Application of CD47 detection reagent in preparing colorectal cancer anti-EGFR monoclonal antibody drug resistance diagnostic agent
A detection reagent and detection kit technology are applied in the field of diagnostic kits for tumor resistance to anti-EGFR monoclonal antibodies to achieve the effect of reducing curative effect and high-efficiency treatment
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Embodiment 1
[0045] Example 1. Establishment of Cetuximab-resistant colorectal cell line LIM1215R
[0046] Colorectal cancer cells were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin and streptomycin; the incubator conditions for the cells were 37°C, 5% CO 2 , saturated humidity and constant temperature. When the cell confluency reached 80-90%, the cells were digested and passaged with 0.25% trypsin. The establishment steps of cetuximab-resistant colorectal cell line LIM1215R are as follows:
[0047] 1. Select the colorectal cancer cell line LIM1215 (European Collection of Authenticated Cell Cultures) sensitive to Cetuximab, and culture the cells in RPMI-1640 medium containing a small concentration of Cetuximab.
[0048] 2. After the cells adapt to the drug concentration environment, gradually add a higher concentration of Cetuximab to the medium until the Cetuximab concentration is 10 μM. At this time, the cells are the colorectal cell line LIM1215R resistant to Cetux...
Embodiment 2
[0051] Example 2. Detection of CD47 RNA expression in Cetuximab-sensitive and drug-resistant colorectal cancer cells
[0052] The RNA of LIM1215 and LIM1215R was extracted, reversed into cDNA, and its expression was detected by qPCR.
[0053] The sequences of CD47 primers used in this experiment are as follows:
[0054] SEQ ID NO.1 forward primer: 5'-AGAAGGTGAAACGATCATCGAGC-3'
[0055] SEQ ID NO.2 reverse primer: 5'-CTCATCCATACCACCGGATCT-3'
[0056] The experimental steps are as follows:
[0057] (1) Remove the culture medium in the cell culture plate, wash with PBS 3 times, then add 1mL Trizol reagent to lyse the cells, pipette evenly until clear, transfer the lysate into a 1.5ml centrifuge tube, and place it at room temperature for 5min ;
[0058] (2) Add 200 μL chloroform to each tube, pipette evenly, and let stand at room temperature for 5 minutes;
[0059] (3) Place the centrifuge tube in a centrifuge at 4°C and centrifuge at 12,000 rpm for 10 minutes, and absorb the...
Embodiment 3
[0080] Example 3. Detection of CD47 Membrane Protein Expression in Cetuximab-sensitive and Drug-resistant Colorectal Cancer Cells
[0081] 1. Remove the medium in the cell culture plate, wash with PBS 3 times, and then digest with trypsin for 2 minutes.
[0082] 2. After terminating the digestion, transfer the cells into a 15mL centrifuge tube with a pipette, centrifuge at 2000rpm for 5min, and wash twice with PBS.
[0083] 3. Resuspend the cells in PBS and adjust the cell density to 1×10 7 / mL, pipette 100 μL into a new centrifuge tube, add 5 μL of FITC anti-human CD47 antibody, incubate at 4°C in the dark for 20 min, and set an isotype control tube for each cell at the same time.
[0084] 4. Add 1mL PBS to each tube, blow and resuspend with a pipette, place the centrifuge tube in a centrifuge and centrifuge at 2000rpm for 5min, discard the supernatant.
[0085] 5. Repeat step 4.
[0086] 6. After the cells were resuspended in 500 μL PBS, the expression level of the cell m...
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