The Expression And Regulation Of The MEK/ERK Signaling Pathway In Hemangioma Vascular Endothelial Cell In Vitro

ObjectiveTo observe the expression and regulation of MEK,ERK and p-ERK in hemangioma derivedendothelial cells(HemECs),to investigate the effect of MEK/ERK signal pathway indifferent phases of hemangioma from infantile.Methods1. HemECs were isolated from freshly resected hemangioma specimens.We observedthe proliferation of HemECs,drew the growth curve.With immunohistochemistrytechnique,we observed the expression of the Factor Ⅷantigen which only existed inendothelial cells to identify HemECs.2. When the cells were in the logarithmic phase,we changed the culture medium toserum-free and made the cell synchronized in48hours. HemECs were treated with50um/L Propranolol,0.1ng/L Estradiol for treatment group and culture medium withserum for control group after24hours,then we observed the protein levels ofMEK,ERK and p-ERK,the cell cycle distribution and apoptosis. Finally we analyzedthe relationship between apoptosis,cell cycle and the protein levels of MEK,ERK andp-ERK.Results1. The HemECs migrated from the hemangioma tissue after three or four days, only asmall number of cells spread after one week.After three weeks,the cells began to growfast and cover the bottom plate one month later.With inverted phase contrastmicroscope,we found that the isolated endothelial cells presenting epithelial cellmorphology and showing the slabs of appearance. Factor Ⅷrelated antigen waspositive.2. After HemECs were treated with Propranolol and Estradiol for24hours, The totalrate of apoptosis cells of Propranolol group (2.87±0.57)%increased,compared to the control group(1.77±0.21)%,the difference was statistically significant(t=3.14,P<0.05).The total rate of apoptosis cells of Estradiol(0.70±0.10)%declined comp-ared to the control group(1.77±0.21)%,the difference was statistically significant (t=-8.00,P<0.05).The cell cycle of Propranolol were arrested in G0/G1phase. Comparedto the control group(94.02±0.85)%,the propranolol group (96.42±1.16)%was higherthan the control group,which showed significant difference(t=-2.89,P<0.05).TheEstradiol group (90.52±1.28)%was lower than the control group (94.02±0.85)%,which showed significant difference(t=-3.98,P<0.05).The protein level of MEK(0.06±0.01),ERK1/2(0.87±0.05/0.14±0.01) and p-ERK1/2(0.02±0.01/0.11±0.05) trea-ted with propranolol was lower than the control group MEK(0.08±0.00),ERK1/2(1.16±0.06/0.29±0.04) and p-ERK1/2(0.06±0.02/0.42±0.08),which showed signi-ficant difference(t＝-2.88,-6.79/-6.20,-3.96/-5.74,P<0.05).The protein level of MEK(0.14±0.01),ERK1/2(1.36±0.11/0.52±0.10),p-ERK1/2(0.12±0.03/0.85±0.10) treatedwith Estradiol was lower than the control group MEK(0.08±0.00),ERK1/2(1.16±0.06/0.29±0.04) and p-ERK1/2(0.06±0.02/0.42±0.08),which showed significant difference(t＝10.01,2.86/3.45,3.57/5.95,P<0.05).Conclusion1. Protein MEK,ERK and p-ERK are expressed in HemECs in vitro.2. MEK/ERK signaling pathway might involve in cell cycle and apoptosis of HemECsin vitro.3.Estradiol,propranolol might regulate the proliferation and apoptosis of HemECsthrough MEK/ERK signaling pathway.