CN106983713B - Stem cell skin care composition and preparation method and using method thereof - Google Patents
Stem cell skin care composition and preparation method and using method thereof Download PDFInfo
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- CN106983713B CN106983713B CN201710209077.3A CN201710209077A CN106983713B CN 106983713 B CN106983713 B CN 106983713B CN 201710209077 A CN201710209077 A CN 201710209077A CN 106983713 B CN106983713 B CN 106983713B
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Abstract
The invention discloses a stem cell skin care composition, which consists of a component A and a component B, wherein the component A is 1-800 parts by mass and comprises a stem cell extract, serum albumin, mannitol, Arg9 membrane-penetrating peptide, and the component A is obtained by freeze drying, wherein the stem cell extract is a stem cell culture medium after stem cells are cultured and a freeze-thaw lysate of the cultured stem cells; the component B comprises the following components in parts by mass: glycerin, sodium citrate, ellagic acid, vitamin E, and water, and when in use, the A component and the B component are mixed. The invention also discloses a preparation method and a using method thereof. The factors of the invention have synergistic effect, and can effectively realize that the factors penetrate the skin, enhance the metabolism of the skin from the deep layer, delay the skin aging, and achieve the purposes of whitening, preventing or improving skin spots and wrinkles, removing pox and preventing allergy.
Description
Technical Field
The invention relates to the technical field of skin care products, in particular to a stem cell skin care composition and a preparation method and a using method thereof.
Background
With the continuous improvement of living standard, people pay more and more attention to health, especially to skin care. The skin tissue is the largest tissue organ of the human body, covers the surface of the human body and blocks the damage effect of all harmful substances in the nature on living organisms. However, with the increase of age, the functions of skin tissues and organs are gradually aged, and from the aesthetic point of view, the enthusiasm of people for life is seriously influenced; from a physiological point of view, effective protection of the internal organs of the human body is affected. Thus, various beauty and skin care products are produced.
Stem cells are cells with proliferative and differentiative potential, have the ability to self-renew and replicate, and are capable of producing highly differentiated functional cells. In addition to having a high differentiation potential, stem cells can secrete a variety of cytokines including Epidermal Growth Factor (EGF), platelet-derived growth factor (PDGF), Transforming Growth Factor (TGF), basic fibroblast growth factor (b-FGF), insulin-like growth factor (IGF), and Nerve Growth Factor (NGF), among others. The cytokines can promote the metabolism of human skin, promote the regeneration and repair of the skin, delay the aging of the skin and restore the elasticity and luster of the skin. However, most of the related beauty skin care products at present are one or two cytokines added into the beauty skin care products. The cell factor repair of the skin is a complex systematic regulation process, one or two cell factors cannot meet the requirements of skin repair and cell renewal, and a plurality of factors can be combined to promote the epidermal renewal. Moreover, these factors are not modified and cannot penetrate the skin, and thus do not actually function.
Disclosure of Invention
The invention aims to provide a stem cell skin care composition, a preparation method and a using method thereof, so as to solve the defects of the prior art.
The invention adopts the following technical scheme:
a skin care composition containing stem cells comprises component A and component B,
the component A comprises 1-800 parts by mass of stem cell extract, serum albumin, mannitol and Arg9 membrane-penetrating peptide, wherein the stem cell extract is obtained by freeze-drying, and the stem cell extract is a stem cell culture medium after stem cells are cultured and a freeze-thaw lysate of the cultured stem cells;
the component B comprises the following components in parts by mass:
when in use, the component A and the component B are mixed.
Furthermore, the serum albumin accounts for 1 percent of the total mass of the stem cell extract, the mannitol accounts for 5 percent of the total mass of the stem cell extract, and the Arg9 membrane-penetrating peptide accounts for 0.05 percent of the total mass of the stem cell extract.
