CN114099547B - Stem cell exosome preparation for skin repair - Google Patents
Stem cell exosome preparation for skin repair Download PDFInfo
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- CN114099547B CN114099547B CN202111488358.XA CN202111488358A CN114099547B CN 114099547 B CN114099547 B CN 114099547B CN 202111488358 A CN202111488358 A CN 202111488358A CN 114099547 B CN114099547 B CN 114099547B
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Classifications
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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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/40—Nucleotides, nucleosides or bases
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
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- C12N2501/30—Hormones
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- C12N2501/998—Proteins not provided for elsewhere
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
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Abstract
The invention provides a stem cell exosome preparation for skin repair, which comprises a basic additive and a stem cell exosome serving as an active ingredient, wherein the stem cell exosome is prepared by the following method: inoculating stem cells into an induction culture medium for induction culture, wherein an induction culture reagent added into the induction culture medium is active peptide, plant thiopeptide hormone-alpha, epidermal growth factor and 3-isobutyl-1-methylxanthine, and inoculating the culture medium with mesenchymal stem cells and 5% CO 2 Culturing at 37 ℃ for 48-72 hours, recovering culture supernatant, filtering, and separating and purifying by an ultracentrifugation method to obtain stem cell exosomes. The invention cultures stem cells by adopting specific components as the induction reagent, can influence the activity and purity of exosomes generated by the stem cells, and the preparation added with the exosomes is more suitable for skin repair and is more beneficial to scar desalination.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a stem cell exosome preparation for skin repair.
Background
The skin is the protective membrane that protects the body, the first line of defense against disease, and the epidermis acts as a barrier against microbial invasion. Thus, in the treatment of wounds, burns, abrasions, scratches, and other skin injuries, the primary objective is to rapidly suture to prevent infection, as well as to treat wounds.
The most mild wounds are limited to the skin epidermis, with skin and subcutaneous tissue breaks in the slightly heavier individuals, and wounds appear; severe trauma may be a break in muscles, muscle bonds, nerves, and fractures. The mild wounds can be healed through epithelial regeneration, the last two wounds can cause the appearance form and histopathology of normal skin tissues to change to form scars, the scars grow beyond a certain limit, various complications such as the damage of the appearance, dysfunction and the like can occur, and huge physical and mental pains, especially scars left after burns, scalds and severe trauma are brought to patients.
Various wounds can cause varying degrees of cellular degeneration, necrosis and tissue defects, and tissue repair must be performed by cell proliferation and intercellular matrix formation. In this repair process, fibroblasts play a very important role, and in the later stages of wound repair, fibroblasts participate in the reconstruction of the repaired tissue by secreting collagenase.
Exosomes (exosomes) are vesicle-like small bodies secreted by cells to the outside of cells, having diameters of 30-150 nanometers, and having a typical lipid bilayer membrane structure. The stem cell-derived exosomes contain not only receptors, i.e., proteins, but also nuclear components, and thus can function as intercellular communication. Therefore, how to use stem cell exosomes to provide a better formulation for repairing skin is a problem that needs to be solved at present.
Disclosure of Invention
In view of the above, the present invention aims to propose a stem cell exosome formulation for skin repair to solve the above-mentioned problems.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
a stem cell exosome preparation for skin repair, comprising a basic additive and a stem cell exosome as an active ingredient, wherein the stem cell exosome is prepared by the following method:
1) Performing subculture on human umbilical cord blood mesenchymal stem cells in a culture medium, selecting 3-5 generations of mesenchymal stem cells to culture in a DMEM/F12 culture medium until the cell fusion rate reaches 90-95%, and digesting and recovering the cells;
2) Inoculating the digested cells into an induction culture medium for induction culture, wherein the induction culture reagent added into the induction culture medium comprises 10-15ng/ml active peptide, 1-5ng/ml plant thiopeptide hormone-alpha, 50-100ng/ml epidermal growth factor, 0.1-0.3 mu mol/ml 3-isobutyl-1-methylxanthine, and inoculating mesenchymal stem cellsCulture medium with 5% CO 2 Culturing at 37 ℃ for 48-72 hours, recovering culture supernatant, filtering, and separating and purifying by an ultracentrifugation method to obtain stem cell exosomes.
Further, the stem cell exosomes are used in a mass percentage of 0.007-0.012% by mass of protein.
Further, the mass percentage of the stem cell exosomes is 0.0095% based on the mass of protein.
