WO2023045117A1 - Exosomes of umbilical cord mesenchymal stem cells, and use thereof - Google Patents

Exosomes of umbilical cord mesenchymal stem cells, and use thereof Download PDF

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WO2023045117A1
WO2023045117A1 PCT/CN2021/138172 CN2021138172W WO2023045117A1 WO 2023045117 A1 WO2023045117 A1 WO 2023045117A1 CN 2021138172 W CN2021138172 W CN 2021138172W WO 2023045117 A1 WO2023045117 A1 WO 2023045117A1
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exosomes
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mscs
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赵顺英
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周桂英
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Priority claimed from CN202111122500.9A external-priority patent/CN113832108A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • 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
    • 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
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of stem cells, in particular to umbilical cord mesenchymal stem cell exosomes and applications thereof.
  • Mesenchymal stem cells are a kind of cells with high self-renewal, multi-lineage differentiation and unique immune regulation ability, which can regulate the immune system by interacting with cells of innate and adaptive immune system and secreting cells. Sex factors play an immunomodulatory role.
  • Mesenchymal stem cell exosomes (exosomes from mesenchymal stem cells, MSCs-Exo) are a secreted substance that MSCs function. Studies have shown that MSCs-Exo possess multiple activities, such as tissue repair, immune regulation, and disease treatment, which have been paid more and more attention.
  • Umbilical cord-mesenchymal stem cells have the characteristics of sufficient sources, convenient material acquisition, simple culture, and no ethical issues, so they have been extensively studied.
  • Umbilical cord mesenchymal stem cell exosomes (UC-MSCs-Exo) are also easier to be applied and promoted than other stem cell exosomes.
  • Skin anti-aging medical cosmetology research is a long-term and arduous subject. With the deepening of the aging problem, human beings have a strong demand for a young and healthy appearance, and the demand for anti-aging will increase in the future. Research on effective anti-aging technology and anti-aging products will help improve the quality of human life , especially for the serious problem of aging.
  • the skin barrier function is one of the most important functions of the skin. It plays an important role in protecting the body from environmental damage, preventing microbial invasion, regulating body temperature, avoiding water loss in the body, and maintaining a relatively stable internal environment.
  • UC-MSCs-Exo has anti-aging activity of the skin, but the activity needs to be improved, so as to overcome the shortage of UC-MSCs-Exo in a small amount, achieve high efficiency in a small amount, and be easy to be applied and promoted.
  • the present invention aims to overcome the deficiencies of the prior art, and provides an umbilical cord mesenchymal stem cell exosome and its application.
  • an umbilical cord mesenchymal stem cell exosome is an exosome secreted by a human umbilical cord mesenchymal stem cell whose expression of miR-328-3p or miR-485-5p is upregulated.
  • exosomes from umbilical cord mesenchymal stem cells with upregulated miR-485-5p expression to promote skin barrier repair.
  • the present invention found that, compared with the exosomes secreted by normal human umbilical cord mesenchymal stem cells, the exosomes secreted by human umbilical cord mesenchymal stem cells with up-regulated miR-328-3p expression can more effectively improve human dermal fibroblasts on the one hand. On the other hand, it can more effectively increase the content of type I collagen and elastin in the extracellular matrix of human dermal fibroblasts, and has the prospect of being developed into anti-aging skin care products or medicines.
  • the present invention also found that compared with the exosomes secreted by normal human umbilical cord mesenchymal stem cells, the exosomes secreted by human umbilical cord mesenchymal stem cells with up-regulated miR-485-5p expression can more effectively improve human immortalization.
  • the proliferative activity of epidermal HaCaT cells can more effectively improve the migration activity of HaCaT cells, and has the prospect of being developed into skin care products or medicines that promote skin barrier repair.
  • A is the observation image of UC-MSCs with an inverted microscope
  • B is the image of flow cytometry detection of UC-MSCs
  • C-1 is the comparison of miR-328-3p content in UC-MSCs of each group
  • D-1 is Transmission electron microscope observation images of UC-MSCs-Exo in each group (correlated with miR-328-3p)
  • E-1 is the Western Blot detection results of UC-MSCs-Exo in each group (correlated with miR-328-3p)
  • C-2 is The content comparison of miR-485-5p in UC-MSCs of each group
  • D-2 is the transmission electron microscope observation image of UC-MSCs-Exo in each group (related to miR-485-5p)
  • E-2 is the UC-MSCs-Exo of each group Western blot detection results of Exo (related to miR-485-5p).
  • A is the comparison of proliferation activity of HDF-a in each group
  • B is the comparison of COL I and Elastin contents.
  • A is a comparison of the proliferation activity of HaCaT cells in each group
  • B is a comparison of the migration activity of HaCaT cells in each group.
  • Fetal bovine serum and DMEM/F12 medium were purchased from Gibco, and double antibodies, PBS and trypsin were purchased from Beyontib.
  • miR-328-3p mimic and miR-328-3p NC mimic were purchased from Shanghai Sangon Bioengineering Co., Ltd.
  • FITC or PE-labeled mouse anti-human CD90, CD105, CD73, CD14, CD34 and CD45 antibodies and related isotype controls were purchased from eBioscince, USA.
  • Lipofectamine 2000 was purchased from Invitrogen.
  • TRIzol kit was purchased from Thermo Fisher. Reagent materials related to Western blot were purchased from Biyuntian Biology, etc.
  • the umbilical cord tissues of healthy full-term neonates were collected under aseptic conditions according to conventional methods, washed several times with PBS containing 1% double antibody to remove residual blood, and the umbilical arteries and veins were removed, leaving Wharton glue-like tissues, which were cut Crush to form small pieces of about 1 mm 3 , then culture in DMEM/F12 containing 10% fetal bovine serum and 1% double antibody at 37°C, 5% CO 2 , and saturated humidity, and change the medium every 3-4 days . Cell morphology and growth were observed under an inverted microscope. When the cells grew to 90% confluent, trypsinized and subcultured. The third generation UC-MSCs were used to detect surface markers by flow cytometry.
  • the method of flow cytometry is as follows: take the third-generation UC-MSCs, digest with 0.25% trypsin and adjust the cell density to 1 ⁇ 106 cells/mL, add 1 mL of cell suspension to each centrifuge tube, wash twice with sterile PBS, 1000 Centrifuge at ⁇ g for 5 minutes, discard the supernatant, resuspend the cells in 100 ⁇ L PBS, add appropriate amount of CD90, CD105, CD73, CD14, CD34 and CD45 antibodies and each isotype control to each tube of single cell suspension, incubate at room temperature for 30 minutes in the dark, 1000 Centrifuge at ⁇ g for 5 min, wash with PBS twice, add 500 ⁇ L PBS to resuspend to make a single cell suspension, and detect by flow cytometry.
  • miR-328-3p NC mimic was transfected, and miR-328-3p was normally expressed
  • miR-328-3p mimic was transfected, and miR-328-3p was highly expressed.
  • the cells were transfected according to the instructions of Lipofectamine 2000 and the above groups, and the miR-328-3p mimic (mimic) and the negative control mimic (NC mimic) were respectively transfected into the UC-MSCs of the corresponding group, 48h After washing, the cells of each group were collected for subsequent experiments.
  • miR-328-3p mimic mimic
  • NC mimic negative control mimic
  • RT-PCR method was used to detect the expression level of miR-328-3p gene after transfection: the transfected cells were collected and the total RNA of each group was extracted with TRIzol kit, the RNA concentration and purity were measured, cDNA was synthesized by reverse transcription kit, PCR amplification Using U6 as the internal reference, the relative expression level of miR-328-3p in each group was calculated according to the formula miR-328-3p/U6 according to the operation method of the kit, and the relative expression level of miR-328-3p in the Blank group was calculated as 1.00.
  • the primers for miR-328-3p and U6 are as follows:
  • miR-328-3p upstream primer 5'-GATAGCCGTACTCTCGAGG-3';
  • miR-328-3p downstream primer 5'-GTAGAGGCTAGAGGGAACCC-3';
  • U6 upstream primer 5'-CCCGGACACGTGGGCTCCC-3';
  • U6 downstream primer 5'-CACATCCCTGGACACAGTCCTAG-3'.
  • Exosomes were collected by conventional centrifugation.
  • the specific method is as follows: take the transfected UC-MSCs and make a cell suspension with DMEM/F12 medium and inoculate them in a culture dish. After 72 hours, collect the supernatant and transfer it to a centrifuge tube, and centrifuge at 300 ⁇ g at 4°C. Remove residual cells for 10 minutes, centrifuge the supernatant at 2000 ⁇ g for 10 minutes at 4°C to remove dead cells, centrifuge the supernatant at 10,000 ⁇ g for 30 minutes at 4°C to remove cell debris, and collect the supernatant at 100,000 ⁇ g for 70 minutes at 4°C Precipitate to obtain exosomes.
  • the pellet was resuspended in sterile PBS, and then centrifuged at 100,000 ⁇ g for 70 min at 4°C to collect the pellet to obtain washed exosomes. Finally, the washed exosomes were resuspended in sterile PBS, and a small amount was taken for the determination of exosome concentration by BCA method, transmission electron microscope observation and Western blot detection, and the rest were frozen at -80°C for later use.
  • the Western blot detection method is as follows: after taking an appropriate amount of exosomes in each group and adjusting the protein concentration to be consistent, add 5 ⁇ loading buffer, boil at 100°C for 10 minutes, transfer to PVDF membrane after 10% SDS-polyacrylamide gel electrophoresis, 5% skimmed milk powder was blocked at room temperature for 2 hours, CD9, CD63, and CD81 primary antibodies were added, and incubated overnight at 4°C. After washing the membrane repeatedly with TBST, the HRP-labeled secondary antibody was incubated for 1 h. After washing the membrane, the color was developed with chemiluminescence reagent. The gel imaging system was used to take pictures, and the gray value was analyzed by Image J software.
