CN111826340B - Preparation method and application of pilose antler stem cell exosome for resisting skin aging - Google Patents
Preparation method and application of pilose antler stem cell exosome for resisting skin aging Download PDFInfo
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- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Abstract
The application discloses a preparation method and application of an anti-skin-aging pilose antler stem cell exosome, and belongs to the technical field of tissue engineering. The application provides an exosome aiming at solving the problem of skin aging resistance, wherein the exosome is an pilose antler stem cell exosome. The application comprises a plurality of components such as a large number of cytokines, growth factors, miRNAs, mRNAs and the like in the pilose antler stem cell exocrine, which are easy to fuse with the cell membranes of adjacent cells, selectively deliver biological active substances to receptor cells, transfer information among different cells and regulate the signal transmission among the cells. Compared with human bone marrow mesenchymal stem cells, the pilose antler stem cell exocrine substance obtained by the method is easy to obtain, has the proliferation speed which is ten times faster than that of human bone marrow mesenchymal stem cells, can remove DPPH free radicals, has an antioxidant function, and has remarkable promotion effect on the proliferation of Hacat cells and NHDF cells.
Description
Technical Field
The application relates to a preparation method and application of an anti-skin-aging pilose antler stem cell exosome, belonging to the technical field of tissue engineering.
Background
Cornu Cervi Pantotrichum is the only mammalian organ known to be completely regenerated, and its growth rate can reach 2cm/d. The complete regeneration and rapid growth ability of deer antler is mainly due to the existence of deer antler stem cells. The deer antler stem cells are identified as a special stem cell type between embryo stem cells and mesenchymal stem cells. Compared with the stem cells such as the bone marrow mesenchymal stem cells, the umbilical cord mesenchymal stem cells and the like which are commonly used at present, the hairy antler stem cells are easier to obtain, have high proliferation speed and secrete a large amount of cytokines and bioactive factors.
In recent years, supplementing human stem cells with stem cell transplantation for anti-aging is considered as a new and effective measure. Various stem cells such as bone marrow mesenchymal stem cells, fat stem cells, umbilical cord mesenchymal stem cells and the like have been demonstrated to improve body aging. However, the direct use of stem cells has disadvantages in terms of inconvenient storage and transportation and immune rejection. And stem cells are easily differentiated into other stromal cells, which may promote tumor cell metastasis and stimulate epithelial-mesenchymal cell transformation, with potential safety hazards.
Exosomes (exosomes) are nanoscale membrane vesicles released into the extracellular environment after fusion of the cell membranes by the multivesicular endosomes (multivesicular bodies, MVBs) of eukaryotic cells, can be secreted by various types of cells, contain substances with biological activities such as different kinds of proteins, lipids, mRNAs, microRNAs, signal molecules and the like, are easily fused with the cell membranes of adjacent cells, selectively deliver the biological active substances to receptor cells, perform information transmission between different cells, regulate the signal transduction between the cells and play a plurality of biological functions. Therefore, it is necessary to provide an anti-aging pilose antler stem cell exosome.
Disclosure of Invention
The application provides an exosome for solving the problem of skin aging resistance, which is an pilose antler stem cell exosome, and a preparation method of the exosome.
