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).