Preliminary Study Of Epidermal Growth Factor Receptor(EGFR) On The Proliferation Of Mammary Gland Epithelial Cells In Dairy Goats

Epidermal growth factor receptor（EGFR）is an importatn member of ErbB tysosine receptor families,which is involeved in the reguation of cell activities,such as cell proliferation,migration and differation.In this experiments,we were used the dairy goat mammary epithelial cells（DMECs）as cell model,and overexpressed the EGFR gene to find the effect of its on proliferation.We were measured the activation of EGFR signaling pathway and the protein level of genes related to cell cycle after given EGF or EGFR inhibitors treatment.The main results of research are as follows:（1）The appropriate concentration of EGF was obtained by adding different concentrations of EGF（0,12.5,25,50,100 ng/ml）into the primary mammary epithelial cells of dairy goats.The results showed that 12.5-50 ng/ml EGF could promote cell proliferation.The proliferation of cell were inhibited by the AG1478（10 umol/L）,but not significantly death.The eukaryotic expression vector of EGFR were successfully transfected into DMECs.RT-PCR showed that the mRNA level of EGFR was increased by about above 25 times.Western blot results showed that the phosphorylation of EGFR tyrosine kinased were increased by the overexpression of EGFR and then it was significantly higher than control group after given EGF treatment（P<0.01）.The results of Hoechst/PI staning showed that EGF significantly promoted the proliferation of overexpressed-EGFR mammary epithelial cells,while AG1478signifcantly reduced the number of cells and the viability of cells.To detemined whether the effect of EGF or AG1478 on DMECs growth,cell cycle progression was analyzed by flow cytometry analysis.The results showed that EGF（50 ng/ml）induced the cell passed G₁ phase into S and G₂ phase.Conversly,DMECs treated AG1478 had an obvious cell cycle arrest at the S phase.It is suggested that the promotion of cell cycle by EGF is directly related to the phsphorylation of EGFR.（2）The EGFR and its downstream signaling proteins are activeted by EGF and AG1478.After stravation of 16 h,the phosphorylation of EGFR protein level was higher with the time of EGF treatment.EGF-mediated AKT and ERK1/2 phosphorylation leves were greatly increased;AG1478（10 umol/L）made ERK and AKT phosphorylation level of DMECs down after 1 hour.The protein level of P21 was increased by 10 umol/Ll AG1478 or 10 umol/L Gefitinib,but the protein level of CDK2 and cyclin E1 were decreased.CDK2 level in EGF-treated cells was upregulated with the time of serum starvation extending.CDK2 expression in response to EGF（50 ng/ml）was blocked by 500 nmol/ml MK2206 or 1 nmol/ml U0126.This demonstrated that EGFR-mediated function of CDK2 in cell cycle by AKT and ERK1/2signaling pathways.In summary,the effect of EGFR signaling pathway on mammary epithelial cells of dairy goat was studies.It was proved that EGFR might participated in the regualtion of cell proliferation of DMECs by CDK2 kinase.ERK1/2 and AKT signaling pathways can regulate the expression of CDK2 kinase,which provides a basic theoretic basis for clarifying the role of EGFR signaling pathway in the development of dairy goat mammary gland.