Application of dandelion sterol in preparation of anti-HBV medicine
A technology of dandelion sterols, drugs, applied in the field of medicine
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Embodiment 1
[0036] 1. MTT method was used to detect the toxic effects of target compounds on HepG2.2.15 cells (ATCC)
[0037] The compounds were intervened in HepG2.2.15 cells at 48μg / ml, 24μg / ml, 12μg / ml, 6μg / ml, 3μg / ml, 1.5μg / ml for 3 days, and the toxic effects of the compounds on HepG2.2.15 cells were detected by MTT method. The specific steps are as follows: 1) Collect and adjust the concentration of the cell suspension, add 100 μl to each well of a 96-well plate, and plate the cells to be tested to a density of 10 4 cells / well, 5% CO2 , and incubated overnight at 37°C; 2) the next day, add the drug with the above concentration gradient, and set up 5 duplicate wells in 5% CO 2 , and incubate at 37°C for 72 hours; 3) Add 20ul MTT solution (5mg / ml, Sigma) and continue to incubate for 4h; 4) Carefully aspirate and discard the culture medium in the well, and terminate the culture; 5) Add 150ul dimethyl sulfoxide to each well, shake The crystals were fully dissolved, and the absorbance v...
Embodiment 2
[0040] 2. The effect of the target compound on HBV DNA replication
[0041] The compounds were intervened in HepG2.2.15 cells at concentrations of 48 μg / ml, 24 μg / ml, 12 μg / ml, 6 μg / ml, 3 μg / ml and 1.5 μg / ml for 3 days, and the level of HBV DNA in the cell supernatant was detected by fluorescent quantitative PCR. The instrument used is ABI7500 real-time fluorescent quantitative PCR system. The specific steps are operated in accordance with the instructions of the Hepatitis B Virus Nucleic Acid Quantitative Detection Kit, and the brief description is as follows: the viral DNA in the cell culture supernatant is extracted with the nucleic acid extraction solution of the kit, and then the viral DNA is used as a template, and the prepared reaction system includes: Fluorescent quantitative PCR reaction solution, primers and probes and other components. The PCR reaction conditions were pre-denaturation at 95°C for 3min; denaturation at 95°C for 10s, annealing extension and fluoresce...
Embodiment 3
[0050] 5. The effect of the target compound on the expression of PTB protein
[0051] The expression level of PTB protein was detected by Western Blot. Total cell protein was extracted and quantitatively analyzed according to the operating instructions of the BCA protein quantification kit. 40 μg of the above-mentioned total cellular protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 1% bovine serum albumin, the membrane was incubated with antibodies PTB (self-made) and β-actin (Cell signaling technology) at 4°C overnight, and the secondary antibody was incubated for 1 hour and detected with chemiluminescence reagent ECL (Pierce).
[0052] Such as Figure 5 As shown, compared with the blank control group, dandelion sterol can significantly down-regulate the expression of PTB protein in cells, and there is a significant difference.
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