in thymoma
Total RNA of thymoma and PBMC was prepared with TRIzol reagent
(Invitrogen-Life Technologies, Carlsbad, CA, USA). cDNA was reverse
transcribed from total RNA (2 μg). cDNA was amplified with SYBR Premix
Ex Taq™ (Takara Biotechnology, Dalian, China). The sequences of the PCR
primer pairs for RORC (RORγt), FOXP3 , and GAPDHwere designed by GeneRunner, as shown in Table 1 (Aoke Biological
Technology LLC, Beijing, China).
The amplification was performed at 95 °C for 1 min for pre-denaturation;
40 cycles of 94 °C for 30 s for denaturation, 56 °C for 15 s for
annealing, and 72 °C for 30 s for extension; followed by a final
extension at 72 °C for 10 min. The relative expression levels of target
genes were determined using the 2−ΔΔCq method.
Thymoma
cell line culture and cytokines ELISA assay in culture medium
The thymoma cell line Thy0517 provided by cardiothoracic surgery
department of Tianjin Medical University General Hospital (Patent
number: ZL 2014 1 0312866.6), established from type AB thymoma with
myasthenia gravis patient. Cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM)/high glucose medium (HyClone, Utah, USA) with 10%
heat-inactivated fetal bovine serum (FBS) (NQBB, Victoria, Australia),
100 U/mL penicillin, 100 ug/mL streptomycin (Sigma-Aldrich, Missouri,
USA) and in 5% CO2 incubator (Thermo, California, USA) at 37°C, the
doubling time was 37 hours [17].
Thy0517 cells were subcultured into three culture bottles at the same
time, the initial cell density was 20%. 1ml of culture medium was token
from the 1st to 5th day after subculture, and blank culture medium was
the control as the 0th day. The contents of IL-1β, IL-4, IL-6, IL-12,
IL-21, IL-23 and TGF-β were quantified using commercially available
ELISA kits according to the manufacturer’s instructions (Proteintach
Group Inc, Chicago, USA).