Further, the active peptide is glutathione.
Further, the basic additive comprises 0.5-0.8% of sea fennel extract, 0.1-0.3% of ceramide, 0.05-0.2% of astaxanthin, 1-3% of lycium barbarum polysaccharide, 0.5-1% of vitamin E and 0.5-1% of carbomer by weight percent.
Further, the separation and purification method comprises the following steps:
1) Centrifuging 500-700g of the collected culture supernatant for 8-10min, retaining the supernatant, discarding the precipitate, and removing cells in the culture solution;
2) Centrifuging 1500-2000g of the obtained supernatant for 15-20min, retaining the supernatant, discarding the precipitate, and removing cell debris;
3) Centrifuging 10000-20000g of the obtained supernatant for 50-90min, retaining the supernatant, discarding the precipitate, and removing cell debris;
4) Centrifuging 120000-200000g of the obtained supernatant for 80-130min, discarding the supernatant, and retaining the precipitate as extracted stem cell exosomes.
The invention also provides an application of the stem cell exosome preparation for skin repair in preparing skin repair medicines.
Compared with the prior art, the stem cell exosome preparation for skin repair has the following advantages:
the invention cultures stem cells by adopting specific components as an induction reagent, can influence the activity and purity of exosomes generated by the stem cells, and the stem cell exosomes prepared by the invention can effectively promote the secretion of collagen, and the preparation added with the exosomes is more suitable for skin repair and is more beneficial to scar desalination.
Detailed Description
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
The present invention will be described in detail with reference to examples.
Example 1
The stem cell exosomes are prepared by the following method:
1) Human umbilical cord blood mesenchymal stem cells are inoculated in DMEM/F12 medium for subculture, and the inoculation density is 4 multiplied by 10 4 Individual cells/ml, culture conditions were 5% CO 2 37 ℃; then selecting 3-5 generations of mesenchymal stem cells to be cultured in a DMEM/F12 culture medium again, and after the cell fusion rate reaches 90-95%, digesting the cells for about 1min by using 0.25% pancreatin, and blowing and beating the cells by adding a stopping solution;
2) The digested cells with good growth state are selected and inoculated into an induction culture medium, wherein the induction culture medium is DMEM/F12 culture medium added with 11ng/ml glutathione, 2.5ng/ml plant thiopeptide hormone-alpha, 100ng/ml epidermal growth factor and 0.15 mu mol/ml 3-isobutyl-1-methylxanthine, and the DMEM/F12 culture medium is placed in 5% CO 2 Culturing at 37deg.C for 72 hr, changing culture medium every 24 hr, and recovering culture supernatant after culturing;
3) Separating and purifying the filtered stem cell exosomes by an ultracentrifugation method; the method comprises the following specific steps: centrifuging 500g of the collected culture supernatant for 10min, reserving the supernatant, discarding the precipitate, and removing cells in the culture solution; centrifuging 2000g of the obtained supernatant for 20min, retaining the supernatant, discarding the precipitate, and removing cell debris; centrifuging 20000g of the obtained supernatant for 90min, retaining the supernatant, discarding the precipitate, and removing cell debris; the resulting supernatant was centrifuged at 150000g for 120min, the supernatant was discarded, and the pellet was retained as extracted stem cell exosomes and stored at below 4 ℃ for further use.
Human umbilical cord blood mesenchymal stem cells were purchased from the marsupenario life technologies, inc., cat: CP-H165.
Example 2
On the basis of the above example 1, the induction culture reagent added to the induction culture medium was 10ng/ml glutathione, 4ng/ml plant thiopeptide hormone-. Alpha.80 ng/ml epidermal growth factor, 0.3. Mu. Mol/ml 3-isobutyl-1-methylxanthine.
Example 3
On the basis of the above example 1, the induction culture reagent added to the induction culture medium was 15ng/ml glutathione, 1ng/ml plant thiopeptide hormone-. Alpha., 90ng/ml epidermal growth factor, 0.2. Mu. Mol/ml 3-isobutyl-1-methylxanthine.
Example 4
On the basis of the above example 1, the induction culture reagent added to the induction culture medium was 15ng/ml glutathione, 3ng/ml plant thiopeptide hormone-. Alpha., 50ng/ml epidermal growth factor, 0.3. Mu. Mol/ml 3-isobutyl-1-methylxanthine.