  • a in Figure 1 is an inverted microscope observation of UC-MSCs. It can be seen that the cells are spindle-shaped, relatively uniform in size, and grow in parallel or spiral, which is consistent with the biological characteristics of UC-MSCs.
  • B in Figure 1 is the flow cytometry detection image, CD73, CD90, and CD105 are positively expressed, and CD14, CD34, and CD45 are negatively expressed, which is consistent with the characteristics of UC-MSCs surface markers.
  • C-1 in Table 1 and Figure 1 is the comparison of miR-328-3p content in UC-MSCs in each group, and the miR-328-3p content in PG group was significantly higher than that in Blank group and NC group, indicating high expression of miR-328-3p
  • the UC-MSCs model was successfully established.
  • D-1 in Figure 1 is the transmission electron microscope observation image (100 ⁇ ) of UC-MSCs-Exo in each group
  • E-1 in Figure 1 is the Western blot detection image of UC-MSCs-Exo marker proteins CD9, CD63, and CD81 in each group , the morphology and markers of exosomes in each group were consistent with the characteristics of UC-MSCs-Exo, indicating that exosomes were prepared in each group.
  • Embodiment 2 anti-skin aging test
  • Human dermal fibroblasts (HDF-a) were purchased from Shenzhen Haodi Huatuo Biotechnology Co., Ltd. Fetal calf serum and DMEM high-glucose medium were purchased from Gibco, and double antibodies, MTT and trypsin were purchased from Beyontib. Rabbit anti-human type I collagen antibody and rabbit anti-human elastin antibody were purchased from Abcam, and donkey anti-rabbit fluorescent secondary antibody was purchased from Shanghai Yaji Biotechnology Co., Ltd.
  • HDF-a was cultured in DMEM high glucose containing 10% fetal bovine serum and 1% double antibody based on the conditions of 37°C, 5% CO 2 , and saturated humidity, and the medium was changed every 2-3 days.
  • the proliferative activity of HDF-a was determined by the conventional MTT method, and divided into blank control group and exosome A, B, and C groups, wherein the exosomes added in exosome A, B, and C groups corresponded to Example 1. Exosomes in the Blank group, NC group, and PG group.
  • the specific method is as follows: Take HDF-a cells in the logarithmic growth phase in good growth state, adjust the cell concentration to 1 ⁇ 10 5 cells/mL, mix them and inoculate them in a 96-well plate, 100 ⁇ L per well, and set 6 replicates in each group. hole. After the cells were completely adhered to the wall, the experimental group was replaced with complete medium containing different concentrations of exosomes (low concentration of 2 ⁇ g/mL, high concentration of 5 ⁇ g/mL), and the culture medium of the blank control group did not contain exosomes. .
  • the exosome A, B, and C groups were cultured with complete medium containing 5 ⁇ g/mL different exosomes, and the culture medium of the blank control group contained no Contains exosomes.
  • HDF-a cells were collected and washed 3 to 5 times with PBS, and cultured in DMEM high-glucose medium without fetal bovine serum at a cell concentration of 1 ⁇ 10 5 cells/mL.
  • the purpose of culturing in a medium that does not contain fetal bovine serum is to reduce the impact of proteins in fetal bovine serum on the detection of target proteins.
  • the cell culture supernatant was collected, the total protein was extracted according to the instructions of the BCA kit and the protein concentration was calculated, and the protein concentration of the experimental group was adjusted based on the protein concentration of the blank control group.
  • A is the comparison of the proliferation activity of HDF-a in each group.
  • the HDF-a proliferation activity of exosome A, B, and C groups was significantly higher than that of the blank control group, and the HDF-a proliferation activity of exosome C group Significantly higher than exosomes A and B groups, exosomes A group and B group HDF-a proliferation activity was not significantly different.
  • B in Fig. 2 is the comparison of COL I and Elastin contents in each group.
  • the contents of COL I and Elastin in exosome A, B, and C groups were significantly higher than those in the blank control group, and the contents of COL I and Elastin in exosome C group were significantly higher than those in exosome A and B groups, and exosome A group There was no significant difference in the content of COL I and Elastin compared with group B.
  • Decreased proliferative activity of dermal fibroblasts leads to skin renewal and decreased metabolic capacity, which is one of the important manifestations of skin aging.
  • the degradation of the extracellular matrix is another important manifestation of skin aging.
  • Collagen mainly type I collagen, is the most important structural protein in the extracellular matrix, which plays a key role in maintaining the mechanical strength of the skin;
  • An important protein, elastin plays a key role in maintaining skin elasticity and preventing deformation. Therefore, improving the proliferative activity of dermal fibroblasts and inhibiting the degradation of extracellular matrix by increasing the content of collagen and elastin in the extracellular matrix can effectively inhibit skin aging.
  • exosomes secreted by human umbilical cord mesenchymal stem cells with upregulated miR-328-3p expression could improve the proliferation of human dermal fibroblasts on the one hand.
  • it can increase the content of type I collagen and elastin in the extracellular matrix of human dermal fibroblasts, and has the prospect of being developed into anti-aging skin care products or medicines.
  • Fetal bovine serum and DMEM/F12 medium were purchased from Gibco, and double antibodies, PBS and trypsin were purchased from Beyontib. miR-485-5p mimic and miR-485-5p NC mimic were synthesized and provided by Shanghai Gemma Biotechnology Co., Ltd.
  • FITC or PE-labeled mouse anti-human CD90, CD105, CD73, CD14, CD34 and CD45 antibodies and related isotype controls were purchased from eBioscince, USA.
  • Lipofectamine 2000 was purchased from Invitrogen.
  • TRIzol kit was purchased from Thermo Fisher. Reagent materials related to Western blot were purchased from Biyuntian Biology, etc.
  • the umbilical cord tissues of healthy full-term neonates were collected under aseptic conditions according to conventional methods, washed several times with PBS containing 1% double antibody to remove residual blood, and the umbilical arteries and veins were removed, leaving Wharton glue-like tissues, which were cut Crush to form small pieces of about 1 mm 3 , then culture in DMEM/F12 containing 10% fetal bovine serum and 1% double antibody at 37°C, 5% CO 2 , and saturated humidity, and change the medium every 3-4 days . Cell morphology and growth were observed under an inverted microscope. When the cells grew to 90% confluent, trypsinized and subcultured. The third generation UC-MSCs were used to detect surface markers by flow cytometry.
  • the method of flow cytometry is as follows: take the third-generation UC-MSCs, digest with 0.25% trypsin and adjust the cell density to 1 ⁇ 106 cells/mL, add 1 mL of cell suspension to each centrifuge tube, wash twice with sterile PBS, 1000 Centrifuge at ⁇ g for 5 minutes, discard the supernatant, resuspend the cells in 100 ⁇ L PBS, add appropriate amount of CD90, CD105, CD73, CD14, CD34 and CD45 antibodies and each isotype control to each tube of single cell suspension, incubate at room temperature for 30 minutes in the dark, 1000 Centrifuge at ⁇ g for 5 min, wash with PBS twice, add 500 ⁇ L PBS to resuspend to make a single cell suspension, and detect by flow cytometry.
  • miR-485-5p NC mimic was transfected, and miR-485-5p was normally expressed
  • miR-485-5p mimic was transfected, and miR-485-5p was highly expressed.
  • the cells were transfected according to the instructions of Lipofectamine 2000 and the above groups, and the miR-485-5p mimic (mimic) and the negative control mimic (NC mimic) were respectively transfected into the UC-MSCs of the corresponding group, 48h After washing, the cells of each group were collected for subsequent experiments.
  • miR-485-5p mimic mimic
  • NC mimic negative control mimic
  • RT-PCR method was used to detect the expression level of miR-485-5p gene after transfection: the transfected cells were collected and the total RNA of each group was extracted with TRIzol kit, the RNA concentration and purity were measured, cDNA was synthesized with reverse transcription kit, and PCR amplification was carried out. Using U6 as the internal reference, the relative expression level of miR-485-5p in each group was calculated according to the formula miR-485-5p/U6 according to the operation method of the kit, and the relative expression level of miR-485-5p in the Blank group was calculated as 1.00.
  • the primers for miR-485-5p and U6 are as follows:
  • miR-485-5p upstream primer 5'-ACACTCCAGCTGGGAGAGGCTGGCCGTGATGAATTC-3';
  • miR-485-5p downstream primer 5'-CTCGATTCGTCACTCACA-3';
  • U6 upstream primer 5'-CCCGGACACGTGGGCTCCC-3';
  • U6 downstream primer 5'-CACATCCCTGGACACAGTCCTAG-3'.
  • Exosomes were collected by conventional centrifugation.
  • the specific method is as follows: take the transfected UC-MSCs and make a cell suspension with DMEM/F12 medium and inoculate them in a culture dish. After 72 hours, collect the supernatant and transfer it to a centrifuge tube, and centrifuge at 300 ⁇ g at 4°C. Remove residual cells for 10 minutes, centrifuge the supernatant at 2000 ⁇ g for 10 minutes at 4°C to remove dead cells, centrifuge the supernatant at 10,000 ⁇ g for 30 minutes at 4°C to remove cell debris, and collect the supernatant at 100,000 ⁇ g for 70 minutes at 4°C Precipitate to obtain exosomes.
  • the pellet was resuspended in sterile PBS, and then centrifuged at 100,000 ⁇ g for 70 min at 4°C to collect the pellet to obtain washed exosomes. Finally, the washed exosomes were resuspended in sterile PBS, and a small amount was taken for BCA method to determine exosome concentration, transmission electron microscope observation and Western Blot to detect exosome marker proteins, and the rest were frozen at -80°C for later use.
  • the Western blot detection method is as follows: after taking an appropriate amount of exosomes in each group and adjusting the protein concentration to be consistent, add 5 ⁇ loading buffer, boil at 100°C for 10 minutes, transfer to PVDF membrane after 10% SDS-polyacrylamide gel electrophoresis, 5% skimmed milk powder was blocked at room temperature for 2 hours, CD9, CD63, and CD81 primary antibodies were added, and incubated overnight at 4°C. After washing the membrane repeatedly with TBST, the HRP-labeled secondary antibody was incubated for 1 h. After washing the membrane, the color was developed with chemiluminescence reagent. The gel imaging system was used to take pictures, and the gray value was analyzed by Image J software.
