摘要目的 探讨基质细胞衍生因子1(SDF-1)在肝卵圆细胞增殖过程中的效应、影响因素及作用机制.方法 流式细胞术检测大鼠肝卵圆细胞株WB-F344细胞表面趋化性细胞受体因子4(CXCR4)的表达情况.Western blot法检测细胞中β连环素蛋白磷酸化水平.激光共聚焦显微镜观察细胞内β连环素的分布情况.荧光定量PCR检测经典Wnt通路下游基因Ccnd1、c-Myc的表达将细胞分为对照组、SDF-1组、转化生长大因子β1(TGF-β1)组、SDF-1+TGF-β1组、SDF-1+TGF-β1+CXCR4抑制剂组,噻唑蓝法检测各组细胞增殖情况.结果 WB-F344细胞中CXCR4的表达量很低,而TGF-β1可上调WB-F344细胞CXCR4的表达(CXCR4阳性率上升39.5％).SDF-1结合于CXCR4后可促进肝卵圆细胞中β连环素磷酸化,抑制β连环素入核,下调下游基因Ccnd1、c-Myc的表达.200 μg/L的SDF-1单独作用72 h后WB-F344细胞的增殖情况(0.512 ±0.010)与对照组(0.513±0.008)相比差异无统计学意义(t=0.337,P＞0.05),5μg/L的TGF-β1可轻度抑制其增殖(0.393±0.007,t=28.001,P＜0.05),而SDF-1+ TGF-β1组WB-F344细胞增殖能力较TGF-β1组进一步减弱(0.272±0.009,t=32.204,P＜0.05).结论 TGF-β1可上调肝卵圆细胞中CXCR4的表达,协同SDF-1通过调控β连环素的功能状态,抑制肝卵圆细胞体外增殖.

abstractsObjective To investigate the role of stromal cell derived factor 1 (SDF-1) on the proliferation of hepatic oval cells,and the influencing factors.Methods Flow cytometry was used to detect the expression of CXCR4 on the cell surface when WB-F344 cells were growing in the culture medium with and without transforming growth factor β1 (TGF-β1) respectively.Western bolt was used to detect the expression of β-catenin and its phosphorylation level.The translocation of β-catenin was shown by confocal microscopy analysis.Q-RT-PCR was used in detecting the β-catenin downstream gene expression such as Cend1 and c-Myc.MTT was used to detect the proliferation of WB-F344 cells which were treated by SDF-1 + TGF-β1 and those cells exposed to SDF-1 or TGF-β1 only,as well as of the negative control group.Results WB-F344 cells rarely express CXCR4 under conventional circumstance,but this receptor can be up-regulated when the culture medium contain a modest amount of TGF-β1 (the rate of CXCR4 positive cell increased by 39.5％).The bond of SDF-1 to CXCR4 results in the phosphorylation of β-catenin,and its inactivation.SDF-1 alone didn't affect the proliferation of WB-F344 cells (0.512 ±0.010 vs.0.513 ±0.008,t=0.337,P ＞0.05),while TGF-β1 group show a slight decrease of cell population (0.393 ±0.007,t =28.001,P ＜0.05).But when TGF-β1 combined with SDF-1,the proliferation of WB-F344 was more weakened than TGF-β1 group,and the difference was statistically significant (0.272 ± 0.009,t =32.204,P ＜ 0.05).Conclusions TGF-β1 can up-regulate the expression of CXCR4 in hepatic oval cells,and then inhibit the proliferation of hepatic oval cells via inactivating β-catenin in vitro.