Further, the preparation of the stem cell extract comprises stem cell origin expression, plasmid electrotransfer mediated stem cell factor expression, lentivirus mediated stem cell factor expression or adenovirus mediated stem cell factor expression, wherein when the plasmid electrotransfer mediated stem cell factor expression, the lentivirus mediated stem cell factor expression or the adenovirus mediated stem cell factor expression is prepared, the stem cell factor and the transmembrane peptide gene series are fused to construct a new fusion gene, and then the subsequent steps are carried out, and the cytokine and the transmembrane peptide gene series can be inquired in a codon table through amino acid series.
Further, the stem cell includes umbilical cord mesenchymal stem cells of animal or human origin, bone marrow mesenchymal stem cells, adipose stem cells, hematopoietic stem cells, neural stem cells or hepatic stem cells.
Further, the types of membrane-penetrating peptides and amino acid series include:
transmembrane peptide type 1: tat: RKKRRQRRR;
transmembrane peptide type 2: VP-22: DAATARGRGRSAASRPTERPRAPARSASRPRRPVD, respectively;
transmembrane peptide type 3: NTP: RQIKIWFQNRRMKWKK;
transmembrane peptide type 4: transportan: GWT LNSAGYLLGKINLKALAALAKKIL, respectively;
transmembrane peptide type 5: PEP-1: KETWWETWWTEWSQPKKKRKV;
transmembrane peptide type 6: MPG: GALFLGFLGAAGSTMGAWS QPKKKRKV, respectively;
transmembrane peptide type 7: MAP: KLALKLALKALKAALKLA:
transmembrane peptide type 8: KALA: WEAKLAKALAKALAKHLAKALAKALKACEA, respectively;
transmembrane peptide type 9: ppTG 20: GLFRALLRLLRS LWRLLLRA, respectively;
transmembrane peptide type 10: arg 9: RRRRRRRRR, respectively; or
Transmembrane peptide type 11: hCT (9-32): LGTYTQDFNKFHTFPQTAIGVGAP are provided.
Further, the vectors used for expressing the stem cell factor mediated by plasmid electrotransfer comprise EGFP-N1, N-P3xflag-CMV or pcDNA3.1; vectors for lentiviral-mediated stem cell factor expression include pHBLV-CMVIE-Puro, PLVX-TRE3G, lentiviral packaging system PLVX-Ac-Puro, psPAX2, pMD2.G, pPACK-H1-Gag, pPACK-H1-Rev, pPACK-H1-Vsvg, PLP1, PLP2, PLP-VSVG, pMDLg/pRRE, pRes-Rev, pCMV-VSVG, pCMV-delta R8 or pCMV-VSVG; vectors for adenovirus-mediated stem cell factor expression include pDC315, pDC316, pDC311, pDC312, AdMax (TM) adenovirus vector system or AdMaxTM Hi-IQ adenovirus vector system.
Further, the serum albumin includes human serum albumin, animal serum albumin, recombinant human serum albumin, or recombinant animal serum albumin.
Further, the ellagic acid source includes red pomegranate.
The preparation method of the stem cell skin care composition comprises the following steps:
step 1, mixing a stem cell culture medium after stem cell culture, a freeze-thaw lysate of the cultured stem cells, serum albumin, mannitol and Arg9 membrane-penetrating peptide, and freeze-drying to obtain freeze-dried powder;
step 2, mixing glycerol, sodium citrate, ellagic acid, vitamin E and water uniformly to obtain a solution;
and 3, respectively packaging the freeze-dried powder prepared in the step 1 and the solvent liquid obtained in the step 2, and mixing when in use.
The application method of the stem cell skin care composition comprises the following steps: uniformly mixing the freeze-dried powder and the solvent liquid, and lightly shaking until the freeze-dried powder is completely dissolved, and then using; is taken in the morning and at night.
