In-vitro culture and differentiation of preadipocytes
The study was approved by the Institutional Review Boards of Xiangya 3th Hospital of Central-south Uniersity (Changsha, Hunan, China) and written informed consent was obtained from each subject. Orbital adipose/connective tissues of 5 patients with TAO underwent orbital decompression were obtained, including 3 females and 2 males; 37-52 years old (mean age 45 years old), inclusion criteria: 1) meet the Tao diagnostic criteria, 2) have normal thyroid function for more than 6 months, 3) have never received iodine 131 treatment or orbital radiotherapy. Normal orbital adipose tissues were obtained from four patients who underwent ophthalmic surgery due to trauma, including 2 females and 2 males; 38-45 years old (average age 42 years old). The inclusion criteria of the control group were: 1) no autoimmune disease, 2) no history of thyroid disease or orbital inflammation disorders.
Orbital tissues were minced and placed directly in plastic culture dishes, allowing preadipocytes to proliferate. Cells were cultured with DMEM /F-12(1:1) containing 20% FBS, penicillin (100 U/mL) and streptomycin (100 U/mL) in a humidified 5% CO2 incubator at 37 ℃. The preadipocyte differentiation was induced as follows. When orbital cells reached confluence after in-vitro culture for 3-5 days, the culture medium were replaced by serum-free DMEM/F12 (1:1) supplemented with biotin (33 mmol/L), pantothenic acid (17 mmol/L), transferrin (10 mg/mL), triiodothyronine (0.2 nmol/L), insulin (1 mmol/L), and (for the first 4 days only) dexamethasone (1 μmol/L) and isobutylmethylxanthine (IBMX; 0.1 mmol/L). The differentiation continued for 13 days and the medium were replaced every 3–4 days.
To evaluate the effect of cytokines on preadipocyte differentiation and HLA-DR expression, cell cultures were treated with IFN-γ (1ng/ml, SIGMA) , IL-6(1ng/ml, SIGMA), PPAR-γagonist pioglitazone (10μmol / L) or PPAR-γantagonist GW9662 (10μmol / L) and cultured for 13 days. The morphological changes of TAO and normal preorbital adipocytes after differentiation were observed.
Oil Red O staining
After differentiation, cells were washed with 1×PBS, fixed in 10% formalin overnight at room temperature and rinsed in distilled water. Cells were stained with filtered 0.21% Oil Red O for 2 h and then again rinsed in distilled water. Then cells were exposed to isopropanol to eliminate extra dye, rinsed with PBS and visualized under a microscope.
Intracytoplasmic lipid (triglyceride accumulation) detection
Cells were grown in 96-well plates for density of (3-5) ×107/L. After differentiation, cells were fixed in 10% formalin for 1 h, and rinsed with distilled water. Cells were stained with filtered 0.21% Oil Red O for 2 h and then rinsed in distilled water for several times. Isopropanol was added to extract the dye after water was vaporized in the incubator. The absorbance (OD value) was measured on wavelength of 510nm using the ultraviolet spectrophotometer.
Cells were harvested using 0.25% trypsin/ EDTA and fixed for 30 min in ice-cold 2% formaldehyde. The fixed cells were washed in flow cytometry buffer (PBS, 2% FBS, 0.2% Tween 20) and incubated for 20 min in flow cytometry buffer containing PE-conjugated monoclonal antibodies against CD29 (eBioscience, Clone: TS2/16.2.1), CD44 (eBioscience, Clone: P2A1), CD49d (eBioscience, Clone: BU49) or HLA-DR (eBioscience, Clone: JM-DR), respectively. Control experiments include blank and isotype IgG. The stained cells were analyzed by BD FACSCalibur.
MTT colorimetric assay
Cells were collected and adjust the cell density to 5 × 104 / L, inoculated on six well plate, cultured for 24 hours. 10 μmol / L pioglitazone and GW9662 were added . After 48 hours of continuous culture, 20ul 5mg / ml MTT solution was added to each well. After 4 hours of culture, the supernatant was discarded, 100ul DMSO was added to fully dissolve, and the absorbance (OD value) was measured at the wavelength of 570nm with an enzyme labeling instrument.
Cells were grown in six-well plates with coverslips. HLA-DR expression was measured using the SP histological assay and HLA-DR antibody (abcam, ab20181). Brown dye in cytoplasm or cell membrane was found in HLA-DR positive cells.
Quantitative Real-time RT-PCR
Total RNA was isolated from cell cultures using Trizol and reverse transcribed into cDNA. Primers were designed using primer premier 5.0 and the sequences were as follows: HLA-DR, 5’- aggcgagtttatgtttg -3’ (sense) and 5’- cagggctgttcgtgag -3’ (antisense). GAPDH, 5’-aatcccatcaccatcttcc-3’ (sense) and 5’-catcacgccacagtttcc-3’ (antisense). Quantitative real-time PCR was performed using SYBR Green method on LightCycler system. The relative expression levels of HLA-DR were calculated with the 2-ΔΔCt formula.
The SPSS software package (version 11.0) was used for the statistical analysis. The data were from at least three separate experiments and expressed as mean ±S.D. Two-sided non-paired t-test was used to analyze the data from two groups. Statistical differences were considered significant when p<0.05.