  • C-2 in Table 3 and Figure 1 is the comparison of miR-485-5p content in UC-MSCs of each group, and the miR-485-5p content in PG group was significantly higher than that in Blank group and NC group, indicating high expression of miR-485-5p
  • the UC-MSCs model was successfully established.
  • D-2 in Figure 1 is the transmission electron microscope observation image (100 ⁇ ) of UC-MSCs-Exo in each group
  • E-2 in Figure 1 is the Western Blot detection image of UC-MSCs-Exo marker proteins CD9, CD63, and CD81 in each group , the morphology and markers of exosomes in each group were consistent with the characteristics of UC-MSCs-Exo, indicating that exosomes were prepared in each group.
  • HaCaT cells Human immortalized epidermal cells
  • Fetal bovine serum and DMEM medium were purchased from Gibco, and double antibodies, MTT and trypsin were purchased from Beyontib.
  • HaCaT cells were cultured in DMEM containing 10% fetal bovine serum and 1% bis-antibody at 37°C, 5% CO 2 , and saturated humidity, and the medium was changed every 2-3 days.
  • the proliferative activity of HaCaT cells was determined by conventional MTT method, and divided into blank control group and exosome A, B, and C groups.
  • the exosomes added in exosome A, B, and C groups corresponded to Blank in Example 1.
  • the specific method is as follows: Take HaCaT cells in the logarithmic growth phase in good growth state, adjust the cell concentration to 1 ⁇ 10 5 cells/mL, mix them and inoculate them in a 96-well plate, 100 ⁇ L per well, and set 6 replicate wells for each group. After the cells were completely adhered to the wall, the experimental group was replaced with complete medium containing different concentrations of exosomes (low concentration of 2 ⁇ g/mL, high concentration of 5 ⁇ g/mL), and the culture medium of the blank control group did not contain exosomes. .
  • the migration activity of HaCaT cells was determined by the conventional scratch method, and divided into blank control group and exosome A, B, C groups, and the exosomes added in exosome A, B, and C groups corresponded to those in Example 1. Exosomes in Blank group, NC group, and PG group.
  • the specific method is as follows: take HaCaT cells in the logarithmic growth phase in a good growth state, adjust the cell concentration to 4 ⁇ 10 5 cells/mL, and inoculate them in a culture dish. After the cells were completely adhered to the wall, the experimental group was replaced with a complete medium containing 5 ⁇ g/mL exosomes, and the culture medium of the blank control group did not contain exosomes. After continuing to culture for 24 h, the digested cells were washed 3 times with PBS, the cell concentration was adjusted to 2 ⁇ 10 5 cells/mL with complete medium, and seeded in a 12-well plate.
  • A is the comparison of the proliferation activity of HaCaT in each group.
  • the proliferation activity of HaCaT in exosome A, B, and C groups was significantly higher than that of the blank control group, and the proliferation activity of HaCaT in exosome C group was significantly higher than that of exosome Groups A and B, there was no significant difference in the proliferation activity of HaCaT between group A and group B of exosomes.
  • Figure 3 B is the comparison of the migration activity of HaCaT in each group.
  • the HaCaT migration activity of exosome A, B, and C groups was significantly higher than that of the blank control group, and the HaCaT migration activity of exosome C group was significantly higher than that of exosomes.
  • the epidermal cell layer is the most important cell layer of the skin barrier, and the proliferation and migration activities of epidermal cells are directly related to the repair ability of the damaged skin barrier.
  • Up-regulation or down-regulation to change the expression level of some genes is an important research method to regulate and improve cell function.
  • the above experiments showed that, compared with exosomes secreted by normal human umbilical cord mesenchymal stem cells, exosomes secreted by human umbilical cord mesenchymal stem cells with upregulated expression of miR-485-5p could more effectively improve human immortalized epidermis on the one hand.
  • the proliferative activity of HaCaT cells on the other hand, can more effectively improve the migration activity of HaCaT cells, and has the prospect of being developed into skin care products or medicines that promote skin barrier repair.

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Abstract

Disclosed in the present invention are exosomes of an umbilical cord mesenchymal stem cells, and the use thereof. The present invention has found that exosomes secreted by human umbilical cord mesenchymal stem cells with the up-regulated expression of miR-328-3p or miR-485-5p have a better anti-aging effect on skin or promoted skin barrier repair activity, and have the prospect of being developed into an anti-aging skin product or a product for promoting skin barrier repair.

Description

脐带间充质干细胞外泌体及其应用Umbilical cord mesenchymal stem cell exosomes and its application 技术领域technical field
本发明属于干细胞领域，具体涉及脐带间充质干细胞外泌体及其应用。The invention belongs to the field of stem cells, in particular to umbilical cord mesenchymal stem cell exosomes and applications thereof.
背景技术Background technique
间充质干细胞(mesenchymal stem cells，MSCs)是一种具有高度自我更新、多向分化及独特免疫调节能力特性的细胞，其可以通过与先天免疫和适应性免疫系统的细胞相互作用以及分泌细胞调节性因子发挥免疫调节作用。间充质干细胞外泌体(exosomes from mesenchymal stem cells，MSCs-Exo)就是MSCs发挥功能的一种分泌物质。研究表明，MSCs-Exo具有多种活性，比如组织修复、免疫调节和疾病治疗，越来越被重视。Mesenchymal stem cells (mesenchymal stem cells, MSCs) are a kind of cells with high self-renewal, multi-lineage differentiation and unique immune regulation ability, which can regulate the immune system by interacting with cells of innate and adaptive immune system and secreting cells. Sex factors play an immunomodulatory role. Mesenchymal stem cell exosomes (exosomes from mesenchymal stem cells, MSCs-Exo) are a secreted substance that MSCs function. Studies have shown that MSCs-Exo possess multiple activities, such as tissue repair, immune regulation, and disease treatment, which have been paid more and more attention.
脐带间充质干细胞(umbilical cord-mesenchymal stem cells，UC-MSCs)具有来源充足、取材方便、培养简单等特点，也不存在伦理学问题，因而得到广泛研究。脐带间充质干细胞外泌体(UC-MSCs-Exo)也较其他干细胞外泌体更易被应用和推广。Umbilical cord-mesenchymal stem cells (UC-MSCs) have the characteristics of sufficient sources, convenient material acquisition, simple culture, and no ethical issues, so they have been extensively studied. Umbilical cord mesenchymal stem cell exosomes (UC-MSCs-Exo) are also easier to be applied and promoted than other stem cell exosomes.
皮肤抗衰老医学美容研究是一项长期而艰巨的课题。随着不断加深的老龄化问题，人类对年轻健康外貌的强烈需求，未来对于抗衰老的需求将会越来越大，研究有效的抗衰老技术和抗衰老产品，有助于提高人类的生活质量，尤其对老龄化的严重问题具有重要意义。Skin anti-aging medical cosmetology research is a long-term and arduous subject. With the deepening of the aging problem, human beings have a strong demand for a young and healthy appearance, and the demand for anti-aging will increase in the future. Research on effective anti-aging technology and anti-aging products will help improve the quality of human life , especially for the serious problem of aging.
皮肤屏障功能是皮肤最重要的功能之一，在保护机体免受环境伤害、防止微生物入侵、调节体温、避免体内水分流失、维持内环境相对稳定等方面发挥重要的作用。The skin barrier function is one of the most important functions of the skin. It plays an important role in protecting the body from environmental damage, preventing microbial invasion, regulating body temperature, avoiding water loss in the body, and maintaining a relatively stable internal environment.
现有技术已经表明，UC-MSCs-Exo具有抗皮肤衰老的活性，但是活性尚待提高，这样才能克服UC-MSCs-Exo量少的不足，实现少量高效，易于被应用和推广。The existing technology has shown that UC-MSCs-Exo has anti-aging activity of the skin, but the activity needs to be improved, so as to overcome the shortage of UC-MSCs-Exo in a small amount, achieve high efficiency in a small amount, and be easy to be applied and promoted.
发明内容Contents of the invention
本发明旨在克服现有技术不足，提供一种脐带间充质干细胞外泌体及其应用。The present invention aims to overcome the deficiencies of the prior art, and provides an umbilical cord mesenchymal stem cell exosome and its application.
本发明目的通过如下技术方案实现：The object of the invention is achieved through the following technical solutions:
一种脐带间充质干细胞外泌体，该脐带间充质干细胞外泌体为miR-328-3p或miR-485-5p表达上调的人脐带间充质干细胞分泌的外泌体。An umbilical cord mesenchymal stem cell exosome, the umbilical cord mesenchymal stem cell exosome is an exosome secreted by a human umbilical cord mesenchymal stem cell whose expression of miR-328-3p or miR-485-5p is upregulated.
上述miR-328-3p表达上调的脐带间充质干细胞外泌体的抗皮肤衰老用途。Anti-aging application of exosomes from umbilical cord mesenchymal stem cells with upregulated miR-328-3p expression.
上述miR-485-5p表达上调的脐带间充质干细胞外泌体的促进皮肤屏障修复用途。The use of exosomes from umbilical cord mesenchymal stem cells with upregulated miR-485-5p expression to promote skin barrier repair.