The invention has the beneficial effects that:
1. based on the research of skin metabolism and the analysis of skin aging, the invention selects proper components, dosage and the synergistic effect of a plurality of factors to recombine the deep elastic structure and function of the skin, quickly hydrolyzes the collagen which is aged and deformed and hardened, reestablishes the balance of the collagen, promotes the skin wrinkles to be quickly lightened, and ensures that the skin shows white, red and healthy color.
2. The invention fuses the gene of the cell factor and the gene series of the membrane-penetrating peptide, expresses a new fusion protein with the membrane-penetrating peptide, and simultaneously cooperates with the artificially synthesized ARG9 membrane-penetrating peptide, so that the factors can effectively realize the purposes of transmitting the factors through the skin, enhancing the metabolism of the skin from the deep layer, delaying the skin aging, whitening, preventing or improving skin spots and wrinkles, removing pox and preventing allergy.
3. The active ingredients (stem cell extract and serum albumin) of the invention are stored in a freeze-dried state and are matched with a solvent solution when in use. The cytokine as protein has long activity retention time in lyophilized state, and can be mixed with solvent solution for dissolving and promoting absorption.
4. The stem cell skin care composition is not added with any chemical harmful substance, heavy metal and hormone, and secondary damage caused by the permeation of the functional components of the skin care product to the skin (the skin becomes thin, the skin becomes speckles, the wrinkles increase and the like after the skin care composition is used for a certain time because the chemical harmful substance, the heavy metal and the hormone permeate into the skin chronically) can not occur. The invention achieves the aims of whitening, preventing or improving skin spots and wrinkles, removing acne and preventing allergy by mainly enhancing the metabolism capability of the skin and delaying skin aging, and has no side effect after long-term use. Is suitable for men and women of all ages.
Detailed Description
The present invention will be further explained with reference to examples. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.
A skin care composition containing stem cells comprises component A and component B,
the component A comprises 1-800 parts by mass of stem cell extract, serum albumin, mannitol and Arg9 membrane-penetrating peptide, wherein the stem cell extract is obtained by freeze-drying, and the stem cell extract is a stem cell culture medium after stem cells are cultured and a freeze-thaw lysate of the cultured stem cells; the serum albumin accounts for 1 percent of the total mass of the stem cell extract, the mannitol accounts for 5 percent of the total mass of the stem cell extract, and the Arg9 membrane-penetrating peptide accounts for 0.05 percent of the total mass of the stem cell extract;
the component B comprises the following components in parts by mass:
when in use, the component A and the component B are mixed.
The preparation of the stem cell extract comprises stem cell origin expression, plasmid electrotransfer mediated stem cell factor expression, lentivirus mediated stem cell factor expression or adenovirus mediated stem cell factor expression and the like, wherein when the plasmid electrotransfer mediated stem cell factor expression, the lentivirus mediated stem cell factor expression or the adenovirus mediated stem cell factor expression is prepared, the stem cell factor and the transmembrane peptide gene series are fused to construct a new fusion gene, and then the subsequent steps are carried out, and the cytokine and the transmembrane peptide gene series can be inquired in a codon table through amino acid series. The stem cells include mesenchymal stem cells of animal or human origin, adipose-derived stem cells, neural stem cells or hepatic stem cells, and the like. Transmembrane peptide species and amino acid series include, but are not limited to:
transmembrane peptide type 1: tat: RKKRRQRRR;
transmembrane peptide type 2: VP-22: DAATARGRGRSAASRPTERPRAPARSASRPRRPVD, respectively;
transmembrane peptide type 3: NTP: RQIKIWFQNRRMKWKK;
transmembrane peptide type 4: transportan: GWT LNSAGYLLGKINLKALAALAKKIL, respectively;
transmembrane peptide type 5: PEP-1: KETWWETWWTEWSQPKKKRKV;
transmembrane peptide type 6: MPG: GALFLGFLGAAGSTMGAWS QPKKKRKV, respectively;
transmembrane peptide type 7: MAP: KLALKLALKALKAALKLA:
transmembrane peptide type 8: KALA: WEAKLAKALAKALAKHLAKALAKALKACEA, respectively;
transmembrane peptide type 9: ppTG 20: GLFRALLRLLRS LWRLLLRA, respectively;
transmembrane peptide type 10: arg 9: RRRRRRRRR, respectively; or
Transmembrane peptide type 11: hCT (9-32): LGTYTQDFNKFHTFPQTAIGVGAP are provided.
