External skin preparation and applications thereof
A technology for external preparations, skins, applied in the field of medicine
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Embodiment 1
[0085] Example 1. In vitro culture of epidermal cells
[0086] Materials and Instruments
[0087] Trypsin was purchased from (GIBCO, USA);
[0088] 0.53mM EDTA was purchased from (GIBCO, USA);
[0089] Pancreatin inhibitor was purchased from (GIBCO, USA);
[0090] Trypan blue was purchased from (Sigma, USA).
[0091] Experimental steps:
[0092] 1) Take materials
[0093] The surgically removed foreskin of children is placed in keratinocyte culture medium containing antibiotics and brought into the cell culture room.
[0094] 2) Separate the epidermis
[0095]Carefully cut off the subcutaneous fat tissue and part of the dermis with scissors, and trim them into strips with a width of about 1-2mm, rinse twice with calcium and magnesium-free PBS, and soak in 0.24U / ml neutral protease (Dispase II, Sigma, USA) overnight at 4°C.
[0096] 3) digestion
[0097] After 18 hours, the epidermis was torn off from the dermis with two ophthalmic tweezers, cut into pieces, and then d...
Embodiment 2
[0103] The effect of embodiment 2.Melittin on epidermal cell proliferation
[0104] Materials and Instruments
[0105] K-SFM culture medium was purchased from GIBCO, USA;
[0106] MTS([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethylcarboxy)-2-(4-sulfophenyl)-2H-tetrazolium inner salt], purchased from Promega;
[0107] Automatic microplate reader was purchased from BioRad, USA.
[0108] experimental method:
[0109] (1) The experiment selects epidermal keratinocytes (Epidermal Keratinocytes, EK) cultured to the third generation according to the method described in Example 1,
[0110] (2) 4000 cells per well were seeded into a 96-well plate at 37°C, 5% CO 2 , cultured in K-SFM medium for 12 hours.
[0111] (3) After 12 hours, the culture medium was replaced in each well, and the culture medium containing different concentrations of Melittin was added again. The final concentrations of Melittin in the culture medium were 1, 5, 10, 20, 50, and 200 μg / mL. Continue incubation...
Embodiment 3
[0119] Example 3. Effect of bee venom polypeptide on epidermal cell proliferation
[0120] For the specific experimental method of this experiment, refer to Example 2, only step 2-a-(3) of Example 2, Melittin is replaced by melittin, wherein melittin is prepared by conventional methods. The final concentrations of mee venom peptides were still 1, 5, 10, 20, 50, and 200 μg / mL, respectively.
[0121] The result is as figure 2 and described in Table 2. As can be seen from the results, each group has different degrees of inhibition. When the final concentration of mee venom polypeptide is 20 μg / mL, the inhibitory effect on epidermal cell proliferation is obvious (P<0.05); when the final concentration of mee venom polypeptide is 50, At 200 μg / mL, the inhibitory effect on the proliferation of epidermal keratinocytes was significantly different from that of the control group (p<0.01).
[0122] Table 2 Effect of mee venom polypeptide on epidermal cell proliferation
[0123] ...
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