技术效果：Technical effect:
本发明发现，与正常人脐带间充质干细胞分泌的外泌体相比，miR-328-3p表达上调的人脐带间充质干细胞分泌的外泌体一方面可以更有效地提高人真皮成纤维细胞的增殖活性，另一方面可以更有效地提高人真皮成纤维细胞的细胞外基质中Ⅰ型胶原蛋白和弹性蛋白的含量， 具有开发成抗皮肤衰老的护肤品或药品的前景。本发明还发现，与正常人脐带间充质干细胞分泌的外泌体相比，miR-485-5p表达上调的人脐带间充质干细胞分泌的外泌体一方面可以更有效地提高人类永生化表皮细胞HaCaT细胞的增殖活性，另一方面可以更有效地提高HaCaT细胞的迁移活性，具有开发成促进皮肤屏障修复的护肤品或药品的前景。The present invention found that, compared with the exosomes secreted by normal human umbilical cord mesenchymal stem cells, the exosomes secreted by human umbilical cord mesenchymal stem cells with up-regulated miR-328-3p expression can more effectively improve human dermal fibroblasts on the one hand. On the other hand, it can more effectively increase the content of type I collagen and elastin in the extracellular matrix of human dermal fibroblasts, and has the prospect of being developed into anti-aging skin care products or medicines. The present invention also found that compared with the exosomes secreted by normal human umbilical cord mesenchymal stem cells, the exosomes secreted by human umbilical cord mesenchymal stem cells with up-regulated miR-485-5p expression can more effectively improve human immortalization. The proliferative activity of epidermal HaCaT cells, on the other hand, can more effectively improve the migration activity of HaCaT cells, and has the prospect of being developed into skin care products or medicines that promote skin barrier repair.
附图说明Description of drawings
图1中A为UC-MSCs的倒置显微镜观察图，B为UC-MSCs的流式细胞仪检测图，C-1为各组UC-MSCs中miR-328-3p的含量比较，D-1为各组UC-MSCs-Exo的透射电镜观察图(miR-328-3p相关)，E-1为各组UC-MSCs-Exo的Western Blot检测结果(miR-328-3p相关)，C-2为各组UC-MSCs中miR-485-5p的含量比较，D-2为各组UC-MSCs-Exo的透射电镜观察图(miR-485-5p相关)，E-2为各组UC-MSCs-Exo的Western blot检测结果(miR-485-5p相关)。In Figure 1, A is the observation image of UC-MSCs with an inverted microscope, B is the image of flow cytometry detection of UC-MSCs, C-1 is the comparison of miR-328-3p content in UC-MSCs of each group, and D-1 is Transmission electron microscope observation images of UC-MSCs-Exo in each group (correlated with miR-328-3p), E-1 is the Western Blot detection results of UC-MSCs-Exo in each group (correlated with miR-328-3p), C-2 is The content comparison of miR-485-5p in UC-MSCs of each group, D-2 is the transmission electron microscope observation image of UC-MSCs-Exo in each group (related to miR-485-5p), E-2 is the UC-MSCs-Exo of each group Western blot detection results of Exo (related to miR-485-5p).
图2中A为各组HDF-a的增殖活性比较，B为COL I、Elastin含量比较。In Figure 2, A is the comparison of proliferation activity of HDF-a in each group, and B is the comparison of COL I and Elastin contents.
图3中A为各组HaCaT细胞的增殖活性比较，B为各组HaCaT细胞的迁移活性比较。In Fig. 3, A is a comparison of the proliferation activity of HaCaT cells in each group, and B is a comparison of the migration activity of HaCaT cells in each group.
具体实施方式Detailed ways
下面结合实施例具体介绍本发明实质性内容，但本领域技术人员应当知道，不应将本发明的保护范围局限于该具体实施例。The substantive content of the present invention will be specifically described below in conjunction with the embodiments, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.
实施例1：UC-MSCs-Exo的制备(miR-328-3p相关)Example 1: Preparation of UC-MSCs-Exo (related to miR-328-3p)
一、实验材料1. Experimental materials
胎牛血清、DMEM/F12培养基购自Gibco公司，双抗、PBS和胰蛋白酶购自碧云天生物。miR-328-3p mimic、miR-328-3p NC mimic购自上海生工生物工程有限公司。FITC或PE标记的小鼠抗人CD90、CD105、CD73、CD14、CD34和CD45抗体及各相关同型对照购自美国eBioscince公司。Lipofectamine 2000购自Invitrogen。TRIzol试剂盒购自赛默飞世尔。Western blot相关试剂材料购自碧云天生物等。Fetal bovine serum and DMEM/F12 medium were purchased from Gibco, and double antibodies, PBS and trypsin were purchased from Beyontib. miR-328-3p mimic and miR-328-3p NC mimic were purchased from Shanghai Sangon Bioengineering Co., Ltd. FITC or PE-labeled mouse anti-human CD90, CD105, CD73, CD14, CD34 and CD45 antibodies and related isotype controls were purchased from eBioscince, USA. Lipofectamine 2000 was purchased from Invitrogen. TRIzol kit was purchased from Thermo Fisher. Reagent materials related to Western blot were purchased from Biyuntian Biology, etc.
二、实验方法2. Experimental method
1、UC-MSCs分离培养1. Isolation and culture of UC-MSCs
按照常规方法在无菌条件下取健康足月顺产新生儿脐带组织，用含1％双抗的PBS洗涤多次以去除残血，并剔除脐动静脉，留下Wharton胶样组织，将其剪碎，形成约1mm 3的小块，然后用含10％胎牛血清和1％双抗的DMEM/F12培养基于37℃、5％CO 2、饱和湿度条件培养，每3～4d换液1次。在倒置显微镜下观察细胞的形态和生长情况。待细胞长至90％融合时，胰蛋白酶消化传代培养。取第3代UC-MSCs用于流式检测表面标志物。流式检测方法为：取第3代UC-MSCs，0.25％胰酶消化并调整细胞密度为1×10 6个/mL，每离心管中加 入1mL细胞悬液，无菌PBS洗涤2次，1000×g离心5min，弃上清，100μL PBS重悬细胞后，向各管单细胞悬液中加入适量CD90、CD105、CD73、CD14、CD34和CD45抗体以及各同型对照，室温避光孵育30min，1000×g离心5min，PBS洗涤2次，加入500μL PBS重悬，制成单细胞悬液，流式细胞仪检测。 The umbilical cord tissues of healthy full-term neonates were collected under aseptic conditions according to conventional methods, washed several times with PBS containing 1% double antibody to remove residual blood, and the umbilical arteries and veins were removed, leaving Wharton glue-like tissues, which were cut Crush to form small pieces of about 1 mm 3 , then culture in DMEM/F12 containing 10% fetal bovine serum and 1% double antibody at 37°C, 5% CO 2 , and saturated humidity, and change the medium every 3-4 days . Cell morphology and growth were observed under an inverted microscope. When the cells grew to 90% confluent, trypsinized and subcultured. The third generation UC-MSCs were used to detect surface markers by flow cytometry. The method of flow cytometry is as follows: take the third-generation UC-MSCs, digest with 0.25% trypsin and adjust the cell density to 1× 106 cells/mL, add 1 mL of cell suspension to each centrifuge tube, wash twice with sterile PBS, 1000 Centrifuge at ×g for 5 minutes, discard the supernatant, resuspend the cells in 100 μL PBS, add appropriate amount of CD90, CD105, CD73, CD14, CD34 and CD45 antibodies and each isotype control to each tube of single cell suspension, incubate at room temperature for 30 minutes in the dark, 1000 Centrifuge at ×g for 5 min, wash with PBS twice, add 500 μL PBS to resuspend to make a single cell suspension, and detect by flow cytometry.
2、UC-MSCs分组、转染和检测2. UC-MSCs grouping, transfection and detection
实验分为空白对照组(Blank)、阴性对照组(Negative Control，NC)和阳性实验组(Positive group，PG)。取生长状态良好的UC-MSCs，用含10％胎牛血清和1％双抗的DMEM/F12培养基于37℃、5％CO 2、饱和湿度条件培养，接种于6孔板中，并分组如下： The experiment was divided into blank control group (Blank), negative control group (Negative Control, NC) and positive experimental group (Positive group, PG). Take UC-MSCs in good growth state, culture them with DMEM/F12 containing 10% fetal bovine serum and 1% double antibody based on 37°C, 5% CO 2 , and saturated humidity conditions, inoculate in 6-well plates, and group as follows :
Blank组：未转染；Blank group: not transfected;
NC组：转染miR-328-3p NC mimic，miR-328-3p正常表达；NC group: miR-328-3p NC mimic was transfected, and miR-328-3p was normally expressed;
PG组：转染miR-328-3p mimic，miR-328-3p高表达。PG group: miR-328-3p mimic was transfected, and miR-328-3p was highly expressed.
24h后，按Lipofectamine 2000说明和上述分组进行细胞转染，将miR-328-3p的模拟物(mimic)及阴性对照模拟物(NC mimic)分别转染至对应组别的UC-MSCs中，48h后洗涤收集各组细胞进行后续实验。After 24 hours, the cells were transfected according to the instructions of Lipofectamine 2000 and the above groups, and the miR-328-3p mimic (mimic) and the negative control mimic (NC mimic) were respectively transfected into the UC-MSCs of the corresponding group, 48h After washing, the cells of each group were collected for subsequent experiments.
RT-PCR法检测转染后miR-328-3p基因表达水平：收集转染后的细胞用TRIzol试剂盒提取各组细胞总RNA，测定RNA浓度和纯度，反转录试剂盒合成cDNA，PCR扩增，以U6为内参，按照试剂盒操作方法根据公式miR-328-3p/U6计算各组miR-328-3p相对表达水平，并以Blank组miR-328-3p相对表达水平计为1.00。RT-PCR method was used to detect the expression level of miR-328-3p gene after transfection: the transfected cells were collected and the total RNA of each group was extracted with TRIzol kit, the RNA concentration and purity were measured, cDNA was synthesized by reverse transcription kit, PCR amplification Using U6 as the internal reference, the relative expression level of miR-328-3p in each group was calculated according to the formula miR-328-3p/U6 according to the operation method of the kit, and the relative expression level of miR-328-3p in the Blank group was calculated as 1.00.
miR-328-3p和U6的引物如下：The primers for miR-328-3p and U6 are as follows:
miR-328-3p上游引物：5'-GATAGCCGTACTCTCGAGG-3'；miR-328-3p upstream primer: 5'-GATAGCCGTACTCTCGAGG-3';
miR-328-3p下游引物：5'-GTAGAGGCTAGAGGGAACCC-3'；miR-328-3p downstream primer: 5'-GTAGAGGCTAGAGGGAACCC-3';
U6上游引物：5'-CCCGGACACGTGGGCTCCC-3'；U6 upstream primer: 5'-CCCGGACACGTGGGCTCCC-3';
U6下游引物：5'-CACATCCCTGGACACAGTCCTAG-3'。U6 downstream primer: 5'-CACATCCCTGGACACAGTCCTAG-3'.
