丁国强 郑春泉 刘颖 田洁
[摘要] 目的 通过对表皮生长因子受体（epidermal growth factor receptor, EGFR）信号通路不同部位的干预，观察体外培养的人鼻腔上皮细胞RPMI-2650中EGFR和黏蛋白5AC（mucin 5AC, MUC5AC）的变化。方法 对人鼻腔上皮细胞RPMI-2650进行体外培养，记录生长曲线，并行扫描电镜观察细胞超微结构。当细胞大部分融合时，将细胞分为四组，A组：在原Eagle最小必需培养基（Eagle΄s Minimum Essential Media，EMEM）培液中继续培养；B组：加入人重组表皮生长因子（epidermal growth factor，EGF）25 ng/ml；C组：加入AG1478（EGFR选择性抑制剂）10 μmol/L，30 min后加入人重组EGF 25 ng/ml；D组：加入PD98059（p44/42MAPK选择性抑制剂）30 μmol/L，30 min后加入人重组EGF 25 ng/ml。以上四组细胞均继续培养24 h后，分别进行细胞免疫和Western Blotting实验，观察EGFR和MUC5AC蛋白在各组细胞中的表达。结果 细胞在培养第3天开始融合，第5～7天大量融合。扫描电镜可见细胞呈圆形或椭圆形，表面覆有大量微绒毛，当细胞融合时可变为梭形或多角形生长。EGFR蛋白在B组细胞中有强表达，A组和D组细胞中有较强表达，而在C组中仅有弱表达，A、B、D组与C组平均吸光度值（A值）相比差异均具有统计学意义（P＜0.01）。而MUC5AC蛋白在B组细胞中有强表达，A组细胞中有较强表达，在C组和D组中仅为弱表达，B组与C、D组间相比吸光度值差异均具有统计学意义（P＜0.01），而C组与D组之间并无显著差异（P＞0.05）。结论 从细胞和蛋白水平上发现，EGFR信号通路在RPMI-2650细胞中发挥作用。对该通路不同部位进行干预，细胞中EGFR和MUC5AC蛋白随之发生相应改变。证实了EGFR信号通路在鼻腔上皮细胞RPMI-2650中对MUC的分泌具有调节作用。
[关键词] 受体，表皮生长因子； 黏蛋白质类；细胞培养技术
Experimental studies for the roles of epidermal growth factor receptor signaling pathway on cultured human nasal epithelial cells RPMI-2650 DING Guo-qiang*, ZHENG Chun-quan, LIU Ying, TIAN Jie. *Department of Otorhinolaryngology-Head and Neck Surgery, Eye and ENT Hospital of Fudan University, Shanghai 200031, China
[Abstract] Objective To explore the roles of epidermal growth factor receptor (EGFR) signaling pathway on cultured human nasal epithelial cells RPMI-2650. Methods RPMI-2650 cells were cultured in vitro, the growth curve was measured and the ultrastructure was observed using scanning electron microscope. When the cells were significantly confluent, they were divided into 4 groups, group A: maintained in Eagle΄s Minimum Essential Media (EMEM) medium without adding any stimulators; group B: added with epidermal growth factor (EGF) 25ng/ml; group C: added with AG1478 (EGFR selective inhibitor) 10 μmol/L followed by EGF 25 ng/ml 30 minutes later; group D: added with PD98069 (p44/42MAPK selective inhibitor) 30 μmol/L followed by EGF 25 ng/ml 30 minutes later. After incubated for 24 hours, the expression of EGFR and MUC5AC proteins in the cells of these 4 groups was studied using cytoimmunity and western blotting. Results RPMI-2650 cells were significantly confluent after incubated for 5 to 7 days. The shape of cells was round or oval, and a large number of microvilli covered to their surface but without cilia under scanning electron microscope. The EGFR protein was expressed in the cells of group A and D, abundantly in group B, while weakly in group C. The values of comparative absorbance had significant difference between group A、B、D and group C, respectively (P﹤0.01). For the MUC5AC protein, its expression was strong in the cells of group A, abundant in group B, and weak in group C and D. Significant difference of the values of comparative absorbance was analyzed between group B and group C、D, respectively (P﹤0.01), while no difference between group C and group D (P﹥0.05). Conclusion The production of MUC5AC in human nasal epithelial cells RPMI-2650 is regulated via the expression and activation of epidermal growth factor receptor signaling pathway.
[Key words] Receptor, epidermal growth factor; Mucoproteins; Cell culture techniques