中文摘要：

目的构建稳定表达人表皮生长因子（EGF）的人永生化角质形成细胞HaCaT细胞株,分析其生物学特性的变化。方法将pcDNA3.1-EGF真核表达载体转入HaCaT细胞,经G418工作液筛选获得稳定表达、分泌EGF的HaCaT-EGF细胞株,同时按照同样步骤将pcDNA3.1空质粒转入HaCaT细胞。分别采用倒置显微镜、PCR仪、ELISA法检测细胞形态变化、EGF的基因和蛋白表达情况,同时对凋亡相关分子Caspase-3、细胞周期相关蛋白分子cyclin D1的mRNA表达水平进行检测分析。结果获得了一HaCaT-EGF细胞株,经检测,HaCaT-EGF细胞株EGF mRNA表达水平为正常HaCaT细胞的147倍,EGF蛋白分泌高达（387.030±25.680）pg/mL。HaCaT-EGF细胞株与空载体转染HaCaT细胞及正常HaCaT细胞相比,细胞集落更加集中和紧密;HaCaT-EGF Caspase-3 mRNA相对表达量低于空载体转染HaCaT细胞,差异具有高度统计学意义（t=5.110,P=0.006）,与正常HaCaT细胞比较差异无统计学意义（t=3.180,P=0.082）;HaCaT-EGF细胞周期相关蛋白cyclin D1 mRNA相对表达量低于空载体组,差异有高度统计学意义（t=9.442,P〈0.01）,与正常HaCaT细胞比较差异无统计学意义（t=2.831,P=0.066）。结论 HaCaT-EGF细胞株可持续分泌EGF,与正常HaCaT细胞及HaCaT空载体细胞相比,HaCaT-EGF细胞生长旺盛,细胞集落更加集中和紧密,是组织工程皮肤构建的良好种子细胞,可用于组织工程相关实验研究。

英文摘要：

Objective To construct the immortalized keratinocytes （HaCaT cell line） with stable expression of the human epidermal growth factor （EGF） and analyze the changes of its biological characteristics. Methods The PCDNA3.1-EGF eukaryotic expression vector was transferred into HaCaT cell, and G418 was utilized to select the HaCaT-EGF cell line. The changes of the cell morphology were detected by using the inverted microscope. The mRNA expression of the EGF, apoptosis related molecules Caspase-3, the cell cycle related proteins cyclin D1were measured with PCR method. The protein expression of the EGF was detected with ELISA. Results The HaCaT-EGF cell line was obtained. The mRNA expression levels of EGF were up regulated 147 times the ordinary HaCaT cell. And the levels of the protein secretion were up to （387.030± 25.680） pg/mL. The colony of the HaCaT-EGF cells was more focused and tighter than the empty vector transfected HaCaT cells and normal HaCaT cells. And its mRNA expression levels of Caspase-3 were lower than the empty vector transfected HaCaT cells, differences being statistically significant （t = 5.110, P = 0.006）, and there was no significant difference compared to the normal HaCaT cells （t = 3.180, P = 0.082 ）. In HaCaT-EGF cell cycle related protein cyclin D1 mRNA relative expression was less than the empty vector group, and difference was statistically significant （t = 9.442, P 0.01）; there was no significant difference compared to the normal HaCaT cells （t = 2.831, P = 0.066）. Conclusions HaCaT-EGF cell can secrete EGF, and the biological characteristics are stable. It can be used for tissue engineering experiment as a nice seed cell for tissue engineered skin constructed.