Gallic acid sulfanilamide derivatives and application thereof in anti-liver cancer
A galloyl sulfonamide and galloyl sulfadiazine technology, applied in the field of gallic acid sulfonamide derivatives and their application in anti-liver cancer, can solve the problem of insufficient anti-cancer research and achieve the effect of expanding the scope of research and development
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Embodiment 1
[0034] Example 1: Determination of the killing effect of gallic acid sulfonamide derivative series compounds on mouse liver cancer cells.
[0035] The liver cancer cell line (H 22 ) into a single cell suspension, with 10 4 Cells / well were seeded in a 96-well plate at 37°C, 5% CO 2 Incubate in saturated humidity for 24 h. Take out the 96-well plate, add the above-mentioned mother solution of different compounds or PBS 100μl / well, the final concentration is 1.25μg / ml, 2.5μg / ml, 5μg / ml, 12.5μg / ml, 25μg / ml, 50μg / ml, each compound 9 replicate wells were set up for each concentration. At 37°C, 5% CO 2 Saturated humidity, after cultured for 24h, 48h and 72h respectively, the death rate of each induced liver cancer cell was detected by Cck8 method.
[0036] The experimental steps are as follows:
[0037] 1) Add 10 μl Cck8 solution to each well;
[0038] 2) Place at 37°C, 5% CO 2 Incubate at saturated humidity for 4 hours;
[0039] 3) Measure the absorbance (OD) at 450nm wav...
Embodiment 2
[0043] Example 2: Determination of activation of IFN R by EJTC + / + Killing effect of NK cells on mouse liver cancer cells
[0044] 1. Add IFN R + / + NK cells were resuspended with serum-free RPMI 1640 at a concentration of 1 × 10 7 , placed in a six-well plate, 1ml per well. Add NDV (25 HU / ml) and EJTC (50 μg / mL) respectively, co-culture at 37°C for 16 hours to stimulate, and set a blank control group without stimulation. After stimulation, the cells were taken out, centrifuged with serum-free RPMI 1640 medium at 1500 rpm, 4°C for 8 min, and washed twice; counted with trypan blue, resuspended cells in serum-free RPMI 1640 medium and adjusted the number of cells to 1×10 5 / ml. as effector cells.
[0045] 2. CFSE-labeled target cells (Hepa1-6): Add CFSE to the cell suspension (final concentration is 2 μmol / L), pipette gently and place in 5% CO 2 , Incubate in a 37°C incubator for 10 min; take out the cells and add 10 times the volume of cold RPMI 1640 complete culture medi...
Embodiment 3
[0051] Example 3: Activated mouse IFN R + / + Detection of GrB protein expression in NK cells
[0052] (1) Western blotting
[0053] Collect IFN R by centrifugation (300 rpm, 10 min, 4°C) + / + NK cells were washed twice with ice-cold PBS. Add cell lysate (100 μl / l × 10 7 ), whose components are (30mM Tris-Hcl pH 7.5, 120 mM NaCl, 10% glycerol, 1% Triton X-100), resuspend the cell pellet. Add protease inhibitors to lyse the cells, place in an ice bath for 30 min, collect the lysate, and heat in a water bath at 100°C for 5 min to denature the protein. The kit determines the content of total protein in the lysate, and the steps are as follows:
[0054] 1. Prepare an appropriate amount of BCA working solution by adding 50 volumes of Bicinchoninic acid (BCA) reagent A and 1 volume of BCA reagent B (50:1), and mix well;
[0055] 2. Dilute the protein standard with 0.9% NaCl to a final concentration of 0.5mg / ml;
[0056] 3. Add 0, 1, 2, 4, 8, 12, 16, and 20 μl of the standard to...
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