CN112891293A - Preparation method and application of human amniotic stem cell culture - Google Patents
Preparation method and application of human amniotic stem cell culture Download PDFInfo
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- CN112891293A CN112891293A CN202110116493.5A CN202110116493A CN112891293A CN 112891293 A CN112891293 A CN 112891293A CN 202110116493 A CN202110116493 A CN 202110116493A CN 112891293 A CN112891293 A CN 112891293A
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- 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
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- 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
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- 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
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
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- 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
- C12N5/06—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
- C12N5/0668—Mesenchymal stem cells from other natural sources
Abstract
The invention relates to the technical field of biomedicine, and discloses a preparation method of a human amniotic stem cell culture, which comprises the steps of firstly culturing human amniotic stem cell culture medium for human amniotic stem cells which are separated and extracted, changing a serum-free culture medium to continuously culture the human amniotic stem cells for 48 hours when the human amniotic stem cells are expanded to 90% of cell confluence degree, then collecting supernatant (namely the culture), and concentrating by 10 times to obtain the human amniotic stem cell culture; the invention also discloses an application. The invention discovers and proves for the first time that the human amniotic stem cell culture has the effects of whitening skin, brightening skin color and keeping skin healthy and natural, has good prevention and treatment effects on melanin deposition caused by endocrine dyscrasia or chemical substance stimulation or physical method stimulation, has safe components, no immunological rejection and tumorigenic effect, has no obvious toxic or side effect on human bodies, and has simple preparation method, controllable operation process and important industrial value.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a preparation method and application of a human amniotic stem cell culture.
Background
Melanin deposition is the excessive deposition of abnormally increased melanin in certain parts of the skin, which results in the formation of pigmented spots, age spots, and the like. Ultraviolet radiation, endocrine dyscrasia, bad living habits and the like can cause skin hypoelasticity, pigmentation and dark whole skin color, seriously affect the beauty of human skin, and particularly under the influence of 'white as beautiful' Asia culture, white skin plays a very important role in aesthetic sense. Therefore, the market of whitening and skin care is huge all over the world, the proportion of whitening and spot-lightening is the highest on the basis of the efficacy type skin care products, and the annual income can reach hundreds of millions of dollars by only selling the whitening products by many multinational companies. Therefore, finding ingredients having melanin deposition inhibiting and whitening effects has been a hot spot of scientific research and product development.
Melanin formation is a complex biochemical process, and an imbalance between melanin synthesis and degradation is a major cause of pigmentation. Tyrosinase plays a crucial role in melanin synthesis, and therefore, inhibition of tyrosinase expression and activity has become the most effective method for inhibiting melanin deposition. Currently, the most common whitening agents in the market are tyrosinase inhibitors such as kojic acid, arbutin and vitamin C, which can reduce the amount of oxidized melanin intermediates. In addition, alpha-hydroxy acids are also commonly used for skin lightening. However, these compounds all suffer from the following significant disadvantages: kojic acid has sensitization and potential carcinogenic risk, arbutin is hydroquinone derivative, hydroquinone is oxidized into toxic semiquinone substance under the action of tyrosinase, and can cause skin erythema, desquamation, pruritus, stabbing pain and allergy, even cause permanent damage to skin, and has carcinogenicity; vitamin C derivatives are effective only at high concentrations and are difficult to stabilize in cosmetic formulations; alpha-hydroxy acids are very irritating. In contrast, there is currently no formulation product that promotes melanin degradation.
In view of the above, there is a need to find a novel whitening preparation which can be applied in whitening skin care products and is safe and effective.
Disclosure of Invention
Based on the problems, the preparation method and the application of the human amniotic membrane stem cell culture provided by the invention have no immunological rejection and oncogenic action, make up for the defects of high sensitization and high carcinogenic risk of the whitening products on the market, fill up the vacancy of the whitening market, are obviously superior to the whitening additives in the skin care products on the market, and have outstanding advantages.