Comparative example 1
Based on example 1 above, only basal DMEM/F12 medium was used as the medium.
Comparative example 2
On the basis of the above example 1, the induction culture reagent added to the induction medium was 11ng/ml glutathione, 100ng/ml epidermal growth factor, 0.15. Mu. Mol/ml 3-isobutyl-1-methylxanthine.
Comparative example 3
Based on the above example 1, the induction culture reagent added to the induction culture medium was 11ng/ml glutathione, 2.5ng/ml plant thiopeptide hormone-. Alpha.and 100ng/ml epidermal growth factor.
Comparative example 4
On the basis of the above example 1, the induction culture reagent added to the induction medium was 2.5ng/ml plant thiopeptide hormone-. Alpha., 100ng/ml epidermal growth factor, 0.15. Mu. Mol/ml 3-isobutyl-1-methylxanthine.
The exosomes obtained in examples 1 to 4 and comparative examples 1 to 4 were quantitatively detected for proteins by BCA method, and the amounts of exosomes were calculated from the quantitative protein values.
Table 1 example and comparative exosome protein content table
| Group of | Protein concentration mg/ml |
| Example 1 | 0.907638 |
| Example 2 | 0.852841 |
| Example 3 | 0.860354 |
| Example 4 | 0.836087 |
| Comparative example 1 | 0.443842 |
| Comparative example 2 | 0.713596 |
| Comparative example 3 | 0.792863 |
| Comparative example 4 | 0.807439 |
As can be seen from Table 1, both examples and comparative examples can obtain mesenchymal stem cell exosomes, but the protein concentration of the stem cell exosomes obtained in the examples is higher than that of the comparative examples, indicating that the purity of the stem cell exosomes prepared by the method of the present invention is higher. Whereas the induction medium of comparative examples 2-4 lacks certain components, the purity of the stem cell exosomes is significantly reduced.
Pharmacological experiment-human fibroblast I, III type collagen secretion influence experiment
And (2) adding 1000 human fibroblasts into each hole of a cell culture pore plate, adding a DMEM/F12 culture medium, changing the liquid once every 48 hours, respectively adding 10 mu L of test liquid after the cells are attached, setting a blank control group, namely adopting an equal volume of PBS buffer solution to culture 10d, measuring the I, III type collagen content by an ELASA method, and carrying out data statistical analysis, wherein the protein content in the test liquid is 30 mu g/mL.
Table 2I type III collagen content Table
| Test solution | Type I collagen ng/mL | Type III collagen ng/mL |
| Example 1 | 458.96±48.78 | 99.51±8.65 |
| Example 2 | 426.15±40.26 | 85.49±7.37 |
| Example 3 | 419.53±42.64 | 86.73±7.31 |
| Example 4 | 431.04±39.57 | 81.65±6.98 |
| Comparative example 1 | 298.34±34.18 | 54.37±4.28 |
| Comparative example 2 | 224.26±31.83 | 48.81±4.06 |
| Comparative example 3 | 247.13±33.49 | 48.26±4.19 |
| Comparative example 4 | 265.67±30.95 | 42.86±3.74 |
| PBS buffer | 156.39±20.64 | 17.86±4.42 |
According to the prior art, the type of collagen in the skin is mainly type I collagen and type III collagen, the type III collagen belongs to reconstruction type collagen, and in the scar repairing process, more type III collagen is expressed, so that the scar repairing can be promoted. As can be seen from Table 2, the stem cell exosomes prepared in examples 1-4 better promote collagen secretion and are more suitable for skin repair.
Example 5
A stem cell exosome preparation for skin repair, comprising a basic additive and the stem cell exosome prepared in example 1 as an active ingredient, the stem cell exosome being used in an amount of 0.0095% by mass based on the mass of protein; the basic additive comprises 0.6% of sea fennel extract, 0.15% of ceramide, 0.2% of astaxanthin, 2% of lycium barbarum polysaccharide, 0.6% of vitamin E, 0.8% of carbomer and the balance of water in percentage by weight.
Example 6
On the basis of the above example 5, the stem cell exosomes were the exosomes prepared in example 2.
Example 7
On the basis of the above example 5, the stem cell exosomes were the exosomes prepared in example 3.
Example 8
On the basis of the above example 5, the stem cell exosomes were the exosomes prepared in example 4.
Comparative example 5
Based on the above example 5, the stem cell exosomes were exosomes prepared in comparative example 1.