The serum albumin comprises human serum albumin, animal serum albumin, recombinant human serum albumin or recombinant animal serum albumin. The ellagic acid source includes pomegranate and the like.
The preparation method of the stem cell skin care composition comprises the following steps:
step 1, mixing a stem cell culture medium after stem cell culture, a freeze-thaw lysate of the cultured stem cells, serum albumin, mannitol and Arg9 membrane-penetrating peptide, and freeze-drying to obtain freeze-dried powder;
step 2, mixing glycerol, sodium citrate, ellagic acid, vitamin E and water uniformly to obtain a solution;
and 3, respectively packaging the freeze-dried powder prepared in the step 1 and the solvent liquid obtained in the step 2, and mixing when in use.
The application method of the stem cell skin care composition prepared by the method comprises the following steps: uniformly mixing the freeze-dried powder and the solvent liquid, and lightly shaking until the freeze-dried powder is completely dissolved, and then using; is taken in the morning and at night. Cleaning skin with clear water or facial cleanser, lightly smearing appropriate amount of product on skin surface, and lightly tapping until all is absorbed. The cosmetic is applied half an hour in the morning, and the skin care product is not applied after the application at night.
Example 1 native expression Using Stem cells
1. Preparing freeze-dried powder:
1.1 Stem cell culture: inoculating 10000000 recovered stem cells and 15 ml stem cell culture medium into a cell culture bottle of T75 in 5% CO2And culturing at 37 deg.C for 48-72 hr. The stem cell culture medium formula is shown in table 1.
TABLE 1
| Reagent | Stock solution concentration | Goods number | Final concentration | The amount required for 100mL of medium |
| KnockOutTM DMEM CTSTM | — | A12861 | 1X | 82.75mL |
| KnockOutTM SR XenoFree CTSTM | — | 12618 | 15％ | 15mL |
| GlutaMAXTM-I CTSTM | 200mM | A12860 | 2mM | 1mL |
| NEAA | 10mM | 11140 | 0.1mM | 1mL |
| bFGF | 10μg/mL | 13256 | 8ng/mL | 80μL |
| 2-Mercaptoethanol | 55mM | 21985 | 0.1mM | 182μL |
1.2 Collection and handling: collecting the stem cell culture medium after culturing the stem cells, and storing the stem cell culture medium in a sterile container for later use; the cultured stem cells were washed twice with sterile physiological saline, 1ml of trypsin was added, the cells were treated at 37 ℃ for 5 minutes, the digested stem cells were aspirated, collected in 2ml of EP tube, treated at 56 ℃ for 10 minutes, frozen in a-80 ℃ freezer for 30 minutes, placed in a 56 ℃ water bath until they were thawed, and the process was repeated three times (freezing, thawing). Centrifuging at 12000 r/min for 10 min, collecting supernatant, mixing with the dry cell culture medium in the sterile container, concentrating in 10K molecular sieve for more than ten times, adding 5% mannitol, 1% serum albumin, and 0.05% Arg9 transmembrane peptide, mixing, placing in-80 deg.C refrigerator overnight, and freeze drying in freeze drying instrument to obtain lyophilized powder.
2. Will 4 × 106ug glycerin, 2 × 106ug sodium citrate, 15ug ellagic acid, 10ug vitamin E, 15ug 15 × 106ug water is mixed evenly to obtain the solution.
3. 400ug of the freeze-dried powder prepared in the step 1 is independently packaged, and the solvent solution prepared in the step 2 is independently packaged.
