Preparation method and product of biological beauty raw material derived from immune cells
A technology of immune cells and raw materials, applied in the direction of blood/immune system cells, animal cells, cosmetic preparations, etc., can solve the problems of lack of modification, low biological activity, functional and biological activity limitations, etc.
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Embodiment 1
[0054] Example 1 Culture of human immune cells
[0055] Collect 100ml of peripheral blood from volunteers (with whom they signed an informed consent form), dilute it with 1×PBS (pH value 7.4), and obtain mononuclear cells after density gradient centrifugation of lymphocyte separation medium, using 1000IU / ml cytokines Interferon γ, 50ng / ml anti-human CD3 monoclonal antibody, 100IU / ml interleukin 1α and 500IU / ml interleukin 2 (all purchased from Germany Miltenyi company) in serum-free medium AIM-V (containing 50μg / ml hydrocortisone pine, 10 μg / ml ascorbic acid and 50 μg / ml human insulin, all purchased from American Life Company) and continuously cultured for 20 days to obtain 5.0×10 9 Cytokines induce killer cells, and 3.0L culture medium.
[0056] Using cytokines 200ng / ml interleukin-15, 100ng / ml interleukin-18 and 1000IU / ml interleukin-2 (both purchased from Miltenyi, Germany) were continuously cultured in serum-free medium AIM-V for 20 days to obtain 3.0×10 9 Natural killer...
Embodiment 2
[0058] Example 2 Preparation of cosmetic raw materials derived from human immune cells
[0059] Take the immune cells obtained in Example 1, add 10ml 1×PBS (pH=7.4) and mix well, then add 1ug protease inhibitor (Dalian Bao Biological Co., Ltd.), place it on ice and crush it in an ultrasonic breaker (Thermo Electric Co., Ltd., USA) After 8 minutes, centrifuge at a high speed of 15,000 rpm for 20 minutes, and take the supernatant and add it to the culture medium in which the immune cells have been cultured.
[0060] The culture solution containing the immune cell lysate was added to a dialysis bag with a molecular weight of 500 (Spectrum, USA), and placed in a refrigerator for dialysis concentration, and the volume was concentrated by 5 times.
[0061] In addition, the culture solution containing the immune cell lysate can also be concentrated by ultrafiltration using a 500 molecular weight cellulose acetate membrane (Spectrum, Inc., USA), and concentrated 5 times in volume.
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Embodiment 3
[0063] Example 3 Detection of cytokine expression and secretion levels in human immune cells
[0064]Take the immune cell culture fluid prepared in Example 1, the culture fluid containing the immune cell lysate and its concentrate, and use the ELISA kit (R&D Systems, Inc., USA) to detect the expression and secretion levels of the cells. A blank control was made with the AIM-V medium in which immune cells had not been cultured. Table 1 shows the expression and secretion levels of various cytokines in the culture solution of immune cells, the culture solution containing immune cell lysate and the concentrated solution, and the unit is ng / ml.
[0065] Table 1
[0066]
[0067] Table 1 shows that immune cell culture fluid, culture fluid containing lysate, and concentrate secrete high levels of anti-inflammatory molecules and antioxidant molecules, and the cell lysate contains Nrf2 molecules with dual functions, indicating that these cytokines play a role in In terms of skin b...
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