Dickkopf-1对人晶状体上皮细胞增生的抑制作用及其生物学机制

Inhibitory effect and biological mechanism of Dickkopf-1 on the proliferation of human lens epithelial cells

目的探讨DKK1(Dickkopf1)通过Wnt/β-catenin信号通路对人晶状体上皮细胞(LECs)增生的影响及其可能的作用机制,为晶状体后囊膜混浊(PCO)的临床治疗提供新靶点。方法将人LECs系(SRA 01/04细胞)分为Wnt3a过表达组、DKK1组和对照组。Wnt3a过表达组采用脂质体介导转染技术将Wnt3a cDNA表达载体瞬时转染入人SRA 01/04细胞构建PCO模型。DKK1组转染Wnt3a cDNA表达载体后48 h在培养基中加入100μg/ml DKK1进行干预,对照组细胞转染pcDNA3-HA表达载体。采用细胞计数试剂盒-8(CCK-8)法检测各组细胞生存率;采用免疫细胞化学法检测各组细胞增生细胞核抗原(PCNA)蛋白的表达;采用Western blot法检各组细胞中Wnt3a、CyclinD1和C-Myc蛋白的表达;采用免疫荧光技术法检测并定位各组细胞中β-catenin的表达。结果Western blot检测发现,Wnt3a过表达组Wnt3a蛋白的相对表达量为0.84±0.06,高于对照组的0.49±0.07,差异有统计学意义(t=3.704,P<0.05)。CCK-8试验显示,不同时间点各组细胞生存率总体比较差异均有统计学意义(F分组=10.910,P<0.05;F时间=6.041,P<0.05),其中Wnt3a过表达组细胞生存率明显高于对照组,DKK1组细胞生存率明显低于Wnt3a过表达组,差异均有统计学意义(均P<0.05)。对照组、Wnt3a过表达组和DKK1组细胞中PCNA阳性表达率分别为(9.4±1.4)%、(43.4±5.4)%和(14.2±2.3)%,总体比较差异有统计学意义(F=28.250,P<0.05),其中DKK1组和对照组细胞中PCNA阳性表达率均低于Wnt3a过表达组,差异均有统计学意义(均P<0.05)。免疫荧光技术检测发现,Wnt3a过表达组β-catenin蛋白主要表达于细胞质和细胞核,对照组β-catenin蛋白仅分布于细胞质,DKK1组β-catenin蛋白分布于细胞质,少量分布于细胞核。Western blot法检测显示,对照组、Wnt3a过表达组和DKK1组C-Myc相对表达量分别为0.59±0.05、0.93±0.02和0.47±0.08,CyclinD1的相对表达量分别为0.64±0.07、0.84±0.03和0.55±0.10,总体比较差异均有统计学意义(F=20.580、5.040,均P<0.05);其中Wnt3a过表达组C-Myc和CyclinD1相对表达量高于对照组和DKK1组,差异均有统计学意义(均P<0.05)。结论DKK1抑制SRA01/04细胞中Wnt3a过表达诱导的Wnt/β-catenin信号通路活化,下游靶蛋白CyclinD1和C-Myc表达下调,可能是DKK1抑制人LECs增生的生物学机制。

Objective To investigate the effects of Dickkopf-1(DKK1)on the proliferation of human lens epithelial cells(LECs)and its possible mechanism in order to search a new target for the treatment of posterior capsular opacification(PCO).Methods Human LECs line(SRA 01/04 cells)were divided into Wnt3a overexpression group,DKK1 group and control group.Wnt3a gene expression vector was transfected into SRA 01/04 cells by liposome mediated transfection to establish a PCO model in the Wnt3a overexpression group,DKK1 of 100μg/ml was added into the medium 48 hours after transfection of Wnt3a gene vector in the DKK1 group,and only pcDNA3-HA vector was transfected in the control group.The survival rate of SRA 01/04 cells was detected with a cell counting kit-8(CCK-8)assay.The expression rate of proliferating cell nuclear antigen(PCNA)in the cells was detecteds by immunocytochemistry.The expression ofβ-catenin in the cells was detected and located by immunofluorescence.The expression of Wnt3a CyclinD1 and C-Myc were detected by Western blot assay.Results The relative expression of Wnt3a protein in the control group was 0.49±0.07,which was significantly lower than that in the Wnt3a overexpression group(0.84±0.06)(t=3.704,P=0.02).The survival rate in the Wnt3a overexpression group,DKK1 group and control group showed significant difference over time(Fgroup=10.910,P<0.05;Ftime=6.041,P<0.05).The survival rate in the Wnt3a overexpression group was significantly increased in comparison with the control group and that in the DKK1 group was significantly reduced in comparison with the Wnt3a overexpression group(all at P<0.05).β-Catenin was expressed mainly in cytoplasm and cell nucleus in the Wnt3a overexpression group and only in cytoplasm in the DKK1 group.in the control group,β-catenin showed a weaked expression in the cytoplasm and nucleus in comparison with the Wnt3a overexpression group.The expression rates of PCNA protein were(9.4±1.4)%,(43.4±5.4)%,and(14.2±2.3)%in the control group,Wnt3a overexpression group and DKK1 group,respectively,with a significant difference among the groups(F=28.250,P<0.05),and the expression rates of PCNA protein were significantly reduced in the control group and DKK1 group compared with the Wnt3a overexpression group(both at P<0.05).β-Catenin protein were expressed mainly in the cytoplasm and nucleus in the Wnt3a overexpression group and only in the cytoplasm in the control group.In the DKK1 group,the expression ofβ-catenin protein was weakened in the cytoplasm and nucleus in comparison with the Wnt3a overexpression group.The relative expressions of CyclinD1 were 0.64±0.07、0.84±0.03 and 0.55±0.10,C-Myc were 0.59±0.05、0.93±0.02 and 0.47±0.08 in the control group,Wnt3a overexpression group and the DKK1 group,respectively with significant differences among the groups(F=20.580,5.040,both at P<0.05).The relative expressions of CyclinD1 and C-Myc in the Wnt3a overexpression group were significantly higher than those in the DKK1 group and the control group(all at P<0.05).Conclusions DKK1 inhibits activation of Wnt/β-catenin signaling pathway induced by Wnt3a overexpression in SRA01/04 cells,and down-regulation of downstream target proteins cyclin D1 and C-Mgc may be the biological mechanism of Dkk1 inhibiting human LECs proliferation.