The Expression And Regulation Of The PI3K/Akt Signaling Pathway In Hemangioma Vascular Endothelial Cell In Vitro

ObjectiveTo observe the expression of PI3Kp110catalytic subunit and p-Akt, Akt in cultured vascular endothelial cells, and its regulation mean while explored the effect of PI3K/Akt signal pathway in the different phases of children hemangioma.Methods1. The hemangioma vascular endothelial cells which came from the sterile hemangioma were cultured by using the tissue culture method and established hemangioma endothelial cell lines, observing the proliferation characteristics, drawing the growth curve.The hemangioma vascular endothelial cell was identified by the immunohistochernistry technique observing the expression of the Factor Ⅷantigen which only existed in endothelial cells.2. Synchronized cultured endothelial cells, PI3Kp110, p-Akt and Akt of them were detected by Western blotting mean while cell cycle and cell apoptosis were examined by flow cytometry technique,then analysis the level of PI3Kp110, p-Akt and Akt expression and the correlation in hemangioma vascular endothelial cell cycle and apoptosis.3. When the cells were in the logarithmic phase,the culture medium was exchanged to serum-free medium to make the cell synchronization in48hours.25umol/L LY294002,10nmol/L rapamycin and1.0g/L triamcinolone acetonide were added to observed the effects on the expressions of PI3Kp110, Akt and p-Akt, the cell cycle distribution and cell apoptosis with the hemangioma vascular endothelial cell when the cells were cultured after24hours,and then the cell apoptosis,cell cycle related of PI3Kp110, p-Akt and Akt expression level of vascular endothelial cells were analyzed.Results1. The hemangioma vascular endothelial cells migrated from the tissue to the postcultural after four or five days, only a small number of cells spread after one week adherent growth, growth and division after two weeks, three weeks after the cells began to grow fast, and around, the cells covered the bottom plate.Inverted phase contrast microscope and found that the isolated endothelial cultured epithelial cell morphology, part of the cells show cricoid arrangement, and has the slabs of appearance.Cultured cells of Factor Ⅷ related antigen was positive.2. The cultured hemangioma vascular endothelial cells expressed p110, p-Akt and Akt protein in vitro.3. After LY294002and rapamycin acts on the cultured hemangioma vascular endothelial cells for24hour, the protein level of p110, p-Akt were lower than control group, control group p110(0.37±0.04), p-Akt(0.22±0.02), intervention group LY294002p110(0.19±0.01),p-Akt(0.09±0.02),the difference was significant(P<O.01).The p110protein expression levels in the triamcinolone acetonide group was declined than control groupp110(0.34±0.04) in the control group, the triamcinolone acetonide group (0.10±0.00),control group and intervention group had significant difference (P<0.01), while the p-Akt protein showed no expression.Intervention group cell cycle were arrested in G0/G1phase.control group(60.88±6.23)%,LY294002(93.81±2.65)%,Rapa(88.30±3.66)%,triamcinolone acetoni-de(92.93±0.01)%. Before and after intervention, the difference was statistically significant (P<0.05). The total of apoptosis cells were increased, control group(0.51±0.03)%, LY294002(1.24±2.34)%,Rapa(14.41±1.77)%.triamcinolone acetonide(9.98±0.67)%. Cont-rol group and intervention group had significant difference(P<0.01). While the total Akt protein expression levels had no changed.Conclusions1. The cultured hemangioma vascular endothelial cells expressed p110, p-Akt and Akt protein in vitro.2. PI3K/Akt signaling pathway may be involved in cultured hemangioma vascular endothelial cells apoptosis in vitro.3. LY294002, Rapa, and triamcinolone acetonide might interfere with PI3K/Akt signaling pathway in the corresponding targets and down regulation p-Akt phosphorylation in cultured vascular endothelial cells in vitro, and the cultured vascular endothelial cells were arrested in G0/G1phase, promote cell apoptosis.