Drugs for ameliorating itch, rough skin or hypersensitive skin or for whitening via inhibition of the production and release of stem cell
A technology of stem cell factor and skin roughness, which is applied in the field of drugs for improving itching, rough skin or skin sensitivity or for whitening by inhibiting the production and release of stem cell factor, which can solve the problems of high cost and time-consuming
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experiment Embodiment 1
[0064] Analysis of SCF released by desiccation-stimulated keratinocytes and screening for inhibitors
[0065] Commercially available human epidermal keratinocytes (Newborn, CryoNHEK-Neo, Sanko Junyaku Co., Ltd.) were cultured using a commercially available serum-free medium (Defined Keratinocyte-SFM, Gibco Industries, Inc.). cells at 1 x 10 5 cells / mL were plated in a 12-well microplate and incubated at about 37°C for about 72 hours to confluence.
[0066] Various plant extracts shown in Figures 1 and 2 extracted with water or ethanol were dissolved in 70% ethanol to 2% w / v.
[0067] Each test plant extract was added to the culture solution at a final concentration of 0.005% w / v, and cultured at 37°C for 24 hours. A culture containing only ethanol (EtOH) was also grown as a control. To check cell viability, i.e. to determine the cytotoxic effect of the plant extracts tested, alamarBlue TM (Biosource International) was added at 10%, and the fluorescence intensity (excitatio...
experiment Embodiment 2
[0074] Accelerated expression of SCF on keratinocyte membrane induced by different stimuli
[0075] After 1.5 million keratinocytes were placed on a 10 cm plate and cultured for 72 hours, the cells were stimulated by one of the following methods: (1) Replace the medium with PBS(-), and use UVB at 20mJ / cm 2 Irradiated cultures were immediately followed by PBS (-) replaced with medium, (2) addition of forskolin, or (3) addition of theophylline. After culturing for 24 hours, the cells were collected and dispersed in 200 μl of 50 mM phosphate buffer (pH 7.8) + protease inhibitor solution, and then disrupted by an ultrasonic disruptor at 4° C. for 5 times for 30 seconds each. After centrifugation at 10,000 g for 20 minutes at 4°C, the supernatant was further centrifuged at 100,000 g for 60 minutes at 4°C. The resulting precipitate was dissolved in 100 μl of 25 mM phosphate buffer (pH 6.8) + 0.1% Triton-X100 solution to obtain a protein extract of the membrane fraction. The protei...
experiment Embodiment 3
[0078] Analysis of SCF expressed on UV-stimulated keratinocyte membranes and screening for inhibitors
[0079] After 1.5 million keratinocytes were placed on a 10cm plate and cultured for 72 hours, the medium was replaced with PBS(-), and UVB was used at 20mJ / cm 2 Cultures were irradiated and PBS(-) was immediately replaced with medium. After adding the plant preparation to be screened and incubating for 24 hours, the cells were collected, then dispersed in 200 μl of 50 mM phosphate buffer (pH 7.8) + protease inhibitor solution, and the cells were disrupted with an ultrasonic disruptor at 4 °C5 times, 30 seconds each time. After centrifugation at 10,000 g for 20 min at 4°C, the supernatant was further centrifuged at 100,000 g for 60 min at 4°C. The resulting precipitate was dissolved in 100 μl of 25 mM phosphate buffer (pH 6.8) + 0.1% Triton-X100 solution to obtain a protein extract of the membrane fraction. The protein content of the solution was measured by well-establish...
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