3、UC-MSCs-Exo收集和检测3. Collection and detection of UC-MSCs-Exo
按照常规离心法收集外泌体。具体方法为：取转染后的UC-MSCs用DMEM/F12培养基制成细胞悬液并接种于培养皿中，72h后收集上清并转移至离心管中，4℃条件下300×g离心10min去除残余细胞，上清于4℃条件下2000×g离心10min去除死细胞，上清于4℃条件下10000×g离心30min去除细胞碎片，上清于4℃条件下100000×g离心70min收集沉淀即得外泌体。用无菌PBS将沉淀重悬，再次于4℃条件下100000×g离心70min收集沉淀即得经过洗涤的外泌体。最后用无菌PBS将经过洗涤的外泌体重悬，取少量用于BCA法测定外泌体浓度、透射电镜观察以及Westernblot检测，其余-80℃冻存备用。Exosomes were collected by conventional centrifugation. The specific method is as follows: take the transfected UC-MSCs and make a cell suspension with DMEM/F12 medium and inoculate them in a culture dish. After 72 hours, collect the supernatant and transfer it to a centrifuge tube, and centrifuge at 300×g at 4°C. Remove residual cells for 10 minutes, centrifuge the supernatant at 2000×g for 10 minutes at 4°C to remove dead cells, centrifuge the supernatant at 10,000×g for 30 minutes at 4°C to remove cell debris, and collect the supernatant at 100,000×g for 70 minutes at 4°C Precipitate to obtain exosomes. The pellet was resuspended in sterile PBS, and then centrifuged at 100,000×g for 70 min at 4°C to collect the pellet to obtain washed exosomes. Finally, the washed exosomes were resuspended in sterile PBS, and a small amount was taken for the determination of exosome concentration by BCA method, transmission electron microscope observation and Western blot detection, and the rest were frozen at -80°C for later use.
Western blot检测方法为：各组外泌体取适量并调整蛋白浓度一致后，加入5×上样缓冲液，100℃煮沸10min，10％SDS-聚丙烯酰胺凝胶电泳后转移至PVDF膜上，5％脱脂奶粉室温封闭2h，加入CD9、CD63、CD81一抗，4℃孵育过夜。TBST反复洗膜后，HRP标记二抗孵育1h，洗膜后化学发光试剂显色，用凝胶成像系统拍照，Image J软件分析灰度值。The Western blot detection method is as follows: after taking an appropriate amount of exosomes in each group and adjusting the protein concentration to be consistent, add 5× loading buffer, boil at 100°C for 10 minutes, transfer to PVDF membrane after 10% SDS-polyacrylamide gel electrophoresis, 5% skimmed milk powder was blocked at room temperature for 2 hours, CD9, CD63, and CD81 primary antibodies were added, and incubated overnight at 4°C. After washing the membrane repeatedly with TBST, the HRP-labeled secondary antibody was incubated for 1 h. After washing the membrane, the color was developed with chemiluminescence reagent. The gel imaging system was used to take pictures, and the gray value was analyzed by Image J software.
4、统计学分析4. Statistical analysis
采用GraphPad Prism 5软件对数据进行统计分析，采用配对t检验对数据进行显著性分析，P<0.05认定为差异显著。GraphPad Prism 5 software was used for statistical analysis of data, and paired t-test was used for significant analysis of data, and P<0.05 was considered significant difference.
三、实验结果3. Experimental results
1、UC-MSCs倒置显微镜下观察结果1. Observation results of UC-MSCs under an inverted microscope
图1中A为UC-MSCs的倒置显微镜观察图，可见细胞呈梭形，大小较均等，呈平行或旋涡状生长，符合UC-MSCs细胞生物学特点。A in Figure 1 is an inverted microscope observation of UC-MSCs. It can be seen that the cells are spindle-shaped, relatively uniform in size, and grow in parallel or spiral, which is consistent with the biological characteristics of UC-MSCs.
图1中B为流式细胞仪检测图，CD73、CD90、CD105阳性表达，CD14、CD34和CD45阴性表达，符合UC-MSCs表面标记特点。B in Figure 1 is the flow cytometry detection image, CD73, CD90, and CD105 are positively expressed, and CD14, CD34, and CD45 are negatively expressed, which is consistent with the characteristics of UC-MSCs surface markers.
2、UC-MSCs转染结果2. UC-MSCs transfection results
表1和图1中C-1为各组UC-MSCs中miR-328-3p的含量比较，PG组miR-328-3p含量显著高于Blank组、NC组，说明高表达miR-328-3p的UC-MSCs造模成功。C-1 in Table 1 and Figure 1 is the comparison of miR-328-3p content in UC-MSCs in each group, and the miR-328-3p content in PG group was significantly higher than that in Blank group and NC group, indicating high expression of miR-328-3p The UC-MSCs model was successfully established.
表1各组UC-MSCs中miR-328-3p相对表达水平Table 1 Relative expression levels of miR-328-3p in UC-MSCs of each group
Figure PCTCN2021138172-appb-000001
Figure PCTCN2021138172-appb-000001
*代表与Blank、NC组相比差异显著。*Represents significant difference compared with Blank and NC groups.
3、UC-MSCs-Exo检测结果3. UC-MSCs-Exo detection results
图1中D-1为各组UC-MSCs-Exo的透射电镜观察图(100×)，图1中E-1为各组UC-MSCs-Exo标志蛋白CD9、CD63、CD81的Western blot检测图，各组外泌体的形态和标志物都符合UC-MSCs-Exo的特点，说明各组均制备得到了外泌体。D-1 in Figure 1 is the transmission electron microscope observation image (100×) of UC-MSCs-Exo in each group, and E-1 in Figure 1 is the Western blot detection image of UC-MSCs-Exo marker proteins CD9, CD63, and CD81 in each group , the morphology and markers of exosomes in each group were consistent with the characteristics of UC-MSCs-Exo, indicating that exosomes were prepared in each group.
实施例2：抗皮肤衰老测试Embodiment 2: anti-skin aging test
一、实验材料1. Experimental materials
人真皮成纤维细胞(HDF-a)购自深圳市豪地华拓生物科技有限公司。胎牛血清、DMEM高糖培养基购自Gibco公司，双抗、MTT和胰蛋白酶购自碧云天生物。兔抗人Ⅰ型胶原蛋白抗体、兔抗人弹性蛋白抗体购自Abcam，驴抗兔荧光二抗购自上海雅吉生物科技有限公司。Human dermal fibroblasts (HDF-a) were purchased from Shenzhen Haodi Huatuo Biotechnology Co., Ltd. Fetal calf serum and DMEM high-glucose medium were purchased from Gibco, and double antibodies, MTT and trypsin were purchased from Beyontib. Rabbit anti-human type Ⅰ collagen antibody and rabbit anti-human elastin antibody were purchased from Abcam, and donkey anti-rabbit fluorescent secondary antibody was purchased from Shanghai Yaji Biotechnology Co., Ltd.
二、实验方法2. Experimental method
1、HDF-a的培养1. The cultivation of HDF-a
将HDF-a用含10％胎牛血清和1％双抗的DMEM高糖培养基于37℃、5％CO 2、饱和湿度条件培养，每2～3d换液1次。 HDF-a was cultured in DMEM high glucose containing 10% fetal bovine serum and 1% double antibody based on the conditions of 37°C, 5% CO 2 , and saturated humidity, and the medium was changed every 2-3 days.
2、HDF-a增殖活性测定2. Determination of HDF-a proliferation activity
采用常规的MTT法测定HDF-a的增殖活性，分为空白对照组和外泌体A、B、C组，其中外泌体A、B、C组添加的外泌体分别对应为实施例1中Blank组、NC组、PG组的外泌体。The proliferative activity of HDF-a was determined by the conventional MTT method, and divided into blank control group and exosome A, B, and C groups, wherein the exosomes added in exosome A, B, and C groups corresponded to Example 1. Exosomes in the Blank group, NC group, and PG group.
具体方法为：取生长状态良好的对数生长期HDF-a细胞，调整细胞浓度至1×10 5个/mL，混匀后接种于96孔板中，每孔100μL，每组设6个复孔。待细胞完全贴壁后，实验组更换为含有不同浓度外泌体(低浓度为2μg/mL、高浓度为5μg/mL)的完全培养基培养，空白对照组的培养基中不含有外泌体。继续培养24h后，每孔加入20μL浓度为5mg/mL的MTT溶液，培养4h，去上清液，加入150μL DMSO轻轻震荡，用酶标仪检测其在490nm处的吸光度(OD值)，通过OD值按照如下公式计算实验组增殖活性，空白对照组增殖活性计为100％：实验组增殖活性＝实验组OD值/空白对照组OD值×100％。吸光度越高代表增殖活性越强。 The specific method is as follows: Take HDF-a cells in the logarithmic growth phase in good growth state, adjust the cell concentration to 1×10 5 cells/mL, mix them and inoculate them in a 96-well plate, 100 μL per well, and set 6 replicates in each group. hole. After the cells were completely adhered to the wall, the experimental group was replaced with complete medium containing different concentrations of exosomes (low concentration of 2 μg/mL, high concentration of 5 μg/mL), and the culture medium of the blank control group did not contain exosomes. . After continuing to cultivate for 24 hours, add 20 μL of MTT solution with a concentration of 5 mg/mL to each well, cultivate for 4 hours, remove the supernatant, add 150 μL of DMSO to shake gently, and detect its absorbance (OD value) at 490 nm with a microplate reader. The OD value was calculated according to the following formula for the proliferative activity of the experimental group, and the proliferative activity of the blank control group was counted as 100%: proliferative activity of the experimental group=OD value of the experimental group/OD value of the blank control group×100%. The higher the absorbance, the stronger the proliferative activity.
