The Effect Of Hepatocyte Growth Factor On Proliferation And Migration Of Human Umbilical Vein Endothelial Cells And Its Related Mechanism Of Action

Objective : To investigate the effect of recombinant human hepatocyte growth factor on proliferation and migration of human umbilical vein endothelial cells and to further study its related mechanism of action. Methods: The cultured endothelial cells in vitro were isolated from human umbilical vein by collagenase. Cells were cultured in Dulbecco 's Modified Eagle's Medium/F12 (DMEM/F12) supplement with 20% FBS,100μg/ml heparin,2mmol/l L- glutamine,100ng/ml penicillin, 100ng/ml streptomycin. HUVECs were identified by the positive expression of FⅧr-Ag through immunohistochemical method and finding Weibel-Palade body through electromicroscope. The cultured HUVECs were treated with rhHGF(1ng/ml, 10ng/ml, 25ng/ml, 50ng/ml, 100ng/ml) for 24h,48h,72h respectively, Hemocytometer was used to detect the growth of HUVECs. MTT assay was used to determine the suitable cell density and to measure the effect of HGF on the proliferation of HUVECs. The method of Trochon was utilized to count the number of cells using reverse microscope when cultured HUVECs were treated with HGF (1ng/ml～100ng/ml) for 24h,Which could calculate the migration ability of cells. The changes of celluar ultrastructure were observed under TEM. FCM was applied to observe the effect of rhHGF on cell cycle and SP was employed to observe the expression of Cyclin E and CDK2. Resulst: HUVECs could be isolated from human umbilical vein by collagenase in vitro successfully and go down to the future successive generation in DMEM/F12. The expression of FⅧr-Ag was positive in HUVECs and the W-P body was found in the plasm of the HUVECs through electromicroscope. rhHGF could promote the proliferation and migration of HUVECs dose-dependently and time-dependently. The proliferation rate of rhHGF groups were significantly greater than that of control group from 24h to 72h.The maximum of proliferation was available in 100 ng/ml rhHGF,but the maximum of proliferation rate was presented in 50 ng/ml rhHGF. rhHGF increased HUVECs migration in a dose-dependent manner by 7.16%,32.31%,75.57%,210.73%,430.25% after 24h in 1ng/ml,10ng/ml,25ng/ml,50ng/ml,100ng/ml HGF respectively. There were obvious changes in celluarstructure ,rhHGF could increase mitochondrion ,rough endoplasmic reticulum and euchromatin. The result of the FCM showed that rhHGF could stimulate HUVECs proliferation by decreasing G0/G1 phase, increasing S and G2/M phase. Proliferation index of 25ng/ml,50ng/ml,100ng/ml rhHGF were 36.26%,41.58%,48.37% respectively.The HSCORE of Cyclin E and CDK2 were increased, which suggested rhHGF could enhance their expression dose- dependently . Conclusions: HGF could promote the proliferation and migration of HUVECs significantly, which suggested that HGF might have the effect of angiogenesis. The increased Cyclin E and CDK2 expression by rhHGF may increase the numbers of cells transferring G0/G1 phase into S and G2/M phase, which might be the regulate and control molecular mechanism of cell cycle inducing proliferation of HUVECs.