Differentiation of HaCaT cells infected with lentivirus / 中国组织工程研究

ABSTRACT

BACKGROUND:YY1 is mainly expressed in the undifferentiated epidermic cells in mouse basal lamina, and the expression level is gradual y down-regulated as the differentiation towards suprabasal lamina. The differential expression indicates that, YY1 is one of the regulators in the process of epidermic cells differentiation. OBJECTIVE:To observe the effects of YY1 over-expression on the differentiation of HaCaT cells infected with lentivirus. METHODS:Lentivirus-YY1 was transferred into the HaCaT cells by using Lipofectamine 2000. After selection of the puromycin, monoclonal celllines were established, and the control group were lentivirus-infected HaCaT cells and uninfected HaCaT cells. The expression of YY1 was detected by using western blot analysis. cells in Lentivirus-YY1-HaCaT group and HaCaT-YY1 group were further divided into two subgroups according to the calcium concentration in culture medium, cells were either cultured in low-calcium medium (0.12 mmol/L) for 48 hours, or cultured in low-calcium medium (0.12 mmol/L) for 24 hours and in high-calcium medium (0.35 mmol/L) for additional 24 hours. Keratin K1, K10, K14, and involucrin, filaggrin and loricrin after over-expression of YY1 were detected with western blot analysis. RESULTS AND CONCLUSION:The HaCaT cells were successful y infected with lentivirus-YY1, and we obtained over-expression of YY1 protein in monoclonal celllines under high-calcium concentrations, the over-expressed YY1 could decrease the expression of K1, involucrin and loricrin, thereby preventing the process of epidermal keratinocytes and maintaining the cells in an undifferentiated state. Lentivirus can efficiently infect human immortalized epidermal cellHaCaT, and YY1 may the important factor of inhibiting the differentiation of basal epidermal cells and maintaining the undifferentiated proliferation status.