人成纤维细胞生长因子1-Ⅲb受体亚型在胰腺导管细胞增殖中的作用及对蛋白激酶的调节

The role of human fibroblast growth factor receptor 1-Ⅲb isoform in proliferation of pancreatic ductal cells and its effects to mitogen-activated protein kinase

目的探讨人类成纤维细胞生长因子受体1的Ⅲb亚型（FGFR1-Ⅲb）在TAKA-1胰腺导管细胞增殖中的作用及对有丝分裂激活的蛋白激酶（MAPK）的调节。方法以脂质体法将人类全长FGFR1-Ⅲb表达质粒pSVK4／FGFR1-Ⅲb稳定转染入TAKA-1胰腺导管细胞，采用Western印迹、Northern印迹、免疫荧光和糖基化分析等方法鉴定FGFR1-Ⅲb在TAKA-1细胞中的表达、分布和结构特征，并以生长因子刺激转染细胞，通过MTY法及MAPK分析，观察FGFR1-HIb受体在胰腺导管细胞中的功能及作用机理。结果FGFR1-Ⅲb受体是位于120000和130～150000之间的糖基化受体，在细胞膜表面和胞浆中为中等强度表达，在胞浆的核周围区为高表达，细胞核内不表达。FGF-1、-2和-4能够明显促进转染FGFR1-Ⅲb基因的TAKA-1细胞的生长，同时能够明显增强p44／p42MAPK的磷酸化。结论人类FGFR1-Ⅲb受体在胰腺导管细胞中为功能性受体，FGF-1、-2和4能够促进转染FGFR1-Ⅲb基因的TAKA-1细胞的增殖，其机制为促进p44／p42MAPK的磷酸化。

Objective To study the role of Ⅲ b isoform of human fibroblast growth factor receptor 1 （FGFR1-Ⅲb） in proliferation of pancreatic ductal cells and its effects on mitogen-activated protein kinase （MAPK）. Methods Human pancreatic ductal cells of the line TAKA-1 were cultured. The plasmid of human full-length FGFR1-Ⅲ b isoform, pSVK4/FGFR1- Ⅲ b, was stable transfected into the cultured TAKA- 1 pancreatic ductal cells facilitated by lipofectamine. Un-transfected TAKA-1 cells and TAKA-1 ductal cells transfected with blank plasmid were used as controls. The expression, distribution and character of protein of FGFR 1-Ⅲ b in the TAKA-1 cells were estimated by Western blotting, Northern blotting, immunofluorescence assay, and glycosylation assay. The function and mechanism of FGFR1-Ⅲ b in the transfected pancreatic ductal cells stimulated by FGF were examined by MTY assay and MAPK assay. Tunicamycin, an inhibitor of N-terminal glycoprotein synthesis, was added into the culture fluid of the FGFR1- IU b transfected TAKA-1 cells to observe the changes of the FGFR1 bands. Results FGFR1-Ⅲ b, a glycosylated receptor at various levels at 120 kDa and between 130 - 150 kDa, was localized at moderate levels at the cell membrane and cytoplasm and at higher level in the perinuclear region of the cytoplasm of the pSVK4/FGFR1-Ⅲ b- transfected cells. FGF-1, -2, and -4 significantly increased the growth of FGFRI-lU b-transfected TAKA-1 cells, and at the same time induced the p44/p42 MAPK phosphorylation. Conclusion Human FGFR1-Ⅲb receptor is a functional receptor in pancreatic ductal cells. FGF-1, -2, and -4 can increase the growth of FGFRl-Ⅲb-transfected pancreatic ductal cells, and the mechanism is that they can induce the p44/p42 MAPK phosphorylation.