Dickkopf-1调控Wnt通路在缺氧诱导的晶状体上皮细胞转分化中的作用

Effect of Dickkopf-1 regulated Wnt pathway on transdifferentiation of hypoxia-induced lens epithelial cells

目的:探讨缺氧条件下Wnt/β-catenin通路在晶状体上皮细胞上皮-间充质转化(EMT)中的作用,分析Dickkopf-1(DKK-1)的表达对晶状体上皮细胞EMT的影响。方法:将体外培养人晶状体上皮细胞(HLEB3细胞)分为正常氧培养组(加入含10%FBS的DMEM培养液)和缺氧培养组(加入含100μmol/L CoCl 2溶液的培养液进行缺氧处理6、12、24、48h)。利用免疫荧光染色法检测Wnt3a和DKK-1蛋白的表达及β-catenin蛋白的表达和定位;利用qRT-PCR法检测DKK-1 mRNA的表达。结果:免疫荧光染色结果显示,随着缺氧时间的延长,HLEB3细胞中Wnt3a和DKK-1蛋白表达水平明显上调,β-catenin蛋白在细胞核内积聚逐渐增多。qRT-PCR检测结果显示,与正常氧培养组相比较,缺氧培养6h组细胞中DKK-1 mRNA无显著差异(P>0.05),而缺氧培养12、24、48h组细胞中DKK-1 mRNA表达明显增加(P<0.001)。结论:缺氧可以激活晶状体上皮细胞Wnt/β-catenin通路,并诱导Dickkopf-1的表达,参与调控Wnt/β-catenin通路,影响EMT进程。

AIM:To scrutinize the role of the Wnt/β-catenin signaling pathway in the epithelial-mesenchymal transition(EMT)of lens epithelial cells under hypoxic conditions,and to further analyze the effect of Dickkopf-1(DKK-1)expression on EMT of lens epithelial cells.METHODS:Human lens epithelial cells(HLEB3 cells)were propagated in vitro and then separated into two groups:one exposed to standard oxygen levels,added DMEM culture solution containing 10%FBS(normoxic group)and another subjected to low oxygen levels(hypoxic group).The hypoxic condition was emulated by applying a concentration of 100μmol/L cobalt chloride(CoCl2)for 6,12,24,and 48h.The utilization of immunofluorescence staining enabled the detection of Wnt3a and DKK-1 expressions,along with the expression and localization ofβ-catenin protein in these groups.The expression of DKK-1 mRNA was discerned by quantitative real-time polymerase chain reaction(qRT-PCR).RESULTS:Immunofluorescence assays indicated an escalating trend in the Wnt3a and DKK-1 protein expression,which corresponded with the increasing duration of hypoxia.Likewise,an intensified nuclear accumulation ofβ-catenin protein was observed to be directly proportional to the length of hypoxia treatment.The qRT-PCR demonstrated that the difference in DKK-1 mRNA expression between the normoxic group and the group exposed to hypoxia for 6h was not statistically significant(P>0.05),whereas the DKK-1 mRNA expression of the 12,24,and 48h hypoxia groups were significantly increased(P<0.001).CONCLUSION:Hypoxia can activate Wnt/β-catenin pathway in lens epithelial cells and induce the expression of DKK-1,thus regulating the Wnt/β-catenin pathway and affecting the EMT process.