4. When in use, the freeze-dried powder and the solvent liquid are mixed.
Example 2 Stem cell factor expression mediated by plasmid electrotransfer
1. Preparing freeze-dried powder:
1.1 respectively fusing 5' ends of genes of Epidermal Growth Factor (EGF), Platelet Derived Growth Factor (PDGF), Transforming Growth Factor (TGF), basic fibroblast growth factor (b-FGF), insulin-like growth factor (IGF) and Nerve Growth Factor (NGF), human keratinocyte growth factor (KGF-2) and interleukin-1 receptor antagonist (IL-1R) with a transmembrane peptide TAT gene series, and respectively cloning the fused genes to EGFP-N1 vectors. The vector for expression of the stem cell factor is not limited to the EGFP-N1 vector, and includes other vectors capable of expression in animal cells such as N-P3xflag-CMV, pcDNA3.1, and the like.
1.2 Stem cell electrotransformation
1.2.1 preparation of Pre-transformed Stem cells: (1) transfer of adherent cell Stem cells to 75cm2The cells are cultured in culture flasks to a confluency of about 50-70% before transformation. The number of cells in this case was about 2-10X106And each power transfer requires about 1-10x105And (4) cells. (2) Cells were washed slowly with 12ml PBS. (3) The PBS was aspirated and the cells were digested with 0.4ml trypsin. (4) 10ml of serum-containing medium was added to neutralize the pancreatin. (5) Cells were collected by 400X centrifugation for 5-7 min. (6) Resuspending the cells in PBS, Hepes, serum-free medium, etc. to a density of 1-5 × 106cell/ml。
1.2.2 electrotransformation: (1) setting a power conversion program or selecting a preset program. (2) The plasmid is added into an electric rotating cup, the required plasmid quantity is determined according to different cells, and the initial concentration is 10-50 ug/ml. (3) Cells were added to the cuvette and the cuvette was inverted and mixed. The use of cell concentrations and volumes is suggested with reference to table 2.
TABLE 2
(4) The electric shock cup is put into an electric shock groove, and pulse electric shock is carried out once. (5) After pulsing, cells were transferred to wells already containing 0.5ml of stem cell medium (6) the well plate was gently shaken to evenly distribute the cells and the cells were cultured in an incubator. (7) After 24-48 hours of electrotransfer, the lyophilized powder was collected and processed as in example 1.
2. 2.5 × 106ug glycerin, 0.1 × 106ug sodium citrate, 0.1ug ellagic acid, 0.1ug vitamin E, 10 × 106ug water is mixed evenly to obtain the solution.
3.1 ug of the freeze-dried powder prepared in the step 1 is independently packaged, and the solvent solution prepared in the step 2 is independently packaged.
4. When in use, the freeze-dried powder and the solvent liquid are mixed.
Example 3 Lentiviral-mediated expression of Stem cell factor
1. Preparing freeze-dried powder:
1.1 respectively fusing 5' ends of genes of Epidermal Growth Factor (EGF), Platelet Derived Growth Factor (PDGF), Transforming Growth Factor (TGF), basic fibroblast growth factor (b-FGF), insulin-like growth factor (IGF) and Nerve Growth Factor (NGF), human keratinocyte growth factor (KGF-2) and interleukin-1 receptor antagonist (IL-1R) with a transmembrane peptide TAT gene series, and respectively cloning the fused genes into pHBLV-CMVIE-Puro vectors. Eight expression vector plasmids were obtained in total and named pHBLV-CMVIE-EGF, pHBLV-CMVIE-PDGF, pHBLV-CMVIE-TGF, pHBLV-CMVIE-b-FGF, pHBLV-CMVIE-IGF, pHBLV-CMVIE-NGF, pHBLV-CMVIE-KGF-2, pHBLV-CMVIE-IL-1R, respectively. Lentiviral expression vectors are not limited to pHBLV-CMVIE-Puro, but also include other lentiviral expression vectors such as PLVX-TRE3G, lentiviral packaging systems PLVX-Ac-Puro, psPAX2, pMD2.G, pPACK-H1-Gag, pPACK-H1-Rev, pPACK-H1-Vsvg, PLP1, PLP2, PLP-VSVG, pMDLg/pRRE, pRes-Rev, pCMV-VSVG, pCMV-delta R8, pCMV-VSVG and the like.