The technical scheme of the application is as follows:
a preparation method of an anti-skin-aging pilose antler stem cell exosome comprises the following steps:
step one, separating and culturing antler stem cells;
the deer antler stem cells are derived from deer antler tissue, deer antler stem tissue and deer antler mesenchymal tissue, and are collected and placed in transportation and transfusion;
step two, primary culture of the deer antler stem cells;
(1) Taking out periosteum tissue, cornucervi periosteum tissue or cornucervi mesenchymal tissue of deer collected in the step one from the transport liquid, repeatedly cleaning with PBS solution until blood stain is completely removed, and cutting the periosteum tissue, the cornucervi periosteum tissue or the cornucervi mesenchymal tissue of deer into 0.7mm in a disposable sterile plate by using a surgical knife 3 Is a fragment of (a);
(2) Taking 2-4g of fragments, washing the fragments with washing liquid, transferring into a 50mL centrifuge tube, centrifuging at 1000rpm for 5min, discarding supernatant, and collecting precipitate;
(3) Adding 30mL of digestion solution into 2-4g of the precipitate obtained in the step (2), vibrating at 37 ℃ for digestion, observing digestion conditions under a microscope every 10min, stopping digestion when hairbrush-like burrs appear at the edges of fragments, centrifuging at 1000rpm for 5min, discarding supernatant, adding 10mL of DMEM primary culture solution for washing, centrifuging at 1000rpm for 5min, discarding supernatant, and collecting precipitate;
(4) Taking 0.5-1g of precipitate obtained in the step (3), uniformly coating the precipitate on the bottom of a cell culture flask, inverting the flask, adding 2-3mL of DMEM primary culture solution, placing at 37 ℃ and 5% CO 2 Culturing in a cell incubator for 2-4h, standing in a culture flask, observing the adhesion and cell divergence of tissue blocks under a microscope, and carrying out trypsin digestion and freeze-preserving on the cells when the cells reach 70% confluence to obtain primary pilose antler stem cells;
step three, extracting the exosomes of the deer antler stem cells;
subculturing the primary cornu Cervi Pantotrichum stem cells obtained in step two on subculture medium, transferring to 3-5 generations, and then culturing 1×10 5 Inoculating the individual cells to 75cm 2 Adding 15mL of culture solution into culture flask, and culturing to 1×10 7 Collecting supernatant when the cells or 90% are pooled, removing cell fragments and apoptotic bodies by adopting a centrifugal or filter membrane filtration mode to obtain a collected sample, mixing the collected sample with a binding buffer solution according to the volume ratio of 1:1, adding 16mL of mixed solution into a binding column, centrifuging for 1min under the condition of 300 Xg, and discarding effluent liquid; then 10mL of washing buffer solution is added, the mixture is centrifuged for 5min under the condition of 5000 Xg, and effluent liquid is discarded; placing the binding column in a collecting tube, adding 400 μl of elution buffer, incubating at room temperature for 1 hr, centrifuging at 5000×g for 5min, collecting the effluent to obtain cornu Cervi Pantotrichum stem cell exosomes, and placing in a refrigerator at-80deg.C.
Further, in the first step, periosteum tissue of the deer antler growth area is collected in an extra crest antler growth area of a 1 year old male deer which does not start to develop in the deer antler growth area.
Further, in the first step, the cornel periosteum tissue is collected from the cornel periosteum of a 2 year old deer.
Further, in the first step, the mesenchymal tissue of pilose antler is collected from pilose antler in 15-60 days of growing period.
Further, the transport solution is DMEM complete culture solution.
Further, the washing solution was DMEM medium containing 200U/mL of the diabody without FBS.
Further, the digestion solution is 50U/mL type I collagenase solution, and the dosage is 20-30mL.
Further, the primary culture broth was DMEM culture broth containing 20% fbs and 1% antibiotic.
Further, the subculture medium is DMEM medium containing 10% fbs and 1% antibiotic; the culture solution in the third step consists of FBS containing 10% of vesicle-free components, DMEM culture solution containing 1% of antibiotics and mTESR TM 1 Medium composition, wherein mTESR TM The mass of the culture medium 1 is 30% of the total mass of the culture solution.
The exosomes prepared by the method are applied to resisting skin aging, and are used for eliminating DPPH free radical activity and promoting proliferation of Hacat cells and NHDF cells.
The application has the following beneficial effects: the pilose antler stem cell exocrine obtained by the method contains a large amount of cytokines, growth factors, miRNAs, mRNAs and other various components, is easy to fuse with the cell membranes of adjacent cells, selectively delivers biological active substances to receptor cells, carries out information transmission among different cells, regulates signal transmission among cells, can clearly obtain DPPH free radicals, has an antioxidant function, and has the advantages that compared with human bone marrow mesenchymal stem cells, the pilose antler stem cell exocrine obtained by the method is easy to obtain, the proliferation speed is ten times that of the human bone marrow mesenchymal cells, and has remarkable promotion effect on the proliferation of Hacat cells and NHDF cells.
Drawings
FIG. 1 shows the effect of the pilose antler stem cell exosomes on Hacat cell proliferation;
FIG. 2 shows the effect of the pilose antler stem cell-exosomes on NHDF cell proliferation.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and apparatus used, without any particular description, are those conventional in the art and are commercially available to those skilled in the art.
Example 1:
1. separating and culturing cornu Cervi Pantotrichum stem cells; the cornu Cervi Pantotrichum stem cells are derived from periosteum tissue, periosteum tissue or mesenchymal tissue of cornu Cervi Pantotrichum.
