Non-small cell lung cancer prognosis detection composition and applications thereof
A technology for non-small cell lung cancer and a composition, which is applied in recombinant DNA technology, biochemical equipment and methods, and microbial determination/testing, etc. Low specificity and low sensitivity, the effect of improving the detection rate
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Embodiment 1
[0045] Cell culture and in vitro experiments: SPC-A1 cell line (purchased from Shanghai Cell Bank of Chinese Academy of Sciences) was grown in RPMI1640 medium (purchased from Hyclone) containing 10% FBS, and log phase cells were seeded in 6-well plates, 2× 10 5 / Well, after the cells grow and adhere to the wall, wash with Hanks solution, add RPMI1640 culture medium containing 5μg / ml cisplatin (purchased from Shandong Qilu Pharmaceutical Factory) for 24h, 48h, 72h, 37℃, 5% CO 2 Cultivate under conditions. At the same time, a control group (without DDP) was set up, and each group was set up with 3 multiple holes. The supernatant corresponding to each time point of each group was collected and stored frozen at -70°C. The independent experiment was repeated 3 times.
[0046] Determination of cell apoptosis rate: Collect SPC-A1 cells at each time point in the above groups, wash twice with cold PBS, and adjust to a final concentration of 1×10 6 / mL of single cell suspension, take 1×10...
Embodiment 2
[0055] SPC-A1 cells were grown in RPMI1640 medium containing 10% fetal bovine serum, 100U / mL penicillin, and 100U / mL streptomycin at 37°C and 5% CO2 in a constant temperature incubator, and grew adherently. SPC-A1 cells were digested with 0.25% trypsin. Collect and count SPC-A1 cells to 1.5×10 6 The concentration of each / mL is added to a 6-well plate, 1mL per well, placed at 37℃, 5% CO 2 Cultivate in a constant temperature incubator. When the cells cover 75%~80% of the bottom of the plate, discard the culture medium, add the culture medium with the final concentration of DDP of 2.5μg / mL, 2mL / well, and set up a control group without DDP for 12h, 24h, 48h respectively And 72h.
[0056] Determination of cell apoptosis rate: the same as in Example 1.
[0057] RNA extraction and RT-PCR: respectively extract RNA from SPC-A1 culture supernatant sample and SPC-A1 intracellular RNA, and use 2720 gene amplification instrument to simultaneously reverse transcribe the target gene miR-27a (mi...
Embodiment 3
[0065] Participants: 26 patients with advanced lung cancer were from the First Affiliated Hospital of Nanjing Medical University from March 2010 to May 2012. There were 16 males and 10 females, aged 31-77 years (median age: 61 years). All patients were newly diagnosed with lung adenocarcinoma, unable to undergo radical surgery, and had not been treated with radiotherapy or chemotherapy. The diagnosis of lung cancer was confirmed by histopathological examination. The patient received first-line or second-line chemotherapy, docetaxel plus cisplatin as the first-line chemotherapy, and pemetrexed as the second-line chemotherapy. Seven of the patients completed 3 to 4 cycles of first-line chemotherapy, and the remaining 15 completed 1 to 2 cycles of short-term chemotherapy. After each chemotherapy cycle, an efficacy evaluation was performed according to RECIST standards, and the patients were followed up until March 2013.
[0066] The evaluation of chemotherapy efficacy is carried o...
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