In order to solve the technical problems, the invention provides the following technical scheme:
the preparation method of the human amniotic membrane stem cell culture comprises the following specific steps:
a. inoculating the human amniotic stem cells into a cell culture dish containing a complete culture medium for culture, and discarding the complete culture medium when the cell confluency is 90%;
b. washing with PBS for 3 times, adding DMEM serum-free culture medium, and culturing for 48 hr;
c. collecting supernatant, namely the primary obtained human amniotic stem cell culture, and centrifuging for 3min at 4 ℃ and 1000rpm to remove cell debris;
d. and (3) placing the supernatant into a protein concentration tube, carrying out centrifugal concentration at 4 ℃ for 70min at 4500g to obtain a final concentrate, namely the human amniotic stem cell culture, and placing the final concentrate at-80 ℃ for storage for later use.
Further, the cell density in the DMEM serum-free medium is at least 2x 10 when the supernatant is collected in step c5one/mL, the concentration in step d is 10-fold.
Further, the human amniotic stem cells are human amniotic epithelial stem cells or human amniotic mesenchymal stem cells.
In order to solve the technical problems, the invention also provides application of the human amniotic stem cell culture in preparing whitening skin care products.
Further, the whitening skin care product can be used for inhibiting melanin deposition caused by endocrine dyscrasia or chemical substance stimulation or physical method stimulation, wherein the inhibition mechanism is to inhibit melanin synthesis and promote melanin degradation.
Further, the chemical substance is alpha-melanocyte stimulating hormone, and the physical method is ultraviolet irradiation.
Compared with the prior art, the invention has the beneficial effects that: the active ingredients in the stem cell culture have the effects of whitening skin, brightening skin color and keeping skin healthy and natural, have good prevention and treatment effects on melanin deposition caused by endocrine dyscrasia or chemical substance stimulation or physical method stimulation, are safe in ingredients, have no immunological rejection and oncogenic effect, have no obvious toxic or side effect on human bodies, make up the defects of high sensitization and high carcinogenic risk of whitening products on the market, fill the vacancy of the whitening market, are obviously superior to whitening additives in skin care products on the market, have outstanding advantages, are simple in preparation method, controllable in operation process and have important industrial value.
Drawings
FIG. 1 is a plate comparison graph and a bar graph of the amount of melanin secretion inhibited by human amniotic mesenchymal stem cells and human amniotic epithelial stem cells according to an embodiment of the present invention;
FIG. 2 is a schematic and bar graph of human amniotic mesenchymal stem cells and human amniotic epithelial stem cells of embodiments of the invention for inhibiting melanin content;
FIG. 3 shows a medium conditioned on human amniotic mesenchymal stem cells and a medium conditioned on human amniotic epithelial stem cells according to an embodiment of the present invention (cell density 2X 10)5Individual cells/mL) histogram of the amount and content of melanin secretion inhibited;
FIG. 4 is a graph showing the results of the inhibition of melanin secretion by the human amniotic mesenchymal stem cell conditioned medium and the epithelial stem cell conditioned medium at different concentrations according to the example of the present invention;
FIG. 5 is a graph showing the results of the inhibition of melanin content by different concentrations of human amniotic mesenchymal stem cell conditioned medium and epithelial stem cell conditioned medium according to the embodiment of the present invention;
FIG. 6 is a graph showing the results of the tyrosinase activity inhibition assay using the human amniotic mesenchymal stem cell conditioned medium and the human amniotic epithelial stem cell conditioned medium according to the embodiment of the present invention;
FIG. 7 is an apparent chromaticity comparison chart of an embodiment of the present invention;
fig. 8 is a comparison chart of apparent luminance (L value) of an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
Example (b):
in this embodiment, human amniotic stem cells, including human amniotic mesenchymal stem cells and human amniotic epithelial stem cells, are obtained by separating and purifying the human amniotic stem cells after human delivery, and after in vitro culture, the supernatant containing various stem cell secretion factors is collected, and after appropriate concentration and other treatments, the final human amniotic stem cell culture is obtained.
