姜黄素通过Wnt/β-catenin信号通路抑制晶状体上皮细胞增殖的实验研究

Curcumin regulation of human lens epithelail cell proliferation through Wnt/β-catenin signaling pathway

[目的]探讨姜黄素通过Wnt/β-catenin信号通路对人晶状体上皮细胞(LECs)增殖的影响及其可能的作用机制,为晶状体后囊膜混浊(PCO)的临床治疗提供新的靶点。[方法]人LECs系SRA 01/04细胞为研究对象,实验分为3组:Wnt3a组,姜黄素组和对照组。Wnt3a组采用脂质体介导转染技术将Wnt3a c DNA表达载体瞬时转染入人SRA 01/04细胞中,构建PCO的细胞模型。姜黄素组在转染Wnt3a c DNA表达载体48 h后加入20μmol/L姜黄素,对照组转染pc DNA3-HA表达载体。采用Western blot技术验证载体在转染细胞中的表达,CKK-8实验检测姜黄素对SRA 01/04细胞增殖能力的影响,采用免疫细胞化学法检测Wnt3a过表达后增生细胞核抗原(PCNA)蛋白表达的变化;Western blot法检测Wnt3a过表达对Wnt/β-catenin下游信号分子cyclin D1和c-Myc蛋白表达的影响;应用免疫荧光法检测Wnt3a过表达后β-catenin表达的定位变化。[结果]Western blot检测证实,转染pc DNA3-HA表达载体后,SRA 01/04细胞内Wnt3a蛋白表达明显升高。CKK-8检测表明,姜黄素组SRA 01/04细胞的增殖率明显低于Wnt3a组(t=2.782,P=0.049)。姜黄组SRA 01/04细胞PCNA蛋白表达阳性率为23.6%±4.0%,明显低于Wnt3a组的43.4%±5.4%,差异有统计学意义(t=2.951,P=0.018)。转染后48 h,β-catenin蛋白密集分布于Wnt3a组细胞核和细胞质中,而在对照组仅出现于细胞之中,姜黄素组β-catenin蛋白主要分布于细胞核中,少量分布于细胞质中。Western blot检测姜黄素组Wnt/β-catenin信号通路靶蛋白Cyclin D1和c-Myc蛋白表达明显低于Wnt3a组。[结论]在SRA01/04细胞中,姜黄素抑制Wnt3a过表达诱导的Wnt/β-catenin信号通路活化,下游靶蛋白Cyclin D1和c-Myc表达下调,抑制人LECs的增生。

[Objective] To investigate the effects of curcumin on proliferation of human lens epithelial cells(LECs) and its mechanism and to provide a new gene target in the prevention and treatment of PCO.[Methods] The research objects were the human LECs SRA 01/04.The experiment was divided into three groups:Wnt3a group,curcumin group and control group.In Wnt3 a group,human Wnt3 a c DNA expressing vector targeted human LECs was constructed to PCO model.In curcumin group,curcumin of 20 μmol/L was added after 48 hs of transfection.pc DNA3-HA expression vector was used as the control group.The expression of Wnt3 a was identified by Western blot assay after transfected.The growth and proliferation of SRA 01/04 cells were detected by CCK-8 test.The expression and localization of proliferating cell nuclear antigen(PCNA) were analyzed by immunocytochemistry for the exploration of the mechanism of curcumin to proliferation of LECs.The expressions of cyclin D1 and c-Myc in the cells were detected by Western blot assay.β-Catenin expression was localized using immunofluorescence assay.[Results] The expression of Wnt3 a was verified in the Wnt3 a transfected group compared with the control group.CCK-8 test indicated that the cell proliferating rate was significantly difference between the curcumin group and Wnt3 a group(t =2.782,P =0.049).The positive expression rate of PCNA protein in SRA 01/04 was 23.6% ±4.0% in the curcumin group and43.4% ±5.4% in the Wnt3 a group with a significant difference between them(t =2.951,P =0.018).After 48 hours of treatment with curcumin,the immunofluorescence was stronger in cell nucleus,and the expressions of cyclin D1 and c-Myc proteins were elevated in SRA 01/04 cells in wnt3 a group,but he immunofluorescence was stronger in cytoplasm,and the expressions of cyclin D1 and c-Myc proteins were less in SRA 01/04 cells in curcumin group and controls.[Conclusion] Curcumin depressed the Wnt/β-catenin signaling pathway and downregulates the expression of a subset of target genes,including cyclin D1 and c-Myc,which plays an important role in inhibiting the proliferation of human LECs.