3、Ⅰ型胶原蛋白(COL I)和弹性蛋白(Elastin)分泌能力测定3. Determination of the secretion capacity of type Ⅰ collagen (COL I) and elastin (Elastin)
按照“2、HDF-a增殖活性测定”中的分组和培养方法，外泌体A、B、C组采用含有5μg/mL不同外泌体的完全培养基培养，空白对照组的培养基中不含有外泌体。干预24h后，收集HDF-a细胞并用PBS洗涤3～5次，使用不含胎牛血清的DMEM高糖培养基培养，细胞浓度1×10 5个/mL。使用不含有胎牛血清的培养基培养是为了降低胎牛血清中蛋白对目标蛋白检测的影响。培养24h后，收集细胞培养上清液，按照BCA试剂盒说明书提取总蛋白并计算蛋白浓度，以空白对照组蛋白浓度为标准调整实验组蛋白浓度。调整蛋白浓度一致后，加入5×上样缓冲液，100℃煮沸10min，10％SDS-聚丙烯酰胺凝胶电泳后转移至PVDF膜上，5％脱脂奶粉室温封闭2h，加入COL I、Elastin、GAPDH一抗，4℃孵育过夜。TBST反复洗膜后，HRP标记二抗孵育1h，洗膜后化学发光试剂显色，用凝胶成像系统拍照，Image J软件分析灰度值。 According to the grouping and culture method in "2. Determination of HDF-a proliferation activity", the exosome A, B, and C groups were cultured with complete medium containing 5 μg/mL different exosomes, and the culture medium of the blank control group contained no Contains exosomes. After 24 hours of intervention, HDF-a cells were collected and washed 3 to 5 times with PBS, and cultured in DMEM high-glucose medium without fetal bovine serum at a cell concentration of 1×10 5 cells/mL. The purpose of culturing in a medium that does not contain fetal bovine serum is to reduce the impact of proteins in fetal bovine serum on the detection of target proteins. After culturing for 24 hours, the cell culture supernatant was collected, the total protein was extracted according to the instructions of the BCA kit and the protein concentration was calculated, and the protein concentration of the experimental group was adjusted based on the protein concentration of the blank control group. After adjusting the protein concentration to be consistent, add 5× loading buffer, boil at 100°C for 10 minutes, transfer to PVDF membrane after 10% SDS-polyacrylamide gel electrophoresis, block with 5% skimmed milk powder at room temperature for 2 hours, add COL I, Elastin, GAPDH primary antibody, incubated overnight at 4°C. After washing the membrane repeatedly with TBST, the HRP-labeled secondary antibody was incubated for 1 h. After washing the membrane, the color was developed with chemiluminescence reagent. The gel imaging system was used to take pictures, and the gray value was analyzed by Image J software.
4、统计学分析4. Statistical analysis
采用GraphPad Prism 5软件对数据进行统计分析，采用配对t检验对数据进行显著性分析，P<0.05认定为差异显著。GraphPad Prism 5 software was used for statistical analysis of data, and paired t-test was used for significant analysis of data, and P<0.05 was considered significant difference.
三、实验结果3. Experimental results
1、HDF-a增殖活性测定结果1. HDF-a proliferative activity assay results
图2中A为各组HDF-a的增殖活性比较，外泌体A、B、C组的HDF-a增殖活性均显著高于空白对照组，且外泌体C组的HDF-a增殖活性显著高于外泌体A、B组，外泌体A组与 B组HDF-a增殖活性无显著性差异。In Figure 2, A is the comparison of the proliferation activity of HDF-a in each group. The HDF-a proliferation activity of exosome A, B, and C groups was significantly higher than that of the blank control group, and the HDF-a proliferation activity of exosome C group Significantly higher than exosomes A and B groups, exosomes A group and B group HDF-a proliferation activity was not significantly different.
表2各组HDF-a的增殖活性Table 2 Proliferation activity of HDF-a in each group
Figure PCTCN2021138172-appb-000002
Figure PCTCN2021138172-appb-000002
#代表与空白对照组相比差异显著；*代表与外泌体A、B组相比差异显著。# represents significant difference compared with blank control group; * represents significant difference compared with exosomes A and B groups.
2、COL I、Elastin含量测定结果2. Determination results of COL I and Elastin content
图2中B为各组COL I、Elastin含量比较。外泌体A、B、C组COL I、Elastin含量均显著高于空白对照组，且外泌体C组的COL I、Elastin含量显著高于外泌体A、B组，外泌体A组与B组COL I、Elastin含量无显著性差异。B in Fig. 2 is the comparison of COL I and Elastin contents in each group. The contents of COL I and Elastin in exosome A, B, and C groups were significantly higher than those in the blank control group, and the contents of COL I and Elastin in exosome C group were significantly higher than those in exosome A and B groups, and exosome A group There was no significant difference in the content of COL I and Elastin compared with group B.
真皮成纤维细胞增殖活性的降低导致皮肤更新、代谢能力降低，是皮肤衰老的重要表现之一。细胞外基质的降解是皮肤衰老的另一重要表现，以Ⅰ型胶原蛋白为主的胶原蛋白是细胞外基质中最主要的结构蛋白，对维持皮肤的机械强度发挥关键作用；细胞外基质中另一种重要蛋白弹性蛋白在保持皮肤弹性、防止变形方面发挥关键作用。因此，提高真皮成纤维细胞的增殖活性、通过提高细胞外基质中胶原蛋白和弹性蛋白的含量抑制细胞外基质的降解可以有效抑制皮肤衰老。上述实验表明，与正常人脐带间充质干细胞分泌的外泌体相比，miR-328-3p表达上调的人脐带间充质干细胞分泌的外泌体一方面可以提高人真皮成纤维细胞的增殖活性，另一方面可以提高人真皮成纤维细胞的细胞外基质中Ⅰ型胶原蛋白和弹性蛋白的含量，具有开发成抗皮肤衰老的护肤品或药品的前景。Decreased proliferative activity of dermal fibroblasts leads to skin renewal and decreased metabolic capacity, which is one of the important manifestations of skin aging. The degradation of the extracellular matrix is another important manifestation of skin aging. Collagen, mainly type I collagen, is the most important structural protein in the extracellular matrix, which plays a key role in maintaining the mechanical strength of the skin; An important protein, elastin, plays a key role in maintaining skin elasticity and preventing deformation. Therefore, improving the proliferative activity of dermal fibroblasts and inhibiting the degradation of extracellular matrix by increasing the content of collagen and elastin in the extracellular matrix can effectively inhibit skin aging. The above experiments showed that, compared with the exosomes secreted by normal human umbilical cord mesenchymal stem cells, exosomes secreted by human umbilical cord mesenchymal stem cells with upregulated miR-328-3p expression could improve the proliferation of human dermal fibroblasts on the one hand. On the other hand, it can increase the content of type I collagen and elastin in the extracellular matrix of human dermal fibroblasts, and has the prospect of being developed into anti-aging skin care products or medicines.
实施例3：UC-MSCs-Exo的制备(miR-485-5p相关)Example 3: Preparation of UC-MSCs-Exo (related to miR-485-5p)
一、实验材料1. Experimental materials
胎牛血清、DMEM/F12培养基购自Gibco公司，双抗、PBS和胰蛋白酶购自碧云天生物。miR-485-5p mimic、miR-485-5p NC mimic由上海吉玛生物技术有限公司合成提供。FITC或PE标记的小鼠抗人CD90、CD105、CD73、CD14、CD34和CD45抗体及各相关同型对照购自美国eBioscince公司。Lipofectamine 2000购自Invitrogen。TRIzol试剂盒购自赛默飞世尔。Western blot相关试剂材料购自碧云天生物等。Fetal bovine serum and DMEM/F12 medium were purchased from Gibco, and double antibodies, PBS and trypsin were purchased from Beyontib. miR-485-5p mimic and miR-485-5p NC mimic were synthesized and provided by Shanghai Gemma Biotechnology Co., Ltd. FITC or PE-labeled mouse anti-human CD90, CD105, CD73, CD14, CD34 and CD45 antibodies and related isotype controls were purchased from eBioscince, USA. Lipofectamine 2000 was purchased from Invitrogen. TRIzol kit was purchased from Thermo Fisher. Reagent materials related to Western blot were purchased from Biyuntian Biology, etc.
二、实验方法2. Experimental method
1、UC-MSCs分离培养1. Isolation and culture of UC-MSCs
按照常规方法在无菌条件下取健康足月顺产新生儿脐带组织，用含1％双抗的PBS洗涤多次以去除残血，并剔除脐动静脉，留下Wharton胶样组织，将其剪碎，形成约1mm 3的小块，然后用含10％胎牛血清和1％双抗的DMEM/F12培养基于37℃、5％CO 2、饱和湿度条件培养，每3～4d换液1次。在倒置显微镜下观察细胞的形态和生长情况。待细胞长至90％融合时，胰蛋白酶消化传代培养。取第3代UC-MSCs用于流式检测表面标志物。流式检测方法为：取第3代UC-MSCs，0.25％胰酶消化并调整细胞密度为1×10 6个/mL，每离心管中加入1mL细胞悬液，无菌PBS洗涤2次，1000×g离心5min，弃上清，100μL PBS重悬细胞后，向各管单细胞悬液中加入适量CD90、CD105、CD73、CD14、CD34和CD45抗体以及各同型对照，室温避光孵育30min，1000×g离心5min，PBS洗涤2次，加入500μL PBS重悬，制成单细胞悬液，流式细胞仪检测。 The umbilical cord tissues of healthy full-term neonates were collected under aseptic conditions according to conventional methods, washed several times with PBS containing 1% double antibody to remove residual blood, and the umbilical arteries and veins were removed, leaving Wharton glue-like tissues, which were cut Crush to form small pieces of about 1 mm 3 , then culture in DMEM/F12 containing 10% fetal bovine serum and 1% double antibody at 37°C, 5% CO 2 , and saturated humidity, and change the medium every 3-4 days . Cell morphology and growth were observed under an inverted microscope. When the cells grew to 90% confluent, trypsinized and subcultured. The third generation UC-MSCs were used to detect surface markers by flow cytometry. The method of flow cytometry is as follows: take the third-generation UC-MSCs, digest with 0.25% trypsin and adjust the cell density to 1× 106 cells/mL, add 1 mL of cell suspension to each centrifuge tube, wash twice with sterile PBS, 1000 Centrifuge at ×g for 5 minutes, discard the supernatant, resuspend the cells in 100 μL PBS, add appropriate amount of CD90, CD105, CD73, CD14, CD34 and CD45 antibodies and each isotype control to each tube of single cell suspension, incubate at room temperature for 30 minutes in the dark, 1000 Centrifuge at ×g for 5 min, wash with PBS twice, add 500 μL PBS to resuspend to make a single cell suspension, and detect by flow cytometry.