The separation and extraction method of the human amniotic mesenchymal stem cells and the human amniotic epithelial stem cells comprises the following steps:
1) under aseptic conditions, the human amniotic membrane is stripped from the human placental tissue after delivery and washed with D-Hank's solution containing 1% double antibody (penicillin/streptomycin) to remove blood filaments and sticky substances from the amniotic membrane;
2) cutting cleaned amnion, adding pancreatin, and digesting at 37 deg.C for 45 min until amnion turns white;
3) taking the supernatant, centrifuging at 1500rpm for 5 minutes;
4) taking the precipitate, re-suspending the precipitate by using a human amniotic epithelial cell culture medium, cleaning, and centrifuging at 1500rpm for 5 minutes;
5) repeating the step (4);
6) taking the sediment, namely human amniotic epithelial stem cells (hAESCs);
7) the remaining amniotic membrane is continuously digested for 45 minutes by pancreatin, and the remaining human amniotic epithelial stem cells are thoroughly removed;
8) the remaining amnion is continuously digested by collagenase 4 until the tissue is melted and is sticky;
9) filtering with 200 mesh sieve, collecting filtrate, centrifuging at 3000rpm for 5 min;
10) taking the precipitate, resuspending and cleaning the precipitate by using an amniotic mesenchymal stem cell culture medium, and centrifuging the precipitate at 1000rpm for 3 min;
11) repeating the step (10);
12) taking the sediment, using an amniotic mesenchymal stem cell culture medium for heavy suspension and plating, selecting to change the solution and wash the solution for 6 hours according to the adherence rate of the isolated primary human amniotic mesenchymal stem cells, namely discarding the supernatant (containing the cells which are not adhered to the wall), wherein the rest adherent cells are human amniotic mesenchymal stem cells (hAMSCs).
The formula of the amnion mesenchymal stem cell culture medium is as follows:
the formula of the amniotic epithelial stem cell culture medium is as follows:
identification of the surface marker of the human amniotic membrane stem cell: in the embodiment, the human amniotic epithelial stem cells obtained by the method are cultured in vitro to be oval, and the human amniotic mesenchymal stem cells are in a long fusiform shape. The results of RT-PCR, flow and immunofluorescence detection of human amniotic epithelial stem cells and human amniotic mesenchymal stem cells show that the human amniotic epithelial stem cells and the human amniotic mesenchymal stem cells can express embryonic stem cell biomarkers SSEA-4, Nanog and OCT-4, mesenchymal stem cell biomarkers CD73, CD105, CD29 and CD90, hematopoietic stem cell markers CD34 and CD45 are not expressed, major histocompatibility protein HLA-ABC is expressed, and major histocompatibility protein HLA-DR is not expressed. The identification result shows that healthy human amniotic epithelial stem cells and human amniotic mesenchymal stem cells are obtained in the embodiment.
In this embodiment, the whitening effect of the human amniotic epithelial stem cells and the human amniotic mesenchymal stem cells obtained in this embodiment is tested: human amniotic mesenchymal stem cells and human amniotic epithelial stem cells are respectively co-cultured with mouse melanoma cells B16-F10, and after 48 hours, the content and the secretion amount of melanin in the mouse melanoma cells B16-F10 added with hAMSCs and hAMSCs are detected under the pathological condition stimulated by alpha-melanocyte-stimulating hormone (alpha-MSH). The test results are shown in figures 1 and 2, wherein Control is a Control group, hAMSCs are human amniotic mesenchymal stem cells, hAMSCs are human amniotic epithelial stem cells, and alpha-MSH is alpha-melanocyte-stimulating hormone. The results show that both hAMSCs and hESCs can obviously reduce the content of melanin in cells and obviously inhibit the secretion of the melanin, and the culture of the hAMSCs and the hESCs is suitable for being used as a whitening active agent in skin care products.