(1) The specific operation process for collecting periosteum tissue of deer antler generation region comprises the following steps: 1 year old giraffe which does not start to develop in the antler-producing area of the anesthetic deer, preparing skin of the antler-producing area of the additional ridge after anesthesia, sterilizing, cutting the skin of the antler-producing area under a sterile environment, exposing periosteum of the antler-producing area, cleaning loose connective tissue, cutting along the antler-producing area (the lowest part of the additional ridge) by a clean scalpel, tearing the cut periosteum from bones by using a pair of mouse tooth forceps, and rapidly putting into transport and transfusion which is DMEM culture medium containing double antibodies.
(2) The specific operation process for collecting the periosteum tissue of the deer antler stem comprises the following steps: anesthetizing a 2-year-old deer, preparing skin on a horn, sterilizing, cutting the skin of the horn along the horn in a sterile environment, exposing the periosteum of the horn, cleaning loose connective tissue, cutting the periosteum of the horn along the horn by using a clean sterile scalpel in an I shape, cutting the periosteum of the horn into a plurality of strips of about 5mm, tearing off each periosteum of the horn by using a pair of mouse forceps, and rapidly putting the strips into transport and transfusion, wherein the transport and transfusion is a DMEM culture medium containing double antibodies.
(3) The specific operation process for collecting the mesenchymal tissue of the pilose antler comprises the following steps: cleaning cornu Cervi Pantotrichum surface with sterile gauze for 15-60 days, preparing skin, and sterilizing with iodine and 75% alcohol. Cutting off the tip of cornu Cervi Pantotrichum of 3-5cm with a tissue slicing blade in a biosafety cabinet, placing in a sterile culture dish, then dividing the tip into slices of about 0.5cm along the longitudinal direction, placing in a new culture dish, cutting off skin and subcutaneous cornu Cervi Pantotrichum tissue along subcutaneous connective tissue, removing skin, cutting off a layer of arc semitransparent tissue protruding slightly from the skin with a scalpel, and rapidly placing into a transfusion solution which is DMEM culture medium containing double antibodies.
2. Primary culture of pilose antler stem cells;
(1) Taking out the collected periosteum/horn periosteum/deer bone mesenchymal layer tissue of the antler zone from the transport liquid (DMEM liquid) by using sterile forceps, and repeatedly cleaning with PBS until blood stains are completely removed. Cutting periosteum/horn periosteum/deer bone mesenchymal tissue into 0.7mm in a disposable sterile plate by using a self-made sterile small guillotine (application patent) 3 Left and right uniform fragments.
(2) 2-4g of fragments were washed with DMEM solution containing double antibody (200U/mL) without FBS and transferred to a 50mL sterile centrifuge tube, centrifuged at 1000rpm for 5min, and the pellet was collected.
(3) The supernatant was gently removed, 30mL of digestion solution (50U/mL collagenase I) was added, digested with gentle shaking in a 37℃water bath, the digestion was observed under a microscope at 10min intervals, when brush-like burrs were present at the edges of most tissue pieces, the digestion was stopped, centrifuged at 1000rpm for 5min, and the pellet was collected.
(4) The supernatant was then discarded, washed with 10mL of primary culture medium (DMEM+20% FBS+1% antibiotics), centrifuged at 1000rpm for 5min, and the pellet was collected.
(5) The pellet of (4) was then aspirated and spread evenly on the bottom of the cell culture flask, the flask was inverted and 2-3mL of DMEM primary culture medium (DMEM+20% FBS+1% antibiotics) was added.
(6) Placing at 37deg.C, 5% CO 2 The culture is carried out in a cell culture box for 2 to 4 hours, and the stationary culture is continued in a normal culture flask.
(7) Observing the adhesion and cell divergence of the tissue block under a microscope, and when the cells reach 70% confluence, carrying out trypsin digestion (0.25% trypsin, digesting for 1-2 minutes, and stopping digestion after cell shrinkage and rounding) on the cells to obtain the primary pilose antler stem cells.
