Markers for drug resistance of cancer cells, preparation compositions for reversing drug resistance of cancer cells and applications of markers
A cancer cell and marker technology, applied in the field of biomedicine, can solve problems such as PARP inhibitor insensitivity
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experiment example 1
[0044] Experimental example 1 Animal experiment
[0045] NOD-SCID nude mice (female, four weeks old) were purchased from Huafukang Biological Company. Will 1×10 6 Ovarian cancer cells (cancer cell OV2008 (CON, CEBPB) and cancer cell C13* (shCON, shCEBPB (1) and shCEBPB (2)) were mixed with cell PBS and matrigel (Matrigel, purchased from Merck) 0.2ml was inoculated in the armpit of one side of nude mice, and the tumor volume was detected. When the tumor volume reached approximately 200mm 3 start dosing.
[0046] Among them, shCEBPB(1) / shCEBPB(2) is the silencing genotype constructed by CEBPB using a lentiviral vector, and shCON is the silencing gene control type, see the attached figure. In this experiment example, the lentivirus purchased from Jikai Gene (China) was used to silence or overexpress C / EBPβ, and the puromycin resistance gene was inserted when constructing the lentiviral vector, which was used for selection by adding puromycin after transfection. The stably tra...
experiment example 4
[0059] Experimental example 4 promoter luciferase reporter gene experiment
[0060] Routinely, an appropriate amount of cells to be transfected is planted in a six-well plate, and the transfection is performed when the cells adhere to the wall and reach about 60% confluency. About 2 μg of firefly luciferase reporter plasmid (carrying the promoters of the genes of interest (BRCA1, BRIP1, BRIT1, and RAD51), Qingke Biological Co., Ltd.) was transfected in each well, and the Renilla luciferase reporter plasmid pRL-TK was used as the Reference plasmid (does not carry the promoter of the target gene, Viking Biotechnology Co., Ltd.). Using transfection reagent (lipofectamine TM 3000, Thermo Scientific Universal Transfection Reagent) for double transfection, and 24 hours after transfection, firefly luciferase reporter gene detection kit (Dual- Reporter Assay System) for promoter luciferase reporter gene experiment.
experiment example 5We
[0061] Experimental example 5 Western blot detection of protein expression
[0062] Add RIPA Lysis Buffer (purchased from Sewell) to the cell culture medium to be tested for lysis, and use Coomassie Brilliant Blue solution to determine the protein concentration. After adding the protein loading buffer and mixing well, the protein sample was boiled at 100°C for 5 minutes. 10% SDS-PAGE gel was used for electrophoresis, PVDF membrane was transferred, and then blocked with TBST solution containing 5% BSA for 1 hour at room temperature. According to the molecular weight of the target protein, the membrane was cut into several strips and incubated with the primary antibody at 4°C overnight. The next day, the strips were rewarmed and washed three times with TBST for 10 min each time, and incubated with the corresponding secondary antibody at room temperature for 1 hour. Then rinse with TBST for 3 times, 10 min each time, then use ECL developer to develop color. A Bio-rad exposure ...
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