1.2 Lentiviral packaging-Each vector sample required 1 × 106The method is characterized in that 8 different lentiviruses are packaged due to 8 different expression vector plasmids, and the method comprises the following specific steps: (1) 1.5ml of sterilized EP tube was filled with 1.5. mu.g of packaging plasmid mixture (pMD2.G and pspAX2) and 0.5. mu.g of expression plasmid (pHBLV-CMVIE-X, X representing the eight different genes above) and 250. mu.l of Opti MEM without serum. Gently mix well and incubate for 5min at room temperature. (2) Then, 1.5ml of the sterilized EP tube was taken, and 9. mu.l of 2000l of the liposome was dissolved in 250. mu.l of serum-free Opti-MEM I medium. Gently mix well and incubate for 5min at room temperature. (3) And mixing the DNA solution and the liposome solution gently and uniformly. Incubate at room temperature for 20 min. (4) The 293FT cells were trypsinized and counted. Cells were resuspended in serum-containing DMEM media. (5) Adding 1ml of raw serum into each hole of a six-hole plateThe medium was grown and DNA-liposome complex (6) was added, and 1ml of resuspended 293FT cells (1 × 10)6Individual cells/ml) were added to the plate. CO at 37 deg.C2Incubate overnight in the incubator. (7) The medium containing the DNA-liposome complexes was removed and replaced with DMEM (containing sodium pyruvate and optional amino acids). (8) And supernatant containing virus is harvested 48-72h after transfection. Centrifuging at 3000rpm for 20min, removing precipitate, and filtering the supernatant with 0.45um microporous membrane. (9) And the virus supernatant is stored at-80 ℃ for one year. The packaged lentivirus has the infection efficiency of more than 90 percent on NIH/3T3 cells.
1.3 infection and culture of Stem cells: transferring the adherent cell stem cells to eight bottles of 75cm2The cells are cultured in culture flasks to a confluency of about 50-70% before transformation. The number of cells in this case was about 2-10X106. The stem cells were infected with each of the eight lentiviruses collected in 3.2, 1ml of lentivirus medium and 1ml of room temperature-solubilized virus solution were added to the mixture to a final concentration of 4ug/ml polyber, and the cells were re-infected the next day and cultured in fresh stem cell medium for 48 hours.
1.4 Collection and handling according to example 1 to obtain lyophilized powder.
2. Mix 6.5 × 106ug glycerin, 3 × 106ug sodium citrate, 30ug ellagic acid, 20ug vitamin E, 20 × 106ug water is mixed evenly to obtain the solution.
3. And (3) independently packaging 800ug of the freeze-dried powder prepared in the step (1), and independently packaging the solvent liquid prepared in the step (2).
4. When in use, the freeze-dried powder and the solvent liquid are mixed.
Example 4 Adenoviral-mediated expression of Stem cell factor
1. Preparing freeze-dried powder:
1.1, respectively fusing 5' ends of Epidermal Growth Factor (EGF), Platelet Derived Growth Factor (PDGF), Transforming Growth Factor (TGF), basic fibroblast growth factor (b-FGF), insulin-like growth factor (IGF) and Nerve Growth Factor (NGF), human keratinocyte growth factor (KGF-2) and interleukin-1 receptor antagonist (IL-1R) genes with a transmembrane peptide TAT gene series, cloning the fused genes into adenovirus vectors pDC315 and naming the genes as pDC315-EGF, pDC315-PDGF, pDC315-TGF, pDC315-b-FGF, pDC315-IGF, pDC315-NGF, pDC315-KGF-2 and pDC 315-IL-1R. The adenovirus expression vectors include pDC315, pDC316, pDC311, pDC312, and AdMax (TM) adenovirus vector system and AdMax (TM) Hi-IQ adenovirus vector system, etc.