3. Extracting the exosomes of the pilose antler stem cells;
subculturing the primary cornu Cervi Pantotrichum stem cells obtained in step two on subculture medium (DMEM+10% FBS+1% antibiotics), transferring to 3-5 passages, and then culturing 1×10 5 Inoculating the individual cells to 75cm 2 15mL of a culture medium (70% (DMEM+10% FBS without vesicle components+1% antibiotics) +30% (mTESRTM1+10% supplements)) was added to the flask and cultured until the concentration became 1X 10 7 Collecting supernatant when the cells or 90% are pooled, removing cell fragments and apoptotic bodies by adopting a centrifugal or filter membrane filtration mode to obtain a collected sample, mixing the collected sample with a binding buffer solution according to the volume ratio of 1:1, adding 16mL of mixed solution into a binding column, centrifuging for 1min under the condition of 300 Xg, and discarding effluent liquid; then 10mL of washing buffer solution is added, the mixture is centrifuged for 5min under the condition of 5000 Xg, and effluent liquid is discarded; placing the binding column in a collecting tube, adding 400 μl of elution buffer, incubating at room temperature for 1 hr, centrifuging at 5000×g for 5min, collecting the effluent to obtain cornu Cervi Pantotrichum stem cell exosomes, packaging into 1.5mL centrifuge tube, placing in a refrigerator at-80deg.C, and preserving. Wherein the binding buffer is the binding buffer in the exo easy maxi kit kit (Qiagen). The wash buffer was the wash buffer in the exo easy maxi kit kit (Qiagen). The elution buffer was that in the exo easy maxi kit kit (Qiagen)And (3) liquid.
Example 2:
(1) DPPH free radical scavenging activity detection was performed on the pilose antler stem cell exosomes obtained in example 1.
1mg of DPPH is weighed and dissolved in 20ml of absolute ethyl alcohol, 1ml of DPPH solution and 0.01ml of pilose antler stem cell exosome sample are added into each tube of reaction system, the mixture is uniformly mixed, and the absorbance value is measured at 519nm after standing for 30 min.
The blank culture medium is used as a blank control, and the sample control group comprises 1mL of ethanol solution and 0.01mL of pilose antler stem cell exosome sample. DPPH radical scavenging was calculated as follows.
DPPH radical clearance = [1- (a sample group-a sample control group)/a blank control group ] x100%
The measured deer antler stem cell conditioned medium and the freeze-dried powder thereof have obvious effect of eliminating DPPH free radical, and the clearance rate can reach 77.4 percent of APC-EXO, 75.3 percent of PPC-EXO and 79.53 percent of RMC-EXO.
(2) Proliferation promotion test of human keratinocytes (Hacat) and dermal fibroblasts (NHDF) was performed on the culture medium of the deer antler stem cells obtained in example 1.
Hacat and NHDF cells were cultured separately, inoculated into 96-well cell culture plates at a density of 5000 cells/well, 10uL of pilose antler stem cell exosomes were added to each culture well, after 24 hours of culture, 10uL of CCK8, 37 were added, and after 2 hours of incubation, absorbance was measured at 410 nm. The test results are shown in fig. 1 and 2, and the result shows that the deer antler stem cell conditioned medium has obvious effect of promoting the proliferation of Hacat cells and NHDF cells.
