Synergetic Application of nicotinamide phosphoribosyltransferase (NAMPT) depressor and NQO1 substrate to treatment of non-small cell lung cancer
A technology of non-small cell lung cancer and inhibitors, applied in drug combinations, medical preparations containing active ingredients, pharmaceutical formulas, etc.
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Embodiment 1
[0020] Embodiment 1.PCR experiment
[0021] Experimental Materials:
[0022] Human non-small cell lung cancer A549 cells were purchased from ATCC at 37°C, 5% CO 2 Under normal conditions, culture medium was RPMI-1640 (Gibco) containing 10% calf serum (PAA); fetal bovine serum (Fetal bovine serum) was purchased from Hyclone (Logan, Utah, USA); trypsin (Trypsin) was purchased from in Amersco (Solon, Ohio, USA); penicillin, streptomycin, dimethylthiodiphenyltettrazole (MTT), and GAPDH antibodies were purchased from Nanjing Shengxing Bioengineering Company (Jiangsu); Trizol total RNA extraction reagent , RT-PCR kit, and Real-time PCR Master Mix (SYBR Green) were purchased from Takara (Dalian Baobiology).
[0023] experimental method:
[0024] A549 cells were seeded in 6-well plates, and the treatment time was 0h, 2h, 4h, 8h, 12h, 24h; per 1×10 6 Add 1mL Trizol to the cells, blow the liquid with a 1mL pipette until the liquid is clear and free of cell clumps, and transfer it i...
Embodiment 2
[0029] Embodiment 2.MTT experiment
[0030] A549 cells were seeded in a 96-well plate, cultured and grown to 80% full, given FK866 10 nM, and treated with different concentrations of Tanshinone IIA (0, 0.4, 1, 2, 4, 10, 20, 40 μM) for 72 hours after 1 hour; or β-La Paquinone (0, 1.25, 2.5, 5, 10, 20 μM) was treated for 2 hours, and then the drug-free medium was replaced for 24 hours. Replace the serum-free medium and add 20 μL (5 mg / mL) MTT to each well, incubate in a carbon dioxide incubator for 4 hours, remove the MTT solution, add 150 μL of DMSO to dissolve the crystals (37°C shaker, 50r / min), take it out after 10 minutes and place in The absorbance was measured in a microplate reader (measurement wavelength 570nm, reference wavelength 630nm). The cell survival rate of the administration group was calculated by the following formula:
[0031] Survival rate = absorbance of treatment group / absorbance of control group * 100%
Embodiment 3
[0032] Example 3.LC-MS n Detection of intracellular NAD + Level
[0033] A549 cells were inoculated into 6-well plates, cultured and grown to 80% confluence, treated with FK866 10nM, 1 hour later with 40 μM Tanshinone IIA for 48 hours; or 20 μM β-lapachone for 24 hours. Discard the medium, wash the cells with normal saline, add 1ml of 80% methanol containing 50ng / ml 2-chloroadenosine (internal standard) to each well, place at -80°C for 20 minutes, scrape the cells in an ice bath and transfer them to EP tubes, and sonicate Break up, centrifuge at 14000rpm for 5 minutes, transfer the supernatant to a new EP tube, evaporate to dryness, reconstitute and inject in 100μl ultrapure water.
[0034]Chromatographic conditions: the chromatographic column is an amide column (3x100mm i.d., 5 μm, Chrom-Matrix Inc), and the column temperature is 40°C. Mobile phase: the aqueous phase (A) is an aqueous solution containing 5mM ammonium acetate, the organic phase (B) is methanol, the column t...
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