2、UC-MSCs分组、转染和检测2. UC-MSCs grouping, transfection and detection
实验分为空白对照组(Blank)、阴性对照组(Negative Control，NC)和阳性实验组(Positive group，PG)。取生长状态良好的UC-MSCs，用含10％胎牛血清和1％双抗的DMEM/F12培养基于37℃、5％CO 2、饱和湿度条件培养，接种于6孔板中，并分组如下： The experiment was divided into blank control group (Blank), negative control group (Negative Control, NC) and positive experimental group (Positive group, PG). Take UC-MSCs in good growth state, culture them with DMEM/F12 containing 10% fetal bovine serum and 1% double antibody based on 37°C, 5% CO 2 , and saturated humidity conditions, inoculate in 6-well plates, and group as follows :
Blank组：未转染；Blank group: not transfected;
NC组：转染miR-485-5p NC mimic，miR-485-5p正常表达；NC group: miR-485-5p NC mimic was transfected, and miR-485-5p was normally expressed;
PG组：转染miR-485-5p mimic，miR-485-5p高表达。PG group: miR-485-5p mimic was transfected, and miR-485-5p was highly expressed.
24h后，按Lipofectamine 2000说明和上述分组进行细胞转染，将miR-485-5p的模拟物(mimic)及阴性对照模拟物(NC mimic)分别转染至对应组别的UC-MSCs中，48h后洗涤收集各组细胞进行后续实验。After 24 hours, the cells were transfected according to the instructions of Lipofectamine 2000 and the above groups, and the miR-485-5p mimic (mimic) and the negative control mimic (NC mimic) were respectively transfected into the UC-MSCs of the corresponding group, 48h After washing, the cells of each group were collected for subsequent experiments.
RT-PCR法检测转染后miR-485-5p基因表达水平：收集转染后的细胞用TRIzol试剂盒提取各组细胞总RNA，测定RNA浓度和纯度，反转录试剂盒合成cDNA，PCR扩增，以U6为内参，按照试剂盒操作方法根据公式miR-485-5p/U6计算各组miR-485-5p相对表达水平，并以Blank组miR-485-5p相对表达水平计为1.00。RT-PCR method was used to detect the expression level of miR-485-5p gene after transfection: the transfected cells were collected and the total RNA of each group was extracted with TRIzol kit, the RNA concentration and purity were measured, cDNA was synthesized with reverse transcription kit, and PCR amplification was carried out. Using U6 as the internal reference, the relative expression level of miR-485-5p in each group was calculated according to the formula miR-485-5p/U6 according to the operation method of the kit, and the relative expression level of miR-485-5p in the Blank group was calculated as 1.00.
miR-485-5p和U6的引物如下：The primers for miR-485-5p and U6 are as follows:
miR-485-5p上游引物：5'-ACACTCCAGCTGGGAGAGGCTGGCCGTGATGAATTC-3'；miR-485-5p upstream primer: 5'-ACACTCCAGCTGGGAGAGGCTGGCCGTGATGAATTC-3';
miR-485-5p下游引物：5'-CTCGATTCGTCACTCACA-3'；miR-485-5p downstream primer: 5'-CTCGATTCGTCACTCACA-3';
U6上游引物：5'-CCCGGACACGTGGGCTCCC-3'；U6 upstream primer: 5'-CCCGGACACGTGGGCTCCC-3';
U6下游引物：5'-CACATCCCTGGACACAGTCCTAG-3'。U6 downstream primer: 5'-CACATCCCTGGACACAGTCCTAG-3'.
3、UC-MSCs-Exo收集和检测3. Collection and detection of UC-MSCs-Exo
按照常规离心法收集外泌体。具体方法为：取转染后的UC-MSCs用DMEM/F12培养基 制成细胞悬液并接种于培养皿中，72h后收集上清并转移至离心管中，4℃条件下300×g离心10min去除残余细胞，上清于4℃条件下2000×g离心10min去除死细胞，上清于4℃条件下10000×g离心30min去除细胞碎片，上清于4℃条件下100000×g离心70min收集沉淀即得外泌体。用无菌PBS将沉淀重悬，再次于4℃条件下100000×g离心70min收集沉淀即得经过洗涤的外泌体。最后用无菌PBS将经过洗涤的外泌体重悬，取少量用于BCA法测定外泌体浓度、透射电镜观察以及Western Blot检测外泌体标志性蛋白，其余-80℃冻存备用。Exosomes were collected by conventional centrifugation. The specific method is as follows: take the transfected UC-MSCs and make a cell suspension with DMEM/F12 medium and inoculate them in a culture dish. After 72 hours, collect the supernatant and transfer it to a centrifuge tube, and centrifuge at 300×g at 4°C. Remove residual cells for 10 minutes, centrifuge the supernatant at 2000×g for 10 minutes at 4°C to remove dead cells, centrifuge the supernatant at 10,000×g for 30 minutes at 4°C to remove cell debris, and collect the supernatant at 100,000×g for 70 minutes at 4°C Precipitate to obtain exosomes. The pellet was resuspended in sterile PBS, and then centrifuged at 100,000×g for 70 min at 4°C to collect the pellet to obtain washed exosomes. Finally, the washed exosomes were resuspended in sterile PBS, and a small amount was taken for BCA method to determine exosome concentration, transmission electron microscope observation and Western Blot to detect exosome marker proteins, and the rest were frozen at -80°C for later use.
Western blot检测方法为：各组外泌体取适量并调整蛋白浓度一致后，加入5×上样缓冲液，100℃煮沸10min，10％SDS-聚丙烯酰胺凝胶电泳后转移至PVDF膜上，5％脱脂奶粉室温封闭2h，加入CD9、CD63、CD81一抗，4℃孵育过夜。TBST反复洗膜后，HRP标记二抗孵育1h，洗膜后化学发光试剂显色，用凝胶成像系统拍照，Image J软件分析灰度值。The Western blot detection method is as follows: after taking an appropriate amount of exosomes in each group and adjusting the protein concentration to be consistent, add 5× loading buffer, boil at 100°C for 10 minutes, transfer to PVDF membrane after 10% SDS-polyacrylamide gel electrophoresis, 5% skimmed milk powder was blocked at room temperature for 2 hours, CD9, CD63, and CD81 primary antibodies were added, and incubated overnight at 4°C. After washing the membrane repeatedly with TBST, the HRP-labeled secondary antibody was incubated for 1 h. After washing the membrane, the color was developed with chemiluminescence reagent. The gel imaging system was used to take pictures, and the gray value was analyzed by Image J software.
4、统计学分析4. Statistical analysis
采用GraphPad Prism 5软件对数据进行统计分析，采用配对t检验对数据进行显著性分析，P<0.05认定为差异显著。GraphPad Prism 5 software was used for statistical analysis of data, and paired t-test was used for significant analysis of data, and P<0.05 was considered significant difference.
三、实验结果3. Experimental results
1、UC-MSCs倒置显微镜下观察结果1. Observation results of UC-MSCs under an inverted microscope
同实施例1。With embodiment 1.
2、UC-MSCs转染结果2. UC-MSCs transfection results
表3和图1中C-2为各组UC-MSCs中miR-485-5p的含量比较，PG组miR-485-5p含量显著高于Blank组、NC组，说明高表达miR-485-5p的UC-MSCs造模成功。C-2 in Table 3 and Figure 1 is the comparison of miR-485-5p content in UC-MSCs of each group, and the miR-485-5p content in PG group was significantly higher than that in Blank group and NC group, indicating high expression of miR-485-5p The UC-MSCs model was successfully established.
表3各组UC-MSCs中miR-485-5p相对表达水平Table 3 Relative expression levels of miR-485-5p in UC-MSCs of each group
Figure PCTCN2021138172-appb-000003
Figure PCTCN2021138172-appb-000003
*代表与Blank、NC组相比差异显著。*Represents significant difference compared with Blank and NC groups.
3、UC-MSCs-Exo检测结果3. UC-MSCs-Exo detection results
图1中D-2为各组UC-MSCs-Exo的透射电镜观察图(100×)，图1中E-2为各组UC-MSCs-Exo标志蛋白CD9、CD63、CD81的Western Blot检测图，各组外泌体的形态和标志物都符合UC-MSCs-Exo的特点，说明各组均制备得到了外泌体。D-2 in Figure 1 is the transmission electron microscope observation image (100×) of UC-MSCs-Exo in each group, and E-2 in Figure 1 is the Western Blot detection image of UC-MSCs-Exo marker proteins CD9, CD63, and CD81 in each group , the morphology and markers of exosomes in each group were consistent with the characteristics of UC-MSCs-Exo, indicating that exosomes were prepared in each group.
实施例4：促进皮肤屏障修复活性Example 4: Promote skin barrier repair activity
一、实验材料1. Experimental materials
人类永生化表皮细胞(HaCaT细胞)购自ATCC。胎牛血清、DMEM培养基购自Gibco公司，双抗、MTT和胰蛋白酶购自碧云天生物。Human immortalized epidermal cells (HaCaT cells) were purchased from ATCC. Fetal bovine serum and DMEM medium were purchased from Gibco, and double antibodies, MTT and trypsin were purchased from Beyontib.
