Wnt3a诱导人晶状体上皮细胞细胞外基质合成和胶原凝胶收缩的机制研究

Wnt3a-induced extracellular matrix production and gel contraction in human lens epithelial cells

背景晶状体上皮细胞（LECs）的细胞外基质（ECM）的产生、异常沉积和收缩是PCO发生的重要病理机制。Wnt3a蛋白参与机体组织上皮细胞ECM的产生和纤维化过程，但Wnt3a对晶状体组织中上皮间质转化（EMT）相伴行的ECM的产生和收缩的影响尚不清楚。目的研究Wnt3a对体外培养的人LECsECM合成的影响，并探讨其介导胶原凝胶收缩的相关分子机制。方法用脂质体介导转染技术将Writ3acDNA表达载体瞬时转染人LECs系SRAOI／04细胞作为Wnt3a转染组，对照组转染pcDNA3-HA表达载体。细胞转染后48h，采用Westernblot法测定各组细胞中Wnt3a、ECM主要成分I型胶原纤维（Col-I），Ⅳ型胶原纤维（C01．IV）和整合素B1（integrinp1）的表达；采用免疫荧光法检测α-SMA、细胞纤维状肌动蛋白（F-actin）在胞中的表达和分布。将SRAOI／04细胞与I型胶原凝胶混合，观察胶原收缩情况。结果Wnt3a转染48h，SRAOI／04细胞内Writ3a蛋白相对表达量（相对灰度值）明显升高，对照组细胞和Wnt3a转染组细胞中Wnt3a蛋白表达量分别为0．290±0．066和0．703±0．105，差异有统计学意义（t=5．782，P〈0．01）。Westernblot法检测显示，Wnt3a转染组SRAOI／04细胞中的Col-I和integrinp1蛋白的相对表达量分别为0．697±0．021和0．875±0．055，明显高于对照组的0．370±0．020和0．580±0．030，差异均有统计学意义（t=19．600、8．156，均P〈0．01），Wnt3a转染组Col—IV蛋白相对表达量为0．430±O．020，高于对照组的0．383±0．031，但差异无统计学意义（t=2．514，P〉0．05）。免疫荧光检测显示，对照组细胞中F-actin和α-SMA蛋白主要表达于细胞膜，而Wnt3a转染组细胞中F-aetin和α-SMA蛋白表达于细胞膜、细胞质和细胞核周围，阳性染色程度较对照组明显增强。Col-I凝胶收缩试验结果显示，细胞与Col-I胶原凝胶混合后24h凝胶收缩面积比率明显低于混合后8h（64．1％±2．3％与98．9％±1．0％），Wnt3a转染组凝胶收缩面积比率明显低于对照组（64．1％±2．3％与93．9％±3．1％），差异均有统计学意义（均P〈0．01）。结论Wnt3a过表达可诱导SRA01／04细胞ECM合成和细胞骨架重建，促进细胞收缩。

Background Excessive production,deposition and contraction of extracellular matrix （ECM） of human lens endothelial cells （LECs） is one of main causes to posterior capsular opacification （PCO）. Researches indicated that Wnt3a protein was involved in production of ECM and fibrosis in epithelial cells,but its effect on LECs is still unclear. Objective This study aimed to elucidate the roles of Wnt3a in production of ECM and gel contraction of LECs. Methods Lipofectamine-mediated transient transfection technique was used to introduce cDNA of Wnt3a gene and pcDNA3-HA vector into the LEC cell line SRA01/04 to act as Wnt3a transfection group and control group respectively. After 48 h of transfection, Western blot was used to detect the expression of Wnt3a, main components of ECM Col- I ,Col-IV and integrin β1. Immunofluorescence was used to detect the expression and distrubition of α-SMA and F-actin; collagen contraction was observed by mingling SRA01/04 cells with Col-I. Results After 48 hours of transfection using lipofectamine 2000,the expressions of wnt3a protein in SRAO1/04 cells was 0. 703 ± 0. 105 in the wnt3a transfected group, and that in the control group was 0. 290 ±0. 066, showing a significant difference between the two groups （t = 5. 782, P〈0.01 ）. Western blot assay showed that the expression levels of Col- I and Integrin β1 was 0. 697±0. 021 and 0. 875±0. 055 in the Wnt3a transfected group,and which was significantly higher than 0. 370±0. 020 and 0. 580±0. 030 in the control group （ t = 19. 600,8. 156, both P〈0. 01 ）. The expression level of Col 1V in the Wnt3a transfected group was higher than that of the control group （0. 430± 0. 020 vs 0. 383 ±0. 031 ） , but the difference was not significant （ t = 2. 514, P〉0.05 ）. Immunofluorescence assay revealed that F-actin and tx-SMA were weakly expressed in the cell membrane primarily in the control group, while they were strongly expressed in the cell membrane,cytoplasma in the Wnt3a transfected group. Tewnty-four hours after addtion of Col- I ,the gel contraction area ratio appeared to be more obvious in comparison with the 8 hours （64. 1% ± 2.3% vs 98.9% ±1.0% ） ,and gel contraction area ratio was lower in the Wnt3a transfected group than that in the control group （64.1% ±2.3% vs 93.9% ± 3. 1% ）. Conclusions The overexpression of Wnt3a activates the production of ECM,and the remodeling of celluar skeleton and cellular contraction.