Preparation method of stem cell cytokine and its use in preparing medicine or cosmetics
A technology of cytokines and uses, applied in the direction of epidermal cells/skin cells, cytokines/lymphokines/interferons, cosmetics, etc., can solve the problems of insufficient types of cosmetics, insufficient cytokines, etc., and achieve good whitening application effect, Effects of promoting proliferation and reducing secretion
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Embodiment 1
[0027] Example 1 Isolation and culture identification of epidermal stem cells
[0028] Under aseptic conditions, remove the subcutaneous fat layer of the foreskin skin, wash the skin repeatedly 3 times with PBS containing penicillin and streptomycin, cut into small pieces with a size of 0.2cm*0.2cm, digest with neutral protease for 18 hours at 4°C, and discard Supernatant, separate the epidermis and dermis. Shred the epidermis, digest with 0.25% trypsin at 37°C for 15 minutes, add DMEM containing 10% calf serum to terminate the digestion, and filter the suspension with a 200-mesh sieve. The filtrate was centrifuged at 1000r / min for 5min to collect the cells. Discard the supernatant, add K-SFM and gently pipette to make a single cell suspension. Inoculate in a culture flask pre-coated with type IV collagen and incubate at 37°C. The cells adhering to the type IV collagen capsule are mainly epidermal stem cells. Aspirate the cell suspension, add K-SFM, and place at 37°C, 5% CO ...
Embodiment 2
[0030] Example 2 Preparation of Epidermal Stem Cell Cytokines
[0031] The epidermal stem cells prepared in Example 1 were placed in cell culture medium for subculture. The cell culture medium was 89% DMEM + 10% FBS + 1% double antibody stem cell medium, and the culture conditions were 37°C, 5% CO 2 nourish;
[0032] Take well-grown stem cells and adjust the cell concentration to 1X10 6 / mL was inoculated in 6-well plates, and the medium was changed after the cells adhered to the wall. 1 The complete medium of the secretagogue peptide (SEQ ID NO: 1), the control group only added the complete medium, and cultured for 48h (wherein 50mg·L- 1 Secretagogue cultures were further subdivided into 0% O 2 +5%CO 2 +94N 2 and 3%O 2 +5%CO 2 +92%N 2 ), the cell supernatants of each group were collected, and the above two culture supernatants were ultrafiltered with a 1KD ultrafiltration membrane to remove small molecular impurities with a molecular weight below 1000, and the retenta...
Embodiment 3
[0039] Embodiment 3 Fibroblast Proliferation Experiment
[0040] Fibroblasts in the logarithmic growth phase (from Saiye (Suzhou) Biotechnology Co., Ltd., product number: HXXFB-00001) were mixed with serum-free medium DMEM+sodium pyruvate+penicillin and streptomycin, and the cell concentration was adjusted to 4000 cells / 200 μl, to the 96-well plate, the inoculated amount of cells is 4000 cells / 200 μl / well, and the cytokine extracts are added to the In each well, set up three duplicate wells, add culture medium and incubate for 72 hours in an incubator for observation, add CCK-8 solution to continue culturing for 2 hours, measure the absorbance value (OD value) of the cells at a wavelength of 450 nm with a microplate reader, and take 0% wells as For the control wells, the percentage increase in cell proliferation activity was calculated. Calculation formula: (OD experimental well-OD control well) / OD control well.
[0041] The result is as figure 2 As shown, after adding cyt...
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