Application of bergapten in preparation of drugs for treating colorectal cancer
A technology of bergamot lactone, colorectal cancer, applied in the field of medicine
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Embodiment 1
[0025] Example 1 Detection of cell drug resistance
[0026] 1×10 3 A SW1116, LoVo, SW1116 / 5-Fu and LoVo / 5-Fu cells were seeded in a 96-well plate at 37°C, 5% CO 2 After 48 hours of culture in an incubator with saturated humidity; different concentrations of bergamot lactone (0 μmol / L, 1 μmol / L, 10 μmol / L and 100 μmol / L) were applied to the above cells for 48 hours; then SW1116 and LoVo cells were treated with DMEM complete medium with different concentrations of 5-Fu (0mg / L, 1mg / L, 2mg / L, 4mg / L and 8mg / L) was continuously cultured for 48h, and SW1116 / 5-Fu and LoVo / 5-Fu cells were cultured with different Concentration of 5-Fu (0mg / L, 10mg / L, 20mg / L, 40mg / L and 80mg / L) of DMEM complete culture solution continued to culture for 48h. Then use CCK-8 kit and microplate reader to detect the best absorbance value at 450nm, and calculate IC50 value to evaluate cell drug resistance.
[0027] GraphpadPrism 5.0 software was used for statistical analysis, IC50 value was calculated by no...
Embodiment 2
[0031] Example 2 Western blot detection
[0032] 1×10 5 A SW1116 / 5-Fu and LoVo / 5-Fu cells were seeded in a 6-well plate at 37°C, 5% CO 2, after 24 hours of culture in an incubator with saturated humidity, different concentrations of bergamot lactone (1 μmol / L, 10 μmol / L and 100 μmol / L) were applied to the above cells for 48 hours, washed with PBS, added an appropriate amount of RIPA lysate, and placed in After lysing on ice, the total protein was extracted and the protein concentration was determined using the BCA protein concentration detection kit. 10% SDS-PAGE gel electrophoresis (120V, 1h) was used to separate proteins, PVDF membrane was transferred (90V, 2h), and 10% skim milk was blocked for 1h. Then, Bcl-2(1:200), Bax(1:200), EBI3(1:200), STAT3(1:100), p-STAT3(1:100), CYPIB1(1:200) and MEKK2 (1:200) was incubated overnight on a shaker, with β-actin as an internal reference. After washing with TBST, horseradish peroxidase-labeled secondary antibody (Goat Anti Mouse I...
Embodiment 3
[0036] Example 3 Effect of bergamotide combined with EBI3 blocking peptide on drug resistance of CRC cells
[0037] 1×10 3 A SW1116, LoVo, SW1116 / 5-Fu and LoVo / 5-Fu cells were seeded in a 96-well plate at 37°C, 5% CO 2 , cultured in an incubator with saturated humidity for 48 hours, then divided into groups: the control group was cultured only with DMEM complete medium; the EBI3 Bp group was cultured with DMEM complete medium containing 1 μg / mL EBI3 blocking peptide; the Bg group was cultured with 10 μmol / L Bergamot lactone was cultured in DMEM complete medium; Bg combined with EBI3 Bp group was cultured in DMEM complete medium containing 1 μg / mL EBI3 blocking peptide and 10 μmol / L bergamot lactone. Then SW1116 and LoVo cells were continuously cultured with DMEM complete medium with different concentrations of 5-Fu (0mg / L, 1mg / L, 2mg / L, 4mg / L and 8mg / L) for 48h, and SW1116 / 5-Fu and LoVo / 5-Fu cells were continuously cultured with DMEM complete medium with different concentra...
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