Construction of the tissue engineering seed cells(HaCaT-EGF) and analysis of its biological characteristics

Objective:To construct the tissue engineering seed cell(HaCaT cell line) with stable expression of the human epidermal growth factor(EGF),and analyze the changes of its biological characteristics.Methods:PCDNA3.1-EGF eukaryotic expression vector was transferred into HaCaT cell,and G418 was utilized to select the HaCaT-EGF cell line.Using an inverted microscope,PCR,ELISA method to detect the changes of the cell morphology,the expression of the ECF gene and protein,and the mRNA expression levels of apoptosis related molecule Caspase-3,the cell cycle related protein cyclin D1.Results:The mRNA expression levels of the obtained HaCaT-ECF cell were more than 100 times higher than the level of ordinary HaCaT cell.The colony of the HaCaT-EGF cells was more focused and tight compared to the empty vector transfected HaCaT cells and normal HaCaT cells.The expression levels of apoptotic factor Caspase-3 and cyclin Dt in HaCaT-EGF cell were significantly higher than those in the empty vector HaCaT- pcDNA3.1 cell,and the differences were statistically significant(P<0.01),but there was no significant difference compared to the normal HaCaT cells(P>0.05).Conclusions: HaCaT-EGF cell can continuously secrete ECF,and the biological characteristic is stable.It can be used for tissue engineering experiment and is an ideal seed cell for constructing tissue engineered skin.