Design
In vitro comparative observation of cytology.
Time and setting
The experiment was performed in the Medical School of Xi’an Jiaotong University, China in July 2010.
Materials
Human glioma cell line SHG-44 was provided by Shanghai Institute of Cell Biology, Chinese Academy of Sciences.
Methods
SHG-44 cell culture and passaging
Human glioma cell lines SHG-44 were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% calf serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China) at 37°C in a 5% CO2 incubator for passage after 48 hours (1:2).
CoCl2-simulated hypoxia
CoCl2 solution (the final concentration was 150 μM; Sigma, St. Louis, MO, USA) was added into DMEM in which the human glioma cell line SHG-44 was cultured routinely in the 5% CO2 incubator to block oxygen signal transduction to simulate hypoxic signaling[24].
Grouping and intervention
SHG-44 glioma cells of passage three were collected and seeded at 4 × 104 cells/mL. In the normoxic control group, SHG-44 glioma cells were cultured in normoxia (5% CO2) at 37°C for 72 hours. In the normoxic + N-acetylcysteine group, SHG-44 glioma cells were cultured in normoxia (5% CO2) at 37°C for 24 hours and then treated with N-acetylcysteine (10 mM; Sigma) and cultured for another 48 hours. In the hypoxic control group, SHG-44 glioma cells were cultured in hypoxia (CoCl2 150 μM) at 37°C for 72 hours. In the hypoxic + N-acetylcysteine group, SHG-44 glioma cells were cultured in hypoxia (CoCl2 150 μM) at 37°C for 24 hours and then treated with N-acetylcysteine (10 mM) and cultured for another 48 hours.
CoCl2 and/or N-acetylcysteine were added according to the preset experimental groups. A 6-well plate and a double-well slide were included in each group. SHG-44 glioma cells in exponential growth phase were harvested to prepare a cell suspension of 4 × 104 cells/mL. They were inoculated into 6-well plates (3 mL in each well) for each group. A 1 mL volume of DMEM containing CoCl2 (600 mM) was added to each well of the hypoxic control and hypoxic + N-acetylcysteine groups on the next day so that the final concentration of CoCl2 was 150 μM. The same procedure was performed for the normoxic control and hypoxia + N-acetylcysteine groups, except that DMEM containing CoCl2 was replaced with DMEM. The cell suspensions were diluted to 2 × 104 cells/mL and then inoculated into double-well slides (200 μL in each well) for each group. The supernatant was discarded carefully by suction and 200 μL DMEM containing CoCl2 (150 μM) was added to each well of the hypoxic control and hypoxic + N-acetylcysteine groups on the next day. The same procedure was performed for the normoxic control and normoxic + N-acetylcysteine groups, except that DMEM containing CoCl2 was replaced with DMEM. After the cells in all groups were cultured for 24 hours, N-acetylcysteine (the inhibitor of reactive oxygen species) at a final concentration of 10 mM was added into each well of the normoxic + N-acetylcysteine and hypoxic + N-acetylcysteine groups. The cells in each of these groups were cultured for an additional 48 hours.
Flow cytometry for reactive oxygen species expression
After the fluorescent probe (2’,7’-dichlorofluorescin diacetate [DCFH-DA], 10 mM; Beyotime Institute of Biotechnology, Beijing, China) was prepared, a 5 μL aliquot was added to 5 mL of serum-free DMEM (1:1 000). Cells were digested with 0.25% trypsin (Sigma) and dispersed with a pipette. They were then transferred into a 10 mL centrifuge tube and centrifuged at 1 000 r/min for 5 minutes. The supernatant was discarded, the pellet was centrifuged once more, and the supernatant was discarded again. 500 μL fluorescent probe DCFH-DA diluted with serum-free medium (1:1 000) was added into each well, mixed and transferred to a 1.5 mL EP tube for incubation in a cell incubator at 37°C for 20 minutes so that DCFH-DA which had entered into the cells was oxidized to fluorescent DCF by the reactive oxygen species. After the incubation was complete, the cells were washed three times with serum-free cell culture medium (centrifuged at 1 000 r/min for 5 minutes) to fully remove extracellular DCFH-DA, and then the fluorescence of DCF was detected with flow cytometry (BD FACSCalibur, San Jose, CA, USA) using excitation and emission wavelengths of 488 nm and 525 nm, respectively, to detect intracellular reactive oxygen species levels.
Real-time reverse transcription-PCR detection of hypoxia-inducible factor-1α mRNA expression
After culture, the cells from each 6-well plate of the four groups were triturated with a pipette and then transferred into 1.5 mL EP tubes (in an ice bath) to rapidly extract RNA using an RNA extraction kit (RNAfast200; Fermentas, Vilnius, Lithuania). 5 μL total RNA was extracted, and mRNA was used as the template to synthesize cDNA by reverse transcription. Then 1 µL reverse transcription reaction mixture (cDNA) was used as the template for amplification reactions. Primer design for hypoxia-inducible factor-1α and β-actin was performed using Primer 5.0 software, and primers were synthesized by Beijing Sunbiotech Co., Ltd., China (Table 1).
Total reaction volume was 25 µL for each sample and included the following: ddH2O, 10.5 μL; SYBR® Green Realtime PCR Master Mix (Fermentas), 12.5 μL; upstream primer (10 µM), 0.5 μL; downstream primer (10 µM), 0.5 μL; cDNA by reverse transcription, 1 μL. PCR conditions: 50°C for 2 minutes for 1 cycle and 95°C for 10 minutes for 1 cycle; then 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds and 72°C for 30 seconds. The products were placed into a Bio-Rad iQTM5 Multiple Real-time PCR Instrument (Hercules, CA, USA) after setting the reaction conditions, and the experimental data were analyzed while the reaction proceeded. Ct value was obtained, and results were obtained using 2–△△Ct calculation. β-actin was used as reference.
Statistical analysis
The statistical software SPSS 13.0 (SPSS, Chicago, IL, USA) was used for statistical analysis of the experimental data (test level α = 0.05). Intergroup comparison was performed using analysis of variance, and paired comparison was conducted using the least significant difference t-test. A value of P < 0.05 indicated a significant difference.