Comparative example 6
Based on the above example 5, the stem cell exosomes were exosomes prepared in comparative example 1.
Comparative example 7
Based on the above example 5, the stem cell exosomes were exosomes prepared in comparative example 1.
Comparative example 8
Based on the above example 5, the stem cell exosomes were exosomes prepared in comparative example 1.
Wound repair experiments
Male Wistar rats of 4 months of age were purchased from the laboratory animal center at medical university. Laboratory feeding is carried out 1 week before the experiment, the day of the experiment is fasted, 3% pentobarbital sodium is injected into 30mg/kg abdominal cavity for anesthesia, the back of a rat is shaved, the area is 7cm multiplied by 6cm, then a special traumatizer puncher is used for cutting 4 circular ulcers at two sides of the spine in the middle of the back, the diameter is 1.8cm, and the deep is subcutaneous, and hemostasis is achieved. After the experimental animals are anesthetized and revived, the experimental animals are continuously fed in the original environment. Dynamically observing living and wound conditions of each animal, and completely epithelizing the wound surface 28 days after operation to form a hypertrophic scar which is successfully molded.
45 well-conditioned test rats were randomly divided into 9 groups of 5 animals each, experimental groups 1-4, control groups 1-4 and blank groups. The administration method comprises the following steps: the formulations obtained in examples 5 to 8 and comparative examples 5 to 8 were applied to the wound sites of the experimental animals of the respective groups, respectively, 3 times per day, and gently to completely absorbed after the application. The recovery state of the skin was observed after 60 days.
The results showed that the formulations prepared using the exosomes prepared in examples 1-4 showed the best skin recovery effect with a light scar imprint, but no apparent scar, especially with the formulation of example 5, the most minimal scar. The other comparative examples show relatively obvious scars, and especially the preparation prepared in comparative example 5 has the worst effect and the scars are obvious.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (4)
1. A stem cell exosome formulation for skin repair, characterized by: comprises a basic additive and a stem cell exosome as an active ingredient, wherein the stem cell exosome is prepared by the following method:
1) Performing subculture on human umbilical cord blood mesenchymal stem cells in a culture medium, selecting 3-5 generations of mesenchymal stem cells to culture in a DMEM/F12 culture medium until the cell fusion rate reaches 90-95%, and digesting and recovering the cells;
2) Inoculating the digested cells into an induction medium for induction culture, selecting the digested cells with good growth state, inoculating into the induction medium for induction culture, and inoculating with the density of 4×10 4 The induction culture reagent added into the induction culture medium is 10-15ng/ml glutathione, 1-5ng/ml plant thiopeptide hormone-alpha, 50-100ng/ml epidermal growth factor, 0.1-0.3 mu mol/ml 3-isobutyl-1-methylxanthine, and the culture inoculated with the mesenchymal stem cells is based on 5% CO 2 Culturing at 37 ℃ for 48-72 hours, recovering culture supernatant, filtering, and separating and purifying by an ultracentrifugation method to obtain stem cell exosomes;
the mass percentage of the stem cell exosome is 0.007-0.012% of the protein mass;
the basic additive comprises 0.5-0.8% of sea fennel extract, 0.1-0.3% of ceramide, 0.05-0.2% of astaxanthin, 1-3% of lycium barbarum polysaccharide, 0.5-1% of vitamin E and 0.5-1% of carbomer by weight percentage.
2. The stem cell exosome formulation for skin repair of claim 1, wherein: the mass percentage of stem cell exosomes based on the mass of protein is 0.0095%.
3. The stem cell exosome formulation for skin repair of claim 1, wherein: the separation and purification method comprises the following steps:
1) Centrifuging 500-700g of the collected culture supernatant for 8-10min, retaining the supernatant, discarding the precipitate, and removing cells in the culture solution;
2) Centrifuging 1500-2000g of the obtained supernatant for 15-20min, retaining the supernatant, discarding the precipitate, and removing cell debris;
3) Centrifuging 10000-20000g of the obtained supernatant for 50-90min, retaining the supernatant, discarding the precipitate, and removing cell debris;
4) Centrifuging 120000-200000g of the obtained supernatant for 80-130min, discarding the supernatant, and retaining the precipitate as extracted stem cell exosomes.
4. Use of a stem cell exosome formulation for skin repair according to any one of claims 1-3 in the preparation of a skin repair medicament.
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