Application of theabrownin in preparation of anti-melanoma drugs
A technology for melanoma and melanin inhibition, applied in the field of theabrownin in inducing apoptosis of A375 human melanoma cells, which can solve the problems of no research reports and unclear effects
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Embodiment 1
[0025] Example 1: Anti-proliferation effect of theabrownin (TB) on A375 cells
[0026] The effect of TB on the proliferation ability of A375 cells was evaluated by MTT assay and morphological observation. A375 cells in the logarithmic growth phase were taken, and 6×10 3Inoculate in a 96-well plate at a density of one per well, with 200 μl of medium per well. After the cells were completely attached to the wall, different concentrations of TB were given for 24, 48 and 72 hours. TB administration concentrations were 0, 40, 80, 120, 160, 200, 240 and 280 μg / ml. After the drug intervention was completed, the morphology of living cells was observed under a microscope. Add 20 μl of MTT reagent (5.0 mg / ml) to each well and incubate at 37°C for 4 hours in the dark. After incubation, 150 μl DMSO was added to each well, and shaken at room temperature for 10 minutes in the dark. Using a microplate reader, detect the optical density value (OD value) at a wavelength of 490 nm, and cal...
Embodiment 2
[0029] Example 2: Pro-apoptotic effect of theabrownin (TB) on A375 cells
[0030] DAPI staining and flow cytometry were used to evaluate the effects of low, medium and high concentrations of TB on the pro-apoptotic effect of A375 cells after 24 hours. A375 cells were seeded in 24-well plates covered with cell slides, and treated with low, medium and high concentrations of TB. After 24 hours of intervention, the cells were washed 3 times with PBS and fixed with 4% paraformaldehyde for 1 hour. After the cell slides were washed with PBS, they were permeabilized with 0.1% sodium citrate solution containing 0.1% Triton X-100 at 4°C for 2 min. Wash twice with PBS, and seal the slides with DAPI reagent at room temperature in the dark. After the slides were dried, they were placed under a fluorescence microscope to observe the cells and collect image data.
[0031] The result is as figure 2 As shown in A, after DAPI staining, the normal control group had intact nuclei, uniform ch...
Embodiment 3
[0032] Example 3: Effect of theabrownin (TB) on DNA damage in A375 cells
[0033] Comet assay was used to detect the effect of TB on the DNA strand damage of A375 cells. A375 was seeded in 6-well plates and treated with low, medium and high concentrations of TB. Cells were collected by centrifugation into pre-cooled PBS, and the cell concentration was adjusted to 1×10 5 pieces / ml. Prepare 1% normal melting point agarose gel on a frosted glass slide in advance, then preheat and mix 25 μl of cell suspension and 75 μl of 0.75% low melting point single agarose at 37°C, and quickly spread it on the first layer of gel . Place it in a 4°C environment and let it cure for more than 30 minutes. Then, in the pre-cooled alkaline electrophoresis buffer at 4°C, stand in the dark for 30 minutes. Next, lyse overnight in 4°C pre-cooled lysate. The electrophoresis condition is 1.0V / cm, and the time is 30min. After electrophoresis, take out the glass slide and place it in a glass dish, ad...
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