The whitening effect test and the screening experiment of the optimal effective concentration of the human amniotic epithelial stem cell culture and the human amniotic mesenchymal stem cell culture are as follows: collecting culture medium after culturing human amniotic mesenchymal stem cells and epithelial stem cells, concentrating by 10 times, diluting properly, adding the diluted solution into mouse melanoma cells B16-F10, and detecting the change of melanin content and secretion. The results are shown in FIG. 3, FIG. 4 and FIG. 5, wherein 0.1 represents the culture medium concentration of 2X 104Individual cells/mL, 0.2 for medium concentration 4X 104Individual cells/mL, and so on; hAMSC-CM is human amniotic mesenchymal stem cell conditioned medium (human amniotic mesenchymal stem cell culture), hAMSC-CM is human amniotic epithelial stem cell conditioned medium (human amniotic epithelial stem cell culture), and the concentration of the human amniotic stem cell conditioned medium is 1 ml of stem cell culture (namely supernatant) obtained by culturing a plurality of stem cells in a serum-free medium for 48 hours. The results show that the concentration of the culture medium of the human amniotic mesenchymal stem cells and the human amniotic epithelial stem cells is 2x 105The secretion and the generation of melanin can be obviously inhibited when each cell/mL is detected, namely, the whitening effect is very good at the concentration.
According to the screening test, the preparation method of the human amniotic stem cell culture prepared by using the obtained human amniotic stem cells in the embodiment comprises the following steps:
a. inoculating human amniotic stem cells including human amniotic epithelial stem cells or human amniotic mesenchymal stem cells into a cell culture dish (the culture medium is the corresponding culture medium, and can also be other culture media), and culturing, and discarding the complete culture medium when the cell confluence degree is about 90%;
b. washing with PBS for 3 times, adding DMEM basal medium (serum-free medium), and culturing for 48 hr;
c. collecting supernatant (i.e. primary obtained human amniotic stem cell culture), and centrifuging at 4 deg.C and 1000rpm for 3min to remove cell debris;
d. and (3) putting the supernatant into a protein concentration tube, concentrating at 4 ℃, 4500g and 70min to obtain a final concentrate, namely the human amniotic membrane stem cell culture, and storing at-80 ℃ for later use.
In this embodiment, the purpose of concentrating the initially obtained human amniotic membrane stem cell culture is to facilitate subsequent application, and a corresponding solvent or culture medium is selected for dilution during application. The cell density in serum-free medium at the time of supernatant collection in step c above in this example was at least 2X 105The concentration ratio per mL in step d of this embodiment is 10 times, but not limited to 10 times, and the obtained human amniotic stem cell culture is a human amniotic mesenchymal stem cell conditioned medium or a human amniotic epithelial stem cell conditioned medium, which will be mentioned below.
In this example, the tyrosinase activity inhibition rate was tested using the human amniotic mesenchymal stem cell conditioned medium and the human amniotic epithelial stem cell conditioned medium obtained as described above: adding a human amniotic mesenchymal stem cell conditioned medium or a human amniotic epithelial stem cell conditioned medium into a mouse melanoma cell B16-F10, measuring the activity of mushroom tyrosinase after 48 hours, sequentially adding 50 mu L of tyrosinase solution, 90 mu L of phosphate buffer solution, 60 mu L of tested solution and 100 mu L of levodopa solution into a 96-well plate, and measuring the absorbance value at the wavelength of 475nm by using an ultraviolet spectrophotometer, wherein the total reaction system is 300 mu L. The results are shown in figure 6, and show that the tyrosinase activity inhibition rates of the human amniotic mesenchymal stem cell conditioned medium and the human amniotic epithelial stem cell conditioned medium are 70% and 53%, respectively.