Claims (5)
1. A preparation method of an anti-skin-aging pilose antler stem cell exosome is characterized by comprising the following steps:
step one, separating and culturing antler stem cells;
the deer antler stem cells are derived from deer antler tissue, deer antler stem tissue and deer antler mesenchymal tissue, and are collected and placed in transportation and transfusion;
step two, primary culture of the deer antler stem cells;
(1) Taking out periosteum tissue, cornucervi periosteum tissue and cornucervi mesenchymal tissue of deer collected in the step one from the transport liquid, repeatedly cleaning with PBS solution until blood stain is completely taken out, and cutting the periosteum tissue, the cornucervi periosteum tissue and the cornucervi mesenchymal tissue of deer into 0.7mm in a disposable sterile plate by using a surgical knife 3 Is a fragment of (a);
(2) Taking 2-4g of fragments, washing the fragments with washing liquid, transferring into a 50mL centrifuge tube, centrifuging at 1000rpm for 5min, discarding supernatant, and collecting precipitate;
(3) Adding 30mL of digestion solution into 2-4g of the precipitate obtained in the step (2), vibrating at 37 ℃ for digestion, observing digestion conditions under a microscope every 10min, stopping digestion when hairbrush-like burrs appear at the edges of fragments, centrifuging at 1000rpm for 5min, discarding supernatant, adding 10mL of DMEM primary culture solution for washing, centrifuging at 1000rpm for 5min, discarding supernatant, and collecting precipitate;
(4) Taking 0.5-1g of precipitate obtained in the step (3), uniformly coating the precipitate on the bottom of a cell culture flask, inverting the flask, adding 2-3mL of DMEM primary culture solution, placing at 37 ℃ and 5% CO 2 Culturing in a cell incubator for 2-4h, standing in a culture flask, culturing for 4-7 days, observing the adhesion of tissue blocks and cell divergence under a microscope, and performing trypsin digestion and freeze-storage to obtain primary cornu Cervi Pantotrichum stem cells when the cells reach 70% confluence;
step three, extracting the exosomes of the deer antler stem cells;
subculturing the primary cornu Cervi Pantotrichum stem cells obtained in step two on subculture medium, transferring to 3-5 generations, and then culturing 1×10 5 Inoculating the individual cells to 75cm 2 Adding 15mL of culture solution into culture flask, and culturing to 1×10 7 Collecting supernatant when the cells or 90% are combined, removing cell fragments and apoptotic bodies by adopting a centrifugal or filter membrane filtration mode to obtain a collected sample, mixing the collected sample with a binding buffer solution according to the volume ratio of 1:1, and adding 16mL of mixed solution into the binding buffer solutionCentrifuging in column at 300 Xg for 1min, and discarding effluent; then 10mL of washing buffer solution is added, the mixture is centrifuged for 5min under the condition of 5000 Xg, and effluent liquid is discarded; placing the binding column in a collecting tube, adding 400 μl of elution buffer, incubating at room temperature for 1 hr, centrifuging at 5000×g for 5min, collecting the effluent to obtain cornu Cervi Pantotrichum stem cell exosomes, and placing in a refrigerator at-80deg.C;
the culture solution in the third step consists of FBS containing 10% of vesicle-free components, DMEM culture solution containing 1% of antibiotics and mTESR TM 1 Medium composition, wherein mTESR TM 1, the mass of the culture medium is 30% of the total mass of the culture solution;
the transport liquid is double-antibody DMEM complete culture liquid;
the washing liquid is DMEM culture liquid which does not contain FBS and contains 200U/mL of double antibody;
the digestion liquid is collagenase I with the concentration of 50U/mL;
the primary culture solution is DMEM culture solution containing 20% FBS and 1% antibiotics;
the subculture medium is DMEM culture solution containing 10% FBS and 1% antibiotics.
2. The method of claim 1, wherein the periosteum tissue of the deer antler growth area is collected from the additional crest antler growth area of a 1 year old deer which does not develop in the deer antler growth area.
3. The method for preparing an anti-aging deer antler stem cell exosome according to claim 1, wherein the deer horn periosteum tissue is collected from the horn periosteum of a deer aged 2 years in the first step.
4. The method for preparing an anti-skin aging deer antler stem cell exosome according to claim 1, wherein deer antler mesenchymal tissue is collected from deer antler in 15-60 days of growing period in the first step.
5. The use of an exosome according to any one of claims 1-4 for anti-skin aging, which is characterized by scavenging DPPH free radical activity and promoting Hacat and NHDF cell proliferation.
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| CN111671772B (en) * | 2020-05-28 | 2021-04-13 | 中国农业科学院特产研究所 | Application of exosome in preparation of medicine or cosmetic for repairing skin injury |
| CN113208998A (en) * | 2021-05-11 | 2021-08-06 | 奥启(深圳)创投科技有限公司 | Anti-wrinkle essence containing stem cell exosomes |
| CN114642200B (en) * | 2022-03-01 | 2023-03-03 | 长春科技学院 | Cryopreservation method for periosteum in antler-growing area |
| CN115029306A (en) * | 2022-06-21 | 2022-09-09 | 长春科技学院 | Method for efficiently preparing antler stem cell exosomes by applying three-dimensional culture system |
| CN115386542B (en) * | 2022-06-21 | 2024-04-23 | 长春科技学院 | Engineered pilose antler stem cell exosome, preparation method and application thereof in osteosarcoma treatment |
| CN115282166A (en) * | 2022-07-20 | 2022-11-04 | 长春科技学院 | Application of pilose antler stem cells and exosomes thereof in preparing anti-scar products |
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