Wnt/β-catenin信号通路在晶状体后囊膜混浊中晶状体上皮细胞纤维化的调控机制研究

Posterior capsular opacification (PCO) is the most common posteoperative complication of modern cataract surgery causing visual loss, which is lens fibrosis induced by wound healing response in lens epithelial cells.Epithelial mesenchymal transition(EMT) ,proliferation,migration, collagen deposition,and lens fiber regeneration of lens epithelial cells are the main causes of PCO. Wnt ligands is involved in regulation in proliferation, migration and transdifferentiation of diverse cells. However, the role of wnt3a ligand inducing canonical wnt signaling in lens epithelial cells remains unclear. In our previous study, we constructed wnt3a expression vector activating canonical wnt signaling pathway and used transfection technology to achieve wnt3a-overexpressing lens epithelial cells, and found the influence of canonical wnt signaling pathway in EMT,migration, and proliferation of lens epithelial cells .Therefore we hypothesis that Wnt/β-catenin signaling invovled in PCO formation. This investigation will reveal the molecular mechanism of Wnt/β-catenin signaling and its related signaling in PCO by establishing PCO rabbit model ; and the function of Wnt/β-catenin signaling and its related signaling in PCO by investigating its key molecules of Wnt/β-catenin, markers of PCO, functional and molecular biological changes in lens epithelial cells through inducing and blocking the Wnt/β-catenin signaling pathway.To the end of study, we are able to put new insight to the mechanism of PCO formation after cataract surgery, and try to find new target to design drugs.

晶状体后囊膜混浊（PCO）是白内障摘除术后最常见的并发症，其病理机制是晶状体上皮组织纤维化，Wnt/β-catenin信号通路参与许多组织的纤维化进程，但在PCO形成中的具体生物学作用目前研究甚少。本组前期工作发现在HLE B-3中，Wnt/β-catenin信号通路介导晶状体上皮细胞EMT、促进细胞增殖和迁移，据此提出假设Wnt/β-catenin信号通路参与调控PCO的发生发展。本研究拟在白内障手术动物标本中了解Wnt/β-catenin信号通路关键分子的表达，通过观察激活或阻断Wnt/β-catenin信号通路后PCO各项指标的变化和晶状体上皮细胞功能和分子生物学改变，揭示Wnt/β-catenin信号通路在PCO形成中的作用及其作用的分子机制以及与其它调控信号通路的交互作用,试阐明Wnt/β-catenin信号通路调控PCO的分子机制，为PCO的靶向性干预提供理论依据。

晶状体后囊膜混浊（PCO）是现代白内障摘除手术最常见的远期并发症，白内障手术刺激可能诱导晶状体上皮细胞(LECs)增殖、迁移、上皮间充质转化（EMT）、胶原沉积和晶状体纤维组织形成是PCO的主要原因。其机制尚需进一步阐明。Wnt/β-catenin信号通路是细胞纤维化进程的重要调控通路，我们的研究证明Wnt3a能够诱导人LECs的Wnt/β-catenin信号通路活化，促进LECs的EMT和增殖，促进PCO的发生和发展。（1）本研究将兔晶状体囊外摘除手术建立白内障手术模型，术后前房内注入Wnt/β-catenin信号通路抑制剂Dkk1预防PCO形成。体外培养HLE B-3细胞，转染Wnt3a后加入Dkk1，研究发现Dkk1能够维持细胞的上皮表型，逆转Wnt3a诱导EMT相关蛋白的表达，Dkk1还可以抑制LECs迁移和MMP-1表达，减弱MMP-2和MMP-9的活性，此外，Dkk1抑制Wnt/β-catenin信号通路的关键调控分子β-catenin的核积聚。研究结果表明Dkk1能够抑制诱导的人LECs的迁移和EMT，从而预防PCO的发生和发展。(2)人LECs系SRA01/04转染Wnt3a 48小时后，I型胶原纤维、IV型胶原纤维和整合素β1的表达明显增加，α-SMA和细胞纤维状肌动蛋白由细胞膜转入细胞质和细胞核周围，胶原凝胶收缩明显降低，提示Wnt3a诱导Wnt/β-catenin信号通路活化可促进细胞ECM合成和细胞骨架重建，促进细胞收缩。（3）SRA01/04细胞转染Wnt3a后加入姜黄素，细胞增殖率明显降低，PCNA表达的阳性率明显降低，β-catenin的核转移明显减少，且核内靶基因Cyclin D1和c-Myc蛋白表达明显减弱，证明在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。本课题通过三部分的研究证明，Wnt3a诱导的Wnt/β-catenin信号通路活化，通过促进LECs的增殖、EMT和ECM合成，促进PCO的发生和发展；而Dkk1能够逆转Wnt/β-catenin信号通路诱导的EMT过程，姜黄素抑制Wnt/β-catenin信号通路诱导的细胞增殖，为PCO的预防和治疗提供新的思路。