Applications of simvastatin in preparation of hepatitis B virus inhibition drugs
A technology of simvastatin and hepatitis B virus, applied in the field of western medicine, can solve the problems such as the lack of simvastatin, and achieve the effect of strong anti-hepatitis B virus activity and inhibition of replication
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Embodiment 1
[0035] MTT assay to detect cell proliferation ability
[0036] Take HepG2.2.15 liver cancer cells in the logarithmic growth phase, digest the cells with a digestive solution containing 0.25% trypsin (Trypsin) + 0.02% EDTA, centrifuge to discard the supernatant, blow and beat with complete medium, and then use a cell counting board to count. 3×104 / well were inoculated in 96-well culture plates. After 24 hours of incubation, the cells were treated with different concentrations of simvastatin (as shown in Table 1), and 5 auxiliary wells were set for each concentration. After 1, 2, 3, 4, 5, 6, and 7 days of drug action, add 20 μl of 500 μg·ml-1 MTT, incubate at 37°C for 4 hours, discard the culture medium, then add 150 μl of DMSO to each well, shake for 15 minutes and mix well. The absorbance (A) value of each well at 570 nm was detected with a microplate reader. Subtract the background A value (cell-free medium plus MTT reagent) from the A value of each test well; calculate the...
Embodiment 2
[0041] Cell counting assay to detect cell proliferation
[0042] Take HepG2.2.15 liver cancer cells in the logarithmic growth phase, digest the cells with a digestive solution containing 0.25% trypsin (Trypsin) + 0.02% EDTA, centrifuge to discard the supernatant, blow and beat with complete medium, and then use a cell counting board to count. 3×104 / well were inoculated in 96-well culture plates. After 24 hours of incubation, the cells were treated with different concentrations of simvastatin (as shown in Table 1), and 5 auxiliary wells were set for each concentration. After 1, 2, 3, 4, 5, 6, and 7 days of drug action, the cells were digested with 0.25% trypsin, and fully pipetted to prepare a single-cell suspension. The counting plate and coverslip were cleaned with 95% ethanol and dried in air. Cover the coverslip on the counting plate, mix the cell suspension well, suck up the cells with a pipette, and gently drop about 10 μl of the cell suspension from the edge of the cou...
Embodiment 3
[0044] Western-blot analysis
[0045] 1) Preparation of protein samples: Remove the culture medium from the cells that have been cultured and treated accordingly, rinse with pre-cooled (4°C) PBS 3 times, try to blot the remaining PBS as dry as possible, then add pre-cooled protein lysate, Lyse on ice for 30 min. Thereafter, the cells were collected with a cell scraper, centrifuged at 12000 rpm for 2 min at 4°C, and the supernatant was taken. A small amount of the supernatant was taken for protein quantification, and the rest was added to Loading Buffer, boiled at 104°C for 30 minutes, and then stored at -20°C for later use.
[0046] 2) Preparation of electrophoresis gel: prepare 5% stacking gel and 10% separating gel according to Table 2. The amount of total protein loaded was 50 μg, and electrophoresis was performed at 60V. After the leading edge of the sample entered the separation gel, the voltage was adjusted to 100V, and electrophoresis was continued for about 1.5 hours...
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