Application of anxa6 expression inhibitor in preparation of medicine for treating lung cancer
A technology for inhibitors and lung cancer, applied in drug combinations, gene therapy, anti-tumor drugs, etc., can solve the problems of ANXA6 function that have not been reported, and achieve the effect of growth rate inhibition
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Embodiment 1
[0071] Embodiment 1, cell culture and lentivirus infection
[0072] Cell culture conditions are: 37 ° C, 5% CO 2 Static cultivation.
[0073] The basal medium used in the examples is: take serum-free RPMI-1640 medium, add fetal bovine serum to a concentration of 10% v / v, add penicillin to a concentration of 100 U / ml, and add streptomycin to a concentration of 0.1 mg / ml.
[0074] 1. Cell culture, subculture and inoculation
[0075] 1. 801D cells and H1299 cells were inoculated in basal medium.
[0076] 2. Subculture the 90% confluent cells, add trypsin digestion solution, and digest at 37°C for about 5 minutes until the cells are completely digested into single cells.
[0077] 3. Add the basal medium and pipette to mix. After mixing, count with a Muse cell counter.
[0078] 4. Inoculate 801D cells and H1299 cells in a 96-well plate, the number of cells per well is 4000, and each type of cells is inoculated in 12 wells.
[0079] 5. 37°C, 5% CO 2 Incubate overnight.
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Embodiment 2
[0085] Example 2, detection of ANXA6 gene knockout effect
[0086] 1. Extraction of total cell protein
[0087] 1. Cells infected with virus and screened by Puromycin were cultured in basal medium until 70% to 80% confluence.
[0088] 2. Collect two groups of cells, perform trypsinization, centrifuge and discard the supernatant.
[0089] 3. Wash the cells 3 times with pre-cooled normal saline to remove the residual medium, and aspirate the supernatant as much as possible after the last centrifugation.
[0090] 4. Prepare cell lysate: 1ml lysate + 10μl protease inhibitor (Cocktail) + 1μl dithiothreitol (DTT) + 7μl PMSF, mix well and place on ice.
[0091] 5. For the cells collected in step 3, add 100 μl of cell lysate for every 1 million cells, lyse on ice for 10-30 minutes, and mix well every 5 minutes.
[0092] 6. Centrifuge in a low-temperature high-speed centrifuge, the centrifugation conditions are: 13000rpm, 4°C, 10min.
[0093] 7. Aliquot the supernatant and store at...
Embodiment 3
[0113] Example 3, detection of the effect of ANXA6 knockout on cell proliferation
[0114] 1. Monoclonal cell screening
[0115] 1. The 801D and H1299 cells with ANXA6 gene knockout were cultured in basal medium to 70% to 80% confluence.
[0116] 2. Add trypsin digestion solution and digest at 37°C for about 5 minutes until the cells are completely digested into single cells. Add basal medium, mix well, and discard the supernatant after centrifugation. Resuspend with basal medium, and count with Muse cell counter after resuspension.
[0117] 3. Dilute according to the counting results, and the final dilution is 10 cells / ml.
[0118] 4. After mixing, inoculate a 96-well plate with 100 μl of cell suspension per well.
[0119] 5. 37°C, 5% CO 2 Let stand and observe after one week.
[0120] 6. Subculture and amplify according to the growth of monoclonal cells.
[0121] 7. According to the detection method shown in Example 2, the ANXA6 knockout condition of the monoclonal ce...
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