Application of CGA gene as target spot in preparation of drug for diagnosing and treating drug-resistant solid tumors
A technology based on solid tumors and genes, applied in drug combinations, antineoplastic drugs, biochemical equipment and methods, etc., can solve problems such as treatment failure
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Embodiment 1
[0053] Example 1: Real-time quantitative PCR (Real-time qualitative PCR, RT-qPCR) detection of CGAmRNA in gastric cancer drug-resistant cell line SGC7901 ADR and SGC7901 VCR The expression difference between the cell line SGC7901 and the control group
[0054] 1. Research object
[0055] Gastric adenocarcinoma cell line SGC7901 is derived from the Academy of Military Medical Sciences and preserved by the State Key Laboratory of Tumor Biology, Air Force Military Medical University;
[0056] SGC7901 cells were cultured in DMEM medium containing 10% (v / v) fetal bovine serum;
[0057] Drug-resistant gastric cancer cell line SGC7901 ADR and SGC7901 VCR The cells were established by the intermittent induction method. The method was to expose SGC7901 parental cells to lethal doses of doxorubicin and vincristine for a short period of time and induce them successively. The alkali concentration is gradually increased from 1 μg / ml to 5 μg / ml; specifically, SGC7901 ADR For long-term c...
Embodiment 2
[0067] Example 2: WB detection of CGA protein in gastric cancer drug-resistant cell line SGC7901 ADR and SGC7901 VCR The expression difference between the cell line SGC7901 and the control group
[0068] 1. Research object
[0069] Drug-resistant gastric cancer cell line SGC7901 ADR and SGC7901 VCR The cultivation with the control group cell line SGC7901 was the same as in Example 1.
[0070] 2. Experimental method
[0071] Detection of CGA protein in SGC7901 by WB ADR and SGC7901 VCR and the expression level of SGC7901 cells, the specific steps are as follows:
[0072] After the normal culture of each cell for 24 hours, the DMEM medium without fetal bovine serum was replaced to continue the culture for 24 hours, and the culture supernatant and cells were collected respectively;
[0073] Use the Millipore ultrafiltration tube to centrifuge at 4°C and 4000×g for 1 h to collect the secreted protein in the culture supernatant, use the RIPA cell lysate to extract the total...
Embodiment 3
[0077] Example 3: Immunofluorescence (IF) detection of CGA protein in gastric cancer drug-resistant cell line SGC7901 ADR and SGC7901 VCR Differences in expression and subcellular localization between the cell line SGC7901 and the control group
[0078] 1. Research object
[0079] Drug-resistant gastric cancer cell line SGC7901 ADR and SGC7901 VCR The cultivation with the control group cell line SGC7901 was the same as in Example 1.
[0080] 2. Experimental method
[0081] Detection of CGA protein in SGC7901 by IF ADR , SGC7901 VCR and the expression and localization of SGC7901 cells, the specific steps are as follows:
[0082] After the cells were inoculated on chamber slides and cultured for 24 hours, 4% paraformaldehyde was added to fix the cells, 0.5% Triton was added, and the cells were permeabilized at room temperature for 15 minutes; goat serum blocking solution was added dropwise and left at room temperature for 30 minutes ; The primary antibody (rabbit anti-hu...
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