Establishment method of tonalid-induced human normal hepatocyte malignant transformation model
A technology of spitting musk and establishing a method, which is applied in the field of cell biology, can solve the problems that the carcinogenic effect of spitting musk has not been effectively verified, and achieves the effect of strong cell migration ability, simple operation and low cost
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Embodiment 1
[0037]The embodiment of the present invention provides a method for establishing a musk-induced malignant transformation model of human normal liver cells. 10 μg / L, 100 μg / L, 1000 μg / L; (2) Culture L02 normal human liver cells to the logarithmic phase for later use; (3) Divide the L02 normal human liver cells in the logarithmic phase into four groups, respectively group of cells and three groups of transformed cells, among which the cells of the three groups of transformed groups were treated with the prepared 10μg / L, 100μg / L, 1000μg / L musk working solution for 24h, and at the same time, they were treated with DMEM culture solution containing an equal volume of DMSO Treat the control group cells for 24h; the transformed group cells treated by the treated control group cells and the musk working solution of 10 μg / L, 100 μg / L, and 1000 μg / L in step (3) are normally cultured to the 20th generation; ( 4) Get the 10th, 15th, and 20th generations of control cells and transformation ...
Embodiment 2
[0040] In an embodiment of the invention, the above step (1) specifically includes: weighing Tuna musk powder and dissolving it in dimethyl sulfoxide solution (DMSO), preparing a stock solution with a concentration of 1 mg / mL, and suction-filtering the stock solution to obtain Concentration is 1mg / mL Tuna musk mother liquor. The musk musk mother solution with a concentration of 1 mg / mL was diluted with DMEM culture solution into musk musk working solutions with concentrations of 10 μg / L, 100 μg / L, and 1000 μg / L, respectively, and stored at 4°C in the dark for future use.
Embodiment 3
[0042] In the embodiment of the present invention, the above step (3) specifically includes: digesting and centrifuging the L02 normal human liver cells in the logarithmic phase with 0.25% trypsin, collecting the cells, resuspending the normal DMEM culture medium, and dissolving the cells Adjust the density to about 2×10 6 cells per milliliter;
[0043] The cells with adjusted density were divided into 4 groups, one group of control group and three groups of transformed groups were inoculated in 6-well plates, and each group was set up with 3 parallel cells, placed in a 37°C incubator, cultured for 24 hours, discarded Remove the old culture medium;
[0044] To the three groups of transformation group cells that discarded the old culture medium, add the Tuna musk working solution prepared in the step (1) at a concentration of 10 μg / L, 100 μg / L, and 1000 μg / L, and discard the old culture medium. Add DMEM medium containing an equal volume of DMSO to the cells of the control gro...
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