Wnt3a促进人晶状体上皮细胞增生及相关机制的研究

背景 晶状体上皮细胞(LECs)的异常增生是晶状体后囊膜混浊(PCO)的重要病理基础,以往研究证实Wnt3a信号可促进上皮细胞的增生,但其对LECs的作用机制尚不清楚. 目的 研究Wnt3a对人LECs增生的作用,探讨相关分子机制,为PCO的临床治疗提供新的靶点.方法 将人LECs系SRA01/04细胞进行培养后以4×105个/孔的密度接种于6孔板继续孵育.构建Wnt3a cDNA表达载体,采用脂质体介导转染技术将Wnt3a cDNA表达载体瞬时转染人SRA01/04细胞中,对照组转染pcDNA3-HA表达载体.采用Western blot技术验证载体在转染细胞中的表达；MTT法和流式细胞仪检测Wnt3a在SRA01/04细胞中的过表达对细胞增生能力的影响；Western blot法检测Wnt3a过表达对Wnt/β-catenin下游信号分子β-catenin、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；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.结果 本研究成功构建了人Wnt3a基因的表达载体Wnt3a cDNA,获得Wnt3a过表达的Wnt3a/SRA01/04细胞及对照pcDNA3-HA/SRA01/04细胞.Western blot检测证实,与pcDNA3-HA/SRA01/04相比,Wnt3a/SRA01/04细胞内Wnt3a蛋白表达明显升高.MTT法检测表明,Wnt3a cDNA转染组SRA01/04细胞的增生率明显高于对照组(F分组=15.235,P=0.005；F时间=369.677,P=0.000),转染后各时间点2个组间SRA01/04细胞的增生率差异均有统计学意义(t=20.843,P=0.001;t=26.214,P＜0.01;t=25.177,P=0.001;t=35.516,P＜0.01;t=615.056,P＜0.01).细胞周期分析发现,Wnt3a cDNA转染组G1期细胞比例为51.74％,明显少于对照组的79.44％,而S期细胞的比例为36.23％,明显多于对照组的12.34％.Wnt3a cDNA转染组SRA01/04细胞PCNA蛋白表达阳性率为47.00％±7.58％,明显高于对照组的16.00％±3.61％,差异有统计学意义(t=8.256,P＜0.01).转染后48 h,β-catenin蛋白密集分布于Wnt3a/SRA01/04细胞核和细胞质中,而在对照组仅出现于细胞质之中.Wnt3a/SRA01/04细胞中Wnt3a蛋白表达水平上调,β-catenin细胞定位由细胞质转入细胞核；Wnt/β-catenin信号通路靶蛋白Cyclin D1和c-Myc蛋白表达上调. 结论 在SRA01/04细胞中,Wnt3a过表达使Wntβ-catenin信号通路活化,下游靶蛋白Cyclin D1和c-Myc表达上调,可促进人LECs的增生.

Wnt3a-induced proliferation in human lens epithelial cell and its mechanism

Background Aberrant proliferation of residual lens epithelial cells （LECs） is one of main causes of posterior capsular opacifieation （ PCO ）. Researches indicated that Wnt3a signaling pathway promote proliferation of epithelial cells, but its effect on LECs is still unclear. Objective The present study was to investigate the effects of Wnt3a on proliferation of human LECs and its mechanism and to provide a new gene target in the prevention and treatment of PCO. Methods Human LECs line （SRA01/04 cells） was cultured and then incubated to 6-well plate at the density of 4×10^5well. A human Wnt3a eDNA expressing vector targeted human LECs was constructed to increase the Wnt3a expression in SRA01/04 cells, and pcDNA3-HA expression vector was used as the control group. The expression of Wnt3a was identified by Western blot assay after transfected. The growth and proliferation of SRA01/04 cells were detected by MTT and flow cytometry （FCM）. The expressions of β-eatenin, cyclin D1 and c-myc in the cells were detected by Western blot assay. β-Catenin expression was localized using immunofluoreseence assay,and the expression and localization of proliferating cell nuclear antigen （PCNA） were analyzed by immunocytochemistry for the exploration of the active mechanism of Wnt3a to proliferation of LECs. Results Human Wnt3a cDNA expression vector was designed successfully and transiently transfected to SRA01/04 cells,and Wnt3a/SRA01/04 cells and pcDNA3-HA/SRA01/04 cells were obtained. The expression of Wnt3a was verified in the Wnt3a transfected group compared with the control group. MTT indicated that the ceil proliferating rate was significantly different between the Wnt3a transfected group and the control group （ Fgroup = 15. 235, P = 0. 005 ; Frame = 369. 677 ,P= 0. 000）, and that in various time points after transfected was significantly different （t = 20. 843, P=0. 001 ;t = 26. 214,P〈0. 001 ;t= 25. 177,P=0. 001 ;t=35. 516,P〈0. 001 ;t = 615. 056,P〈0. 001 ）. The proportion of SRA01/04 cells in G1 phase was 51.74% in the Wnt3a cDNA transfected group,with a significantly decrease in comparison with 79.44% of the control group. However, the proportion of SRA01/04 cells in S phase in the Wnt3a cDNA transfected group was higher than that of the control group （36.23% versus 12.34% ）. The positive expression rate of PCNA protein in SRA01/04 was （47.00% ±7.58% ） in the Wnt3a cDNA transfected group and （ 16.00%± 3.61% ） in the control group with a significant difference between them （t = 8. 256,P〈0.01 ）. After 48 hours of transfection of the Wnt3a cDNA,the expression amount of β-catenin proteins was higher and the immunofluorescence was stronger in cell nucleus,and the expressions of cyclin D1 and c-myc proteins were elevated in Wnt3a/SRA01/04 ceils. Conclusions The overexpression of Wnt3a activates 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 promoting the proliferation of human LECs.