Materials and Apparatus
HUVECs (GNHu39, purchased from cell bank of Chinese Academy of Sciences, China). Fetal bovine serum (FBS) (Gibco. USA), Dulbecco’s modified eagle medium (DMEM) (Gibco. USA), Penicillin/Streptomycin (Gibco. USA), Pancreatin (Gibco. USA). Sevoflurane (Abbott Laboratories. USA). RevertAid Fist Strand cDNA Synthesis Kit (Thermo Fisher.K1622. USA), Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher.K0221. USA), Trizol reagent (Invitrogen, Carlsbad, CA), RNeasy kit (Qiagen, Dusseldorf, Germany), Quantitative PCR instrument (Bio-RAD. USA). Flow cytometer (BD. USA). BCA Protein Assay Kit (pa115-01, TIANGEN. China), Wide range color pre-stained protein marker (MP206, TIANGEN. China), HRP-DAB substrate reagent kit (PA109, TIANGEN, China), GAPDH antibody (GTX17307, TIANGEN, China). Lipofectamin RNAiMAX reagent (Invitrogen, USA)
HUVECs treatment was performed as previously described. Sterile technique was abided during all experiment operations. Vascular endothelial cells were cultured in DMEM which contains 1% penicillin/streptomycin,5% FBS, and 1% endothelial cell growth supplement, and in a humidified atmosphere contains 5% CO2 and 95% air at 37°C.
Sevoflurane exposure was performed as previously described. A sealed plastic box was prepared in advance, it has an air inlet and an outlet. A Datex infrared gas analyser (Puritan-Bennett, Tewksbury, MA, USA) was necessary to continuously monitor the delivered oxygen, CO2 and sevoflurane concentrations. In the section of sevoflurane exposure, 21% oxygen and 5% CO2, 2% sevoflurane or not, were delivered from an anesthesia machine to a sealed plastic box, and the sealed box was placed in an abacteria incubator at 37°C all the time. Cells were cultured in 6-well plates and when cells grow to 90%-95% confluence, the 6-well plates without lips were put into the sealed plastic box (the box was sterile also) to receive sevoflurane exposure. We treated the cells with 2% sevoflurane for 0.5 h, 1 h, 2 h respectively in serum free media. The first group was the control group which exposed to the same environment with other three groups but without sevoflurane.
HUVECs were planted into 6-well plates at a density of 2×105 cells/well and then cultured in a humidified atmosphere contains 5% CO2 and 95% air at 37°C. Cell suspension was harvested in serum free media, and cell count was used to maintain the density of 8×104/ml for transfection. Added 1.5 μl VE-cadherin siRNA, 500 μl Optimem and 5 μl RNAMAX in per plate and incubated in room temperature. Added 2.5 ml HUVECs suspension in per plate after 20 min later. Then replaced the serum free media with the complete media included 10% FBS after 6 h. Cells were collected and then extracted mRNA and protein after 48 hours. The sequences for VE-cadherin siRNA were as follows (prepared by Shanghai GenePharma Co., Ltd): sense, 5’-GGAACCAGAUGCACAUUGAUU-3’ and anti- sense,5’-UCAAUGUGCAUCUGGUUCCUU-3’.
The scratch assay
After sevoflurane exposure, the scratch assay was performed immediately. Put out the 6-well plates from the sealed plastic box, sterile yellow pipette tips were used to scratch similar sized wounds on monolayer cells on the super clean bench. Scratched monolayer cells were washed three times by PBS to remove cell fragments and then cultured with serum free media in a incubator, in a humidified atmosphere contains 5% CO2 and 95% air at 37°C. A microscope was used to observe the migration distance of HUVECs and taken photos. We took photos after just scratched and 12 h later.
Poured out the media and washed the cells with PBS three times. HUVECs were lysed in 1×lysis buffer (1 ml RIPA and 10 μl PMSF) and kept on ice for 30 min. Then scraped the cells off the plate and centrifugated 12000 rpm in 4 °C for 20 min，then collected the supernatant liquid transferred to new EP tubes for protein quantitation by BCA Protein Assay Kit. The extracted protein was loaded onto 10% SDS-PAGE for electrophoresis with equal amount and then transferred onto a PVDF membrane to transmembrane in 350 mA for 2 h. After transmembrane, the membrane was blocked overnight at 4 °C with 5% skimmed milk. Then the membrane was incubated with primary antibodies for 1 h at 37 °C and overnight at 4 °C later. The membrane was incubated with secondary antibodies at 37 °C for 1 h after washed three times by PBS. Finally, added 1 ml A solution and 1 ml B solution onto the membrane to develop after PBS washed three times again.
RNA was extracted from HUVECs using the Trizol reagent and RNeasy kit. Then these RNA samples were reversely transcribed into single-stranded cDNA by using the first-strand cDNA synthesis kit. These cDNA products were further amplified using qPCR by SYBR Green RT-PCR kit. Sequence specific primers as follows: VE-cadherin,5'-TCT GTC CCA GAA ATG TCA CG-3' (Forward) and 5'-CAC CGG AGT CAT CAA CTG TG-3' (Reverse); GAPDH,5'-CCT CTC TGG CAA AGT CCA AG-3' (Forward) and 5'-GGT CAC GCT CCT GGA AGA TA-3' (Reverse). The PCR cycles were as follows: 5 min at 95°C and 10 sec at 95°C including 40 cycles , 15 sec at 65°C, 10 sec at 72°C and a final 1 min at 65°C. Relative expression of qPCR products was normalized with GAPDH mRNA expression.
Flow cytometry was used to detect the activity of cells. After sevoflurane exposure, HUVECs were seeded in 6-well plate on the logarithm growth phase，then added 2 ml DMEM in each well (4×105 cells/well). After culturing 24 h, HUVECs were fixed by 70% ethanol and were stained. Briefly, cells were mixed with 5 μl propidium iodide (PI) and were incubated in the dark at room temperature for 30 min. The cell activity was detected within 1 h by flow cytometer.
Statistical analysis of the data was conducted with SPSS (version 19.0) software and the statistical histograms were derived from GraphPad Prism (version 5) software. Differences between two groups were tested using an unpaired student t test. Significant difference was received when P <0.05.