut them into tissue blocks with a diameter of about 1-2 mm; digest them with type 2 collagenase and trypsin in turn, centrifuge the supernatant, take the cell pellet and put it into a culture bottle, and culture it in DMEM / F12 medium containing 10% fetal bovine serum, 5% CO2, and 37°C saturated humidity; remove non-adherent cells, and digest them with 0.25% trypsin after 80% fusion of adherent cells for subculture; Step 2: Collection of mesenchymal stem cell culture medium: Take 3-5 generations of mesenchymal stem cells and culture them serum-free for 48 hours; collect the culture supernatant; filter through a 0.22 μm sterile membrane to obtain the mesenchymal stem cell conditioned medium; Step 3: Isolation and purification of exosomes: collect the filtered mesenchymal stem cell conditioned medium and centrifuge it at 4°C, 1000g for 10 min, and collect the supernatant; centrifuge the collected supernatant at 4°C, 2000g for 20 min, and collect the supernatant; The collected supernatant was centrifuged at 10000 g for 30 min at 4 °C, and the supernatant was collected; The collected supernatant was centrifuged at 110,000 g for 70 min, the supernatant was discarded, and the precipitate was resuspended in phosphate buffer; the supernatant was centrifuged again at 110,000 g for 70 min, the supernatant was discarded, the precipitate was resuspended in a small amount of phosphate buffer, and the precipitate was sterilized by filtration with a 0.22 μm filter membrane to obtain exosomes derived from mesenchymal stem cells; Step 4: Identification of exosomes: Scanning electron microscopy to observe the general morphology of exosomes: Take a uniformly suspended exosome solution, dehydrate, dry, stick, and conduct the sample, observe the particle morphology under a scanning electron microscope, take electron microscope photos, randomly select 20 exosomes, and record their diameters according to the ruler. Under scanning electron microscopy, it can be seen that the diameter of the exosomes is about 50-100nm, with a complete lipid membrane, a low electron density component in the center of the body, and a round or oval membrane vesicle structure; take exosome samples and use high performance liquid chromatography and mass spectrometry to analyze the proteins contained in the exosomes; Step 5: Prepare a UVB-induced mouse control group: 6-8 week-old healthy female mice (weight 15-18 g) were randomly divided into an irradiation group and a treatment group in equal numbers. The backs of the mice in the irradiation group and the treatment group were shaved, and then the backs were irradiated with UVB 430 mJ / cm2 for 2 hours; Step 6: Observation of the therapeutic effect of exosomes: After injecting 5 μl (0.5 mg / ml) of exosomes into the treatment group, photos were taken to observe the back conditions of the irradiation group and the treatment group. After a certain period of time, the back skin tissues of the mice in the treatment group and the irradiation group were obtained, and then the tissues were fixed, dehydrated, paraffin-embedded, sliced ​​and stained with HE. The skin thickness and necrosis of the two groups were compared to analyze the application effect of exosomes derived from mesenchymal stem cells in anti-UVB-induced skin photoaging.

2. The use of exosomes derived from mesenchymal stem cells in anti-UVB-induced skin photoaging according to claim 1, characterized in that: In step 1, human placental mesenchymal stem cells and human adipose mesenchymal stem cells can also be selected.

3. The use of exosomes derived from mesenchymal stem cells in preventing UVB-induced skin photoaging according to claim 1, characterized in that: In step 6, a treatment group may be added, and exosomes with different concentrations may be injected into the added treatment group to compare the application of the exosome concentration in resisting UVB-induced skin photoaging.

Images