Application of NO donor compound in preparation of drugs for inhibiting invasion and metastasis capabilities of tumor cells rich in sulfhydryl molecules
A technology of tumor cells and metastatic ability, applied in the direction of anti-tumor drugs, drug combinations, tripeptide components, etc., can solve the problem of not changing the state of sulfhydryl group
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Embodiment 1
[0039] Take the protein molecule "CYR61" rich in sulfhydryl groups as an example: the sulfhydryl nitrosylation reaction of CYR61 protein: MDA-MB-231 cells were washed by adding sulfhydryl nitrosoglutathione (S-nitrosoglutathione, GSNO) to make it The final concentration was 30 μM, and incubated at 37° C. in the dark for 30 minutes. Add 200 μL of HENS buffer solution to the cell suspension, place the centrifuge tube in a small box with crushed ice, and ultrasonically disrupt for 30 min in a cell disruptor. Centrifuge at 10,000 g for 10 min at 4°C, collect the supernatant in a centrifuge tube, and measure the protein concentration by the BCA method. Add 2 μL of 1 M MMTS to the protein sample, vortex for 1 min to fully mix with the protein, and incubate at room temperature for 30 min in the dark. Add 600 μL of pre-cooled acetone, and place in a -20°C refrigerator for 1 h in the dark. Centrifuge at 10,000 g for 10 min at 4°C, pour off the acetone, and dry in the air. Resuspend ...
Embodiment 2
[0041] The effect of CYR61 on the adhesion of breast cancer cell MDA-MB-231 to the cell matrix, and the effect of thiol-rich protein molecules (such as CYR61) on breast cancer cell MDA-MB-231 by thiol-nitrosoglutathione sulfhydryl nitrosylation. This effect reduces the adhesion of cancer cells to the cell matrix.
[0042] Spread gelatin: Take a 24-well plate, add 200 μL gelatin to each well, shake well, and distribute evenly in the wells. Incubate overnight at 37°C in an incubator. Blot the excess gelatin, add 200 μL of PBS containing 2% BSA to each well, and place in an incubator at 37°C for 1 hour to block. Aspirate the liquid and wash three times with PBS. Cell count: MDA-MB-231, OE-CYR61, MDA-MB-231, Si-CYR61 MDA-MB-231 cells were washed and digested, then resuspended twice in PBS. Pipette 2 parts of 12 μL cells into new EP tubes, add 12 μL reagent T to one part, and add 12 μL reagent NC to one part, mix well, and add to the sample well of the cell counting plate. Ins...
Embodiment 3
[0045] CYR61 affects the adhesion of breast cancer cell MDA-MB-231 to endothelial cells, and S-nitrosoglutathione (S-nitrosoglutathione) has an effect on the sulfhydryl of breast cancer cell MDA-MB-231 rich in sulfhydryl molecules (such as CYR61). Nitrosylation modification inhibits the adhesion of tumor cell MDA-MB-231 to endothelial cells.
[0046] Endothelial cell plating: take logarithmically grown umbilical vein endothelial cells, wash and digest them with PBS, spread the cells in a 24-well plate, and wait until they are covered with a layer of endothelial cells. The cells were washed three times with PBS, ECM medium containing 10 μg / mL TNF-α was added, and incubated in a 37°C incubator for 4 h to activate endothelial cells. MDA-MB-231, OE-CYR61 MDA-MB-231, Si-CYR61 MDA-MB-231 cells were stained with rhodamine b, plated and observed. The umbilical vein endothelial cells with logarithmic growth were inoculated in a 24-well plate covered with gelatin. After the endothelial...
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