Application of PF-4708671 to preparation of drugs for reversing osimertinib resistance of non-small-cell lung cancer
1. PF-4708671, non-small cell lung cancer technology, applied in the field of genetic engineering, to improve survival rate, good clinical promotion value, and reduce drug resistance
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Embodiment 1
[0028] The influence of PC-9, PC-9 / G, HCC-827, HCC-827 / OR on Osimertinib sensitivity by CCK-8 method:
[0029] 1) Cell inoculation: Digest monolayer cultured cells with 0.25% trypsin, prepare a single cell suspension with 1640 culture medium containing 10% fetal bovine serum, inoculate 2500 cells per well in a 96-well culture plate, each well Volume 200ul.
[0030] 2) Culture cells: move the culture plate into CO 2 In an incubator at 37°C, 5% CO 2 : and relative humidity conditions, cultivated for 3 days.
[0031] 3) Color development: After the first 1-3 days of culture, add 20ul of CCK-8 solution to each well, and continue to incubate at 37°C for 4h.
[0032] 4) Colorimetry: Measure the optical density (OD) at a wavelength of 490nm, and repeat the experiment three times with 3 replicate wells in each group. Calculate the survival rate. Survival rate = OD value of the experimental group / OD value of the control group × 100%.
[0033] Seed pPC-9, PC-9 / G, HCC-827, HCC-827 / OR...
Embodiment 2
[0037] The activation level of p-s6k1 in PC-9, PC-9 / G, HCC-827, HCC-827 / OR cells was detected by Western blot:
[0038] 1. Method: extraction of total cell protein; SDS-PAGE electrophoresis; semi-dry transfer; western blot; ECL detection
[0039] 2. Results determination: if image 3 shown.
[0040] 3. Results analysis: the expression level of p-s6k in osimertinib-resistant PC-9 / G, HCC-827 / OR cell lines was significantly higher than that in osimertinib-sensitive PC-9, HCC-827 cell lines .
Embodiment 3
[0042] Sensitivity of HCC-827 / OR cells to osimertinib and osimertinib+PF-4708671 detected by CCK-8 method
[0043] 1) Cell inoculation: Digest monolayer cultured cells with 0.25% trypsin, prepare a single cell suspension with 1640 culture medium containing 10% fetal bovine serum, inoculate 2500 cells per well in a 96-well culture plate, each well Volume 200ul.
[0044] 2) Culture cells: move the culture plate into CO 2 In an incubator at 37°C, 5% CO 2 : and relative humidity conditions, cultivated for 3 days.
[0045] 3) Color development: After the first 1-3 days of culture, add 20ul of CCK-8 solution to each well, and continue to incubate at 37°C for 4h.
[0046] 4) Colorimetry: Measure the optical density (OD) at a wavelength of 490nm, and repeat the experiment three times with 3 replicate wells in each group. Calculate the survival rate. Survival rate = OD value of the experimental group / OD value of the control group × 100%.
[0047] HCC-827 / OR treated with osimertin...
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