二、实验方法2. Experimental method
1、HaCaT细胞的培养1. Culture of HaCaT cells
将HaCaT细胞用含10％胎牛血清和1％双抗的DMEM培养基于37℃、5％CO 2、饱和湿度条件培养，每2～3d换液1次。 HaCaT cells were cultured in DMEM containing 10% fetal bovine serum and 1% bis-antibody at 37°C, 5% CO 2 , and saturated humidity, and the medium was changed every 2-3 days.
2、HaCaT细胞增殖活性测定2. Determination of the proliferation activity of HaCaT cells
采用常规的MTT法测定HaCaT细胞的增殖活性，分为空白对照组和外泌体A、B、C组，外泌体A、B、C组添加的外泌体分别对应为实施例1中Blank组、NC组、PG组的外泌体。The proliferative activity of HaCaT cells was determined by conventional MTT method, and divided into blank control group and exosome A, B, and C groups. The exosomes added in exosome A, B, and C groups corresponded to Blank in Example 1. The exosomes of group, NC group and PG group.
具体方法为：取生长状态良好的对数生长期HaCaT细胞，调整细胞浓度至1×10 5个/mL，混匀后接种于96孔板中，每孔100μL，每组设6个复孔。待细胞完全贴壁后，实验组更换为含有不同浓度外泌体(低浓度为2μg/mL、高浓度为5μg/mL)的完全培养基培养，空白对照组的培养基中不含有外泌体。继续培养24h后，每孔加入20μL浓度为5mg/mL的MTT溶液，培养4h，去上清液，加入150μL DMSO轻轻震荡，用酶标仪检测其在490nm处的吸光度(OD值)，通过OD值按照如下公式计算实验组增殖活性，空白对照组增殖活性计为100％：实验组增殖活性＝实验组OD值/空白对照组OD值×100％。吸光度越高代表增殖活性越强。 The specific method is as follows: Take HaCaT cells in the logarithmic growth phase in good growth state, adjust the cell concentration to 1×10 5 cells/mL, mix them and inoculate them in a 96-well plate, 100 μL per well, and set 6 replicate wells for each group. After the cells were completely adhered to the wall, the experimental group was replaced with complete medium containing different concentrations of exosomes (low concentration of 2 μg/mL, high concentration of 5 μg/mL), and the culture medium of the blank control group did not contain exosomes. . After continuing to cultivate for 24 hours, add 20 μL of MTT solution with a concentration of 5 mg/mL to each well, cultivate for 4 hours, remove the supernatant, add 150 μL of DMSO to shake gently, and detect its absorbance (OD value) at 490 nm with a microplate reader. The OD value calculated the proliferative activity of the experimental group according to the following formula, and the proliferative activity of the blank control group was counted as 100%: proliferative activity of the experimental group=OD value of the experimental group/OD value of the blank control group×100%. The higher the absorbance, the stronger the proliferative activity.
3、HaCaT细胞迁移活性测定3. Determination of HaCaT cell migration activity
采用常规的划痕法测定HaCaT细胞的迁移活性，分为空白对照组和外泌体A、B、C组，外泌体A、B、C组添加的外泌体分别对应为实施例1中Blank组、NC组、PG组的外泌体。The migration activity of HaCaT cells was determined by the conventional scratch method, and divided into blank control group and exosome A, B, C groups, and the exosomes added in exosome A, B, and C groups corresponded to those in Example 1. Exosomes in Blank group, NC group, and PG group.
具体方法为：取生长状态良好的对数生长期HaCaT细胞，调整细胞浓度至4×10 5个/mL，接种于培养皿中。待细胞完全贴壁后，实验组更换为含有5μg/mL外泌体的完全培养基培养，空白对照组的培养基中不含有外泌体。继续培养24h后，消化细胞用PBS洗涤3次，用完全培养基调整细胞浓度至2×10 5个/mL，接种于12孔板中。待细胞完全贴壁后，更换为含0.5％胎牛血清的培养基饥饿处理12h，使用无菌10μL枪头在细胞层垂直划痕，用PBS冲洗2～3次去除划痕区残留细胞，加入完全培养基培养12h后于显微镜下观察拍照。 The specific method is as follows: take HaCaT cells in the logarithmic growth phase in a good growth state, adjust the cell concentration to 4×10 5 cells/mL, and inoculate them in a culture dish. After the cells were completely adhered to the wall, the experimental group was replaced with a complete medium containing 5 μg/mL exosomes, and the culture medium of the blank control group did not contain exosomes. After continuing to culture for 24 h, the digested cells were washed 3 times with PBS, the cell concentration was adjusted to 2×10 5 cells/mL with complete medium, and seeded in a 12-well plate. After the cells are completely adhered to the wall, replace with a medium containing 0.5% fetal bovine serum for starvation treatment for 12 hours, use a sterile 10 μL pipette tip to scratch the cell layer vertically, wash with PBS 2 to 3 times to remove residual cells in the scratched area, add The complete culture medium was observed and photographed under a microscope after 12 hours of culture.
4、统计学分析4. Statistical analysis
采用GraphPad Prism 5软件对数据进行统计分析，采用配对t检验对数据进行显著性分析，P<0.05认定为差异显著。GraphPad Prism 5 software was used for statistical analysis of data, and paired t-test was used for significant analysis of data, and P<0.05 was considered significant difference.
三、实验结果3. Experimental results
1、HaCaT细胞增殖活性测定结果1. Determination results of HaCaT cell proliferation activity
图3中A为各组HaCaT的增殖活性比较，外泌体A、B、C组的HaCaT增殖活性均显著 高于空白对照组，且外泌体C组的HaCaT增殖活性显著高于外泌体A、B组，外泌体A组与B组HaCaT增殖活性无显著性差异。In Figure 3, A is the comparison of the proliferation activity of HaCaT in each group. The proliferation activity of HaCaT in exosome A, B, and C groups was significantly higher than that of the blank control group, and the proliferation activity of HaCaT in exosome C group was significantly higher than that of exosome Groups A and B, there was no significant difference in the proliferation activity of HaCaT between group A and group B of exosomes.
表4各组HaCaT的增殖活性Table 4 Proliferation activity of HaCaT in each group
Figure PCTCN2021138172-appb-000004
Figure PCTCN2021138172-appb-000004
#代表与空白对照组相比差异显著；*代表与外泌体A、B组相比差异显著。# represents significant difference compared with blank control group; * represents significant difference compared with exosomes A and B groups.
2、HaCaT细胞迁移活性测定结果2. HaCaT cell migration activity assay results
图3中B为各组HaCaT的迁移活性比较，外泌体A、B、C组的HaCaT迁移活性均显著高于空白对照组，且外泌体C组的HaCaT迁移活性显著高于外泌体A、B组，外泌体A组与B组HaCaT迁移活性无显著性差异。Figure 3 B is the comparison of the migration activity of HaCaT in each group. The HaCaT migration activity of exosome A, B, and C groups was significantly higher than that of the blank control group, and the HaCaT migration activity of exosome C group was significantly higher than that of exosomes. Groups A and B, there was no significant difference in HaCaT migration activity between group A and group B of exosomes.
表皮细胞层是皮肤屏障最重要的细胞层，表皮细胞的增殖活性和迁移活性直接关系到皮肤屏障受损后的修复能力。上调或下调改变部分基因表达水平是调节、改进细胞功能的重要研究方法。上述实验表明，与正常人脐带间充质干细胞分泌的外泌体相比，miR-485-5p表达上调的人脐带间充质干细胞分泌的外泌体一方面可以更有效地提高人类永生化表皮细胞HaCaT细胞的增殖活性，另一方面可以更有效地提高HaCaT细胞的迁移活性，具有开发成促进皮肤屏障修复的护肤品或药品的前景。The epidermal cell layer is the most important cell layer of the skin barrier, and the proliferation and migration activities of epidermal cells are directly related to the repair ability of the damaged skin barrier. Up-regulation or down-regulation to change the expression level of some genes is an important research method to regulate and improve cell function. The above experiments showed that, compared with exosomes secreted by normal human umbilical cord mesenchymal stem cells, exosomes secreted by human umbilical cord mesenchymal stem cells with upregulated expression of miR-485-5p could more effectively improve human immortalized epidermis on the one hand. The proliferative activity of HaCaT cells, on the other hand, can more effectively improve the migration activity of HaCaT cells, and has the prospect of being developed into skin care products or medicines that promote skin barrier repair.
上述实施例的作用在于具体介绍本发明的实质性内容，但本领域技术人员应当知道，不应将本发明的保护范围局限于该具体实施例。The purpose of the above embodiments is to specifically introduce the substantive content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims (3)

  1. 一种脐带间充质干细胞外泌体，其特征在于：该脐带间充质干细胞外泌体为miR-328-3p或miR-485-5p表达上调的人脐带间充质干细胞分泌的外泌体。An umbilical cord mesenchymal stem cell exosome, characterized in that: the umbilical cord mesenchymal stem cell exosome is an exosome secreted by human umbilical cord mesenchymal stem cells whose expression of miR-328-3p or miR-485-5p is upregulated .
  2. 权利要求1中miR-328-3p表达上调的脐带间充质干细胞外泌体的抗皮肤衰老用途。Anti-aging application of umbilical cord mesenchymal stem cell exosomes with up-regulated miR-328-3p expression in claim 1.
  3. 权利要求1中miR-485-5p表达上调的脐带间充质干细胞外泌体的促进皮肤屏障修复用途。Use of umbilical cord mesenchymal stem cell exosomes with up-regulated miR-485-5p expression in claim 1 to promote skin barrier repair.
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CN112143708A (en) * 2020-10-12 2020-12-29 张川 Umbilical cord mesenchymal stem cells, stem cell essence factor and application of umbilical cord mesenchymal stem cells and stem cell essence factor in aspect of resisting skin aging
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