This example also tested the whitening effect on human amniotic mesenchymal stem cell conditioned medium at the animal level: irradiating ears of C57BL/6 mouse with 20W UVB lamp tube at a dose of 150mJ/cm2After 7 days, the reaction mixture was,injecting the human amniotic mesenchymal stem cell conditioned medium concentrated solution subcutaneously, and continuing to irradiate for 7 days by using a UVB lamp tube. The result shows that the concentrated solution of the human amniotic mesenchymal stem cell conditioned medium has an obvious inhibition effect on melanin deposition caused by UVB, and a good whitening effect is achieved.
The 3D melanin skin model is a tool for effectively detecting the influence of a tested medicament on human skin melanin, and the proper stimulus can induce the melanin deposition of the 3D melanin skin model (simulating the melanin deposition in the skin stress process). 50mJ/cm per day on the day of model shipment (defined as day 0)2And (3) UVB stimulation treatment, namely uniformly coating the concentrated solution of the conditioned medium of the human amniotic mesenchymal stem cells on the surface of a melanin skin model in a surface administration mode after continuous stimulation is carried out for 3 days, wherein the administration volume is 10 mu L, the administration is carried out for 1 time every 2 days, and the whitening effect of the conditioned medium of the human amniotic mesenchymal stem cells is determined by detecting the apparent chromaticity and the apparent brightness (L value) on the 7 th day.
The results shown in fig. 7 and fig. 8 show that the human amniotic mesenchymal stem cell conditioned medium can obviously inhibit melanin deposition of the 3D melanin human skin model caused by UVB irradiation, which shows that the apparent chromaticity and apparent brightness of the human skin model are obviously improved, and the whitening and skin-brightening effects are remarkable.
This example shows that the human amniotic membrane stem cell culture of this example can be applied to prepare whitening skin care products, which can be used to inhibit melanin deposition caused by endocrine dyscrasia or chemical substance stimulation or physical method stimulation, wherein the inhibition mechanism is to inhibit melanin synthesis and promote melanin degradation, but not limited to the above-mentioned reasons causing melanin deposition.
The above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the verification process of the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all equivalent structural changes made by using the contents of the specification and the drawings of the present invention should be covered by the scope of the present invention.
Claims (6)
1. The preparation method of the human amniotic membrane stem cell culture is characterized by comprising the following steps of:
a. inoculating the human amniotic stem cells into a cell culture dish containing a complete culture medium for culture, and discarding the complete culture medium when the cell confluency is 90%;
b. washing with PBS for 3 times, adding DMEM serum-free culture medium, and culturing for 48 hr;
c. collecting supernatant, namely the primary obtained human amniotic stem cell culture, and centrifuging for 3min at 4 ℃ and 1000rpm to remove cell debris;
d. and (3) placing the supernatant into a protein concentration tube, carrying out centrifugal concentration at 4 ℃ for 70min at 4500g to obtain a final concentrate, namely the human amniotic stem cell culture, and placing the final concentrate at-80 ℃ for storage for later use.
2. The method for preparing human amniotic stem cell culture according to claim 1, wherein the cell density in the DMEM serum-free medium is at least 2x 10 when the supernatant is collected in step c5one/mL, the concentration in step d is 10-fold.
3. The method of claim 1, wherein the human amniotic stem cell culture is a human amniotic epithelial stem cell or a human amniotic mesenchymal stem cell.
4. Use of the human amniotic stem cell culture according to any one of claims 1 to 3 for preparing a whitening skin care product.
5. The use according to claim 4, wherein the whitening skin care product is used for inhibiting melanin deposition caused by endocrine dyscrasia or chemical substance stimulation or physical method stimulation, wherein the inhibition mechanism is inhibition of melanin synthesis and promotion of melanin degradation.
6. The use according to claim 4, wherein the chemical substance is alpha-melanocyte stimulating hormone and the physical means is ultraviolet irradiation.
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