摘要
背景:研究证实YY1主要在小鼠基底层未分化表皮细胞中表达,且随着表皮细胞向基底上层分化其表达逐渐下降,这种差异性表达方式说明YY1可能是表皮细胞分化过程中重要的调节因子之一。目的:采用慢病毒-YY1感染HaCaT细胞观察YY1过表达对细胞分化的影响。方法:将慢病毒-YY1感染至HaCaT细胞,经puromycin筛选,建立单克隆稳定细胞株,设对照组为慢病毒感染的HaCaT细胞和未感染的HaCaT细胞,Western blot检测分析YY1蛋白的表达水平;将慢病毒-YY1-HaCaT组和HaCaT-YY1组细胞各分成2组,一组在低钙(0.12 mmol/L)培养基条件下培养48 h,另一组在低钙(0.12 mmol/L)培养基条件下培养24 h后在高钙(0.35 mmol/L)培养基条件下再培养24 h,用Western blot法检测YY1过表达对HaCaT细胞分化中的表皮细胞特异性分化角蛋白K1、K10、K14和晚期分化产物外皮蛋白、中间丝相关蛋白、兜甲蛋白的影响。结果与结论:慢病毒-YY1成功感染HaCaT细胞,高钙条件下获得的单克隆细胞株过表达YY1蛋白,可抑制K1、外皮蛋白和兜甲蛋白的合成,从而阻止表皮细胞角质化进程并使细胞处于未分化状态。结果说明慢病毒可高效感染皮肤人永生化表皮细胞株HaCaT细胞,YY1可能是抑制基底表皮细胞分化并维持其未分化增殖状态的重要因子之一。
BACKGROUND： YY1 is mainly expressed in the undifferentiated epidermic cells in mouse basal lamina, and the expression level is gradually 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 cell lines 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 Ioricrin after over-expression of YY1 were detected with western blot analysis. RESULTS AND CONCLUSION： The HaCaT cells were successfully infected with lentivirus-YY1, and we obtained over-expression of YY1 protein in monoclonal cell lines under high-calcium concentrations, the over-expressed YY1 could decrease the expression of K1, involucrin and Ioricrin, thereby preventing the process of epidermal keratinocytes and maintaining the cells in an undifferentiated state. Lentivirus can efficiently infect human immortalized epidermal cell HaCaT, and YY1 may the important factor of inhibiting the differentiation of basal epidermal cells and maintaining the undifferentiated proliferation status.
出处
《中国组织工程研究》
CAS
CSCD
2014年第29期4625-4629,共5页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金资助项目(81171489)~~