1.2, co-transfecting pDC315-EGF, pDC315-PDGF, pDC315-TGF, pDC315-b-FGF, pDC315-IGF, pDC315-NGF, pDC315-KGF-2, pDC315-IL-1R and a vector pBGHloxp △ E3 to 293 cells by using L ipo2fectamine2000, generating virus plaques after 9-14 days of co-transfection, purifying the virus plaques by using 3 times of virus plaques, extracting recombinant adenovirus DNA by using Q IAamppDNA BloodMini Kit, identifying correct proliferation defective adenovirus, and performing large-scale amplification and purification, and performing titer determination.
1.3 infection of Stem cells by adenovirus
Transferring the adherent cell stem cells to eight bottles of 75cm2The cells are cultured in culture flasks to a confluency of about 50-70% before transformation. The number of cells in this case was about 2-10X106. Each vial was infected with pDC-EGF, pDC-PDGF, pDC-TGF, pDC-b-FGF, pDC-IGF, pDC-NGF, pDC-KGF-2, pDC-IL-1R, respectively. The cells were cultured for 72 hours after the addition of the stem cell culture medium.
1.4 collection and handling according to example 1 to obtain lyophilized powder.
2. 2.5 × 106ug glycerin, 3 × 106ug sodium citrate, 10ug ellagic acid, 10ug vitamin E, 10ug 10 × 106ug water is mixed evenly to obtain the solution.
3. 300ug of the freeze-dried powder prepared in the step 1 is independently packaged, and the solvent solution prepared in the step 2 is independently packaged.
4. When in use, the freeze-dried powder and the solvent liquid are mixed.
Claims (6)
1. A stem cell skin care composition is characterized by consisting of a component A and a component B,
1-800 parts of component A, which is prepared by freeze-drying stem cell extract, serum albumin, mannitol and Arg9 membrane-penetrating peptide,
the preparation of the stem cell extract comprises the expression of a plasmid electrotransfer-mediated stem cell factor, the expression of a lentivirus-mediated stem cell factor or the expression of an adenovirus-mediated stem cell factor, wherein when the expression of the plasmid electrotransfer-mediated stem cell factor, the expression of the lentivirus-mediated stem cell factor or the expression of the adenovirus-mediated stem cell factor is prepared, the stem cell factor and the transmembrane peptide gene series are fused to construct a new fusion gene, and then the subsequent steps are carried out, and the cytokine and the transmembrane peptide gene series can be inquired in a codon table through an amino acid series; the stem cell extract is a stem cell culture medium after stem cells are cultured and a freeze-thaw lysate of the cultured stem cells, and is rich in stem cell active factors with membrane penetrating peptides for induced expression;
the types of membrane-penetrating peptides and amino acid series include:
transmembrane peptide type 1: tat: RKKRRQRRR;
transmembrane peptide type 2: VP-22: DAATARGRGRSAASRPTERPRAPARSASRPRRPVD, respectively;
transmembrane peptide type 3: NTP: RQIKIWFQNRRMKWKK;
transmembrane peptide type 4: transportan: GWT LNSAGYLLGKINLKALAALAKKIL, respectively;
transmembrane peptide type 5: PEP-1: KETWWETWWTEWSQPKKKRKV;
transmembrane peptide type 6: MPG: GALFLGFLGAAGSTMGAWS QPKKKRKV, respectively;
transmembrane peptide type 7: MAP: KLALKLALKALKAALKLA:
transmembrane peptide type 8: KALA: WEAKLAKALAKALAKHLAKALAKALKACEA, respectively;
transmembrane peptide type 9: ppTG 20: GLFRALLRLLRS LWRLLLRA, respectively;
transmembrane peptide type 10: arg 9: RRRRRRRRR, respectively; or
Transmembrane peptide type 11: hCT (9-32): LGTYTQDFNKFHTFPQTAIGVGAP, respectively;
