Psoriasis skin repairing agent prepared by mixing stem cell extract and traditional Chinese medicine extract and application thereof

[0036] Example 1 Preparation of skin repairing agent of the present invention

[0037] Step 1: Preparation of umbilical cord mesenchymal stem cell extract

[0038] (1) Take out the umbilical cord tissue of the newborn, remove the blood vessels, epithelium and blood clots, separate the Wharton's block, and cut it into 1mm 3 After sizing, put it into a 10cm sterile petri dish; digest with DMEM medium containing 0.1% collagenase II; after filtration, use DMEM medium containing 10% fetal bovine serum FBS for subculture, and change to serum-free medium on the third day Subculture in DMEM medium, once every 2 days; replace a bottle of cells that have been passaged for 3 generations for subsequent treatment;

[0039] (2) Digest cells with DMEM medium containing 0.05M EDTA, and wash with PBS solution;

[0040] (3) The cells were resuspended in serum-free DMEM medium, and disrupted by ultrasonic lysis (120W, 15S*20 times, 15S interval), the disrupted product was centrifuged at 12000rpm for 15 minutes, and the supernatant was taken;

[0041] (4) the supernatant is purified and concentrated by ultrafiltration with a YM-50 ultrafiltration tube with a pore diameter of 5000 angstroms from Millipore in the United States, the time is 35min, and the pressure is 0.3MPa;

[0042] (5) Quantify TGFB1 in the purified product with Elisa kit, and dilute the purified product with serum-free DMEM medium to a concentration of 40 mg/ml of TGFB1;

[0043] (6) The product is stored at -80 degrees Celsius for a long time.

[0044] Step 2: Preparation of Chinese herbal extracts

[0045] Take fully dried traditional Chinese medicine raw materials by weight: 7 parts of hibiscus flower, 3 parts of licorice, 12 parts of lotus leaf, 8 parts of calamus, pulverize and pass through a 40-mesh sieve, add 75% ethanol by volume to prepare a material-to-liquid ratio of 1 : 30g/mL solution, choose extraction temperature 35 ℃, extraction time 25min, ultrasonic power 200W to carry out extraction, the extract is centrifuged at 6000r/min for 20min, take the supernatant, and make up with ethanol; The water-saturated n-butanol was extracted for a total of 5 times; the recovered n-butanol solution was drained with a negative pressure centrifuge (-30 mm Hg, 45 degrees Celsius), and the residue was dissolved in absolute ethanol; After extraction with petroleum ether, the volume was fixed, and a standard curve was made with the lotus leaf flavonoid standard. The product was accurately quantified by the absorbance method, and the remaining product was diluted with sterile water until the total flavonoid content was 20 mg/L. If this concentration cannot be reached, the extraction is deemed to have failed and the product should be discarded.

[0046] Step 3: Preparation of Skin Repair Agent

[0047] (1) Get 1ml of the above-mentioned traditional Chinese medicine extract and 24ml of umbilical cord mesenchymal stem cell extract, add 25ml of sterile water and mix;

[0048] (2) The above mixed solution was sterilized, filtered and packaged with a 0.7 μm filter, each 1.5 ml.

[0049] Example 2 Preparation of skin repairing agent of the present invention

[0050] 1. Experimental method

[0051] (1) Primary culture of umbilical cord mesenchymal stem cells;

[0052] (2) Umbilical cord mesenchymal stem cells were prepared at 2*10 5 cells/ml were plated into 12-well plates for experiment, and adherent culture was carried out for at least 24h;

[0053] (3) adding the skin repairing agent prepared in Example 1 of the present invention (the mass concentrations are 0%, 0.5%, 1%, 2%, 4%, 10% and 15% respectively), and adding the cell damage agent bleomycin as comparison;

[0054] (4) The cells were detected by flow cytometry to detect cell apoptosis and cell viability (CCK8 method).

[0055] 2. Experimental results

[0056] The CCK8 experiment on mesenchymal stem cells confirmed that the cytotoxicity of the skin repairing agent of the present invention at a concentration of 4% was 6.0%, and the cytotoxicity at a concentration of 10% was 15.2%, indicating that its cytotoxicity was small, and 4% mass was selected. concentration for subsequent cell experiments.

[0057] Example 3 Cell experiments in which the skin repairing agent of the present invention inhibits the growth of fibroblasts and promotes the growth of epidermal stem cells and vascular endothelial cells

[0058] 1. Experimental method

[0059] (1) Primary culture of different cells, including fibroblasts, epidermal stem cells and vascular endothelial cells;

[0060] (2) Divide the cells into 2*10 5 Plated into 12-well plates for experiments, each cell has 4 wells, and adhered to culture for at least 24 hours;

[0061] (3) The skin repairing agent of the present invention (mass concentration 4%) is added to each type of cells, and bleomycin (final concentration is 2 μg/ml) is added as a cell-damaging agent. The specific sample adding method is:

[0062]

[0063] (4) All cells were detected by flow cytometry to detect cell apoptosis and cell viability (CCK8 method).

[0064] 2. Experimental results

[0065] The results of cell apoptosis rate in each group are shown in Table 1. From the results in Table 1, it can be seen that in the presence of 4% skin repair agent, 12.6% of fibroblasts undergo apoptosis, and the difference in apoptosis rate is statistically significant compared with the control group (P<0.05). , while the apoptosis rate of epidermal stem cells and vascular endothelial cells was lower than that of the control group, and the cell viability was better; bleomycin had a significant killing effect on all cells, compared with the apoptosis rate of the control group The differences were statistically significant (P<0.05); in the presence of 4% skin repair agent and bleomycin, the apoptosis rate of fibroblasts was significantly increased compared with the group with only bleomycin. , while the apoptosis rate of epidermal stem cell and vascular endothelial cell group was significantly decreased, and the difference was statistically significant compared with the apoptosis rate of the group with only bleomycin (P<0.05).

[0066] Table 1 Apoptosis rate of cells in each group

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