the carrier used for expressing the stem cell factor mediated by plasmid electrotransfer comprises EGFP-N1, N-P3xflag-CMV or pcDNA3.1; vectors for lentiviral-mediated stem cell factor expression include pHBLV-CMVIE-Puro, PLVX-TRE3G, lentiviral packaging system PLVX-Ac-Puro, psPAX2, pMD2.G, pPACK-H1-Gag, pPACK-H1-Rev, pPACK-H1-Vsvg, PLP1, PLP2, PLP-VSVG, pMDLg/pRRE, pRes-Rev, pCMV-VSVG, pCMV-delta R8 or pCMV-VSVG; vectors for adenovirus-mediated stem cell factor expression include pDC315, pDC316, pDC311, pDC312, AdMax (TM) adenovirus vector system or AdMaxTM Hi-IQ adenovirus vector system;
the serum albumin accounts for 1 percent of the total mass of the stem cell extract, the mannitol accounts for 5 percent of the total mass of the stem cell extract, and the Arg9 membrane-penetrating peptide accounts for 0.05 percent of the total mass of the stem cell extract;
the component B comprises the following components in parts by mass:
when in use, the component A and the component B are mixed.
2. The stem cell skin care composition according to claim 1, wherein the stem cells comprise umbilical cord mesenchymal stem cells of animal or human origin, bone marrow mesenchymal stem cells, adipose stem cells, hematopoietic stem cells, neural stem cells or hepatic stem cells.
3. The stem cell skin care composition of claim 1, wherein the serum albumin comprises human serum albumin, animal serum albumin, recombinant human serum albumin, or recombinant animal serum albumin.
4. The stem cell skin care composition of claim 1, wherein the source of ellagic acid comprises pomegranate.
5. A method of preparing a stem cell skin care composition according to any one of claims 1 to 4 comprising the steps of:
step 1, mixing a stem cell culture medium after stem cell culture, a freeze-thaw lysate of the cultured stem cells, serum albumin, mannitol and Arg9 membrane-penetrating peptide, and freeze-drying to obtain freeze-dried powder;
step 2, mixing glycerol, sodium citrate, ellagic acid, vitamin E and water uniformly to obtain a solution;
and 3, respectively packaging the freeze-dried powder prepared in the step 1 and the solvent liquid obtained in the step 2, and mixing when in use.
6. The method of using the stem cell skin care composition prepared in claim 5, comprising the steps of: uniformly mixing the freeze-dried powder and the solvent liquid, and lightly shaking until the freeze-dried powder is completely dissolved, and then using; is taken in the morning and at night.
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| CN107951820A (en) * | 2017-12-01 | 2018-04-24 | 重庆金时代生物技术有限公司 | A kind of stem cell skin care composition and preparation method thereof |
| CN108771638A (en) * | 2018-07-22 | 2018-11-09 | 张晓南 | A kind of biomass placenta freeze-dried powder preparation and preparation method thereof |
| CN109394563A (en) * | 2018-11-06 | 2019-03-01 | 福建省海西细胞生物工程有限公司 | A kind of preparation method of stem cell media liposome and the application in skin care item |
| CN110878287A (en) * | 2019-12-05 | 2020-03-13 | 伯仕利生物科技发展（盐城）有限公司 | Preparation method and application of stem cell supernatant over-expressing TAT-KGF |
| CN111407717A (en) * | 2020-03-27 | 2020-07-14 | 河北银丰鼎诚生物技术有限公司 | Stem cell factor freeze-dried powder and preparation method and application thereof |
| CN113304063A (en) * | 2021-04-23 | 2021-08-27 | 四川省恩乐生物工程有限公司 | Composition for skin beautifying and anti-aging and preparation method thereof |
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