TGF-β/Smad信号传导通路参与后发障形成的分子生物学调节机制的研究

Posterior capsular opacification (PCO) is a major complication of cataract surgery. TGF-β cytokine plays a key role in the development of PCO via TGF-β/Smad pathway and Smad-independent signaling pathway. Our previous study showed that in HLE B-3 cells, silencing Smad3 could block the effect of TGF-β2 on cell proliferation, production of fibronectin, and type I collagen; silencing Smad2 could block the effect of TGF-β2 on cell migration and production of αSMA; Smad2/Smad 3 depletion enhanced Smad3/Smad 2 activity; silencing Smad2 and Smad3 efficiently blocked the effect of TGF-β2 on cell proliferation, migration, and extracellular matrix production.Therefore we hypothesis that knock out Smad 2 or 3 can block the PCO formation. This investigation will reveal the mechanism of PCO formation in molecular level in vitro and in vivo and explore the possibility of drug development using siRNA technology specific to member of Smad family in order to prevent PCO formation. Three lines of work in this study will be conducted: 1. Collect clinic cataract sample to detect TGF-β signal pathway changes. 2. Using Smad3-/-and conditional Smad2-/-mice to investigate the role of Smad signaling pathway in PCO pathological development in conjunction of PCO model, and the their influence on the activities of TGF-β, regulation of TGF-β/ Smad signaling pathway, TGF-β /Smad-independent signaling pathway, and other activities. 3. Using siRNA technology to conduct Smad2 siRNA, Smad3 siRNA, and Smad2/3 siRNA drug candidates and design delivery method specifically for lens epithelial cells (LECs). The Smad siRNAs drug candidates will be tested in mouse model of PCO in vivo and cultured LEC in a contained IOL culture system mimicking PCO formation in vitro. The inhibitory effects of drug Smad siRNAs on the activities of LEC originated from mouse and human, including the proliferation, migration and epithelial-to-mesenchyaml transition will be investigated and elucidated. To the end of study, we are able to put new insight to the mechanism of PCO formation after cataract surgery, to shed a light on drug research and development and to provide the preclinical data for further investigation of the drug for blocking PCO formation after cataract surgery.

后囊膜混浊（PCO）是白内障术后常见并发症，TGF-β通过Smad信号传导通路在PCO形成中发挥重要作用。但Smad2和3通路分别介导的具体生物学作用并不明确。本组前期工作发现在HLE B-3中， Smad3信号通路介导细胞增殖和ECM产生的调节，Smad2信号通路介导细胞迁移和αSMA产生的调节，据此提出假设阻断二通路能阻止PCO形成。本研究获取白内障手术标本了解TGF-β/Smad通路相关基因表达，并应用Smad2和Smad3基因敲除鼠揭示TGF-β/Smad信号通路在PCO形成中的作用，并构建Smad2和Smad3-shRNA 慢病毒载体，应用于野生型小鼠，探索siRNA阻断TGF-β/Smad信号通路对PCO形成的阻止作用。基于以上假设和前期工作，通过近一步研究以阐明TGF-β对PCO形成的机制，为应用RNA干扰药物阻止PCO形成提供实验理论依据，并初步验证其可行性。

后发性白内障是白内障术后常见并发症。手术残留的晶状体上皮细胞增殖并迁移，发生间充质转化及细胞外基质的形成，最终形成后囊膜混浊。在其发生发展过程中， TGF-β信号通路如何调节晶状体上皮细胞的增殖和迁移以及如何导致EMT等尚不明了。.本组应用siRNA技术干扰Smad3、Smad2的表达结果显示Smad3在TGF-β2的生长抑制作用中起重要作用。TGF-β2／Smad2信号转导通路对αSMA的表达至关重要，Smad3介导纤维素和Ⅰ型胶原的表达。本课题以pcDNA3.1为质粒载体，构建Smad2和Smad3的过表达质粒并转染晶状体上皮细胞HLE B-3，其合成P-Smad2 和P-Smad3蛋白表达均明显升高。Smad3过表达后细胞增殖减少，而Smad2过度表达后促进晶状体上皮细胞迁移增强。Smad2 和Smad3过表达后对HLE B-3的细胞外基质和上皮间质转化的影响，Smad2介导上皮间质转化，而Smad3在细胞外基质表达中起重要作用。本研究采用基因敲除小鼠，分别敲除Smad3， Smad2， Smad2和3，并建立白内障摘除动物模型，得到实验结果与体外实验结果一致。本研究通过构建TAK1-pcDNA3和TAB1-pcDNA3表达载体，使HLE B-3细胞中外源性TAK1和TAB1基因得到了充分表达，免疫印迹及免疫荧光染色结果发现TAK1过表达促使HLE B-3细胞内的E-cadherin表达下调，而纤维连接蛋白、α-SMA和Ⅰ型胶原表达上调。不同剂量的TGF-β2作用于 HLE B-3细胞，促进HLE B-3细胞发生上皮间质转化，并验证HLE B-3细胞内磷酸化TAK1的蛋白水平明显升高即TAK1被激活。通过转染TAK1 siRNA或加入TAK1的化学抑制剂以下调TAK1的表达，发现E-cadherin蛋白表达升高而纤维连接蛋白表达降低，证实阻断TAK1可抑制上皮间质转化的发生。外源性TAK1和TAB1 在HLE B-3细胞过表达使得α-平滑肌肌动蛋白 和Ⅰ型胶原mRNA表达持续增强，说明TAK1参与了促进细胞外基质沉积。.本研究中应用真核生物基因表达调控技术敲除基因或增强基因的表达以明确TGF-β2/Smad信号通路对晶状体的增殖、迁移、EMT的影响，通过体内及体外实验，验证此通路对治疗后发性白内障的有效性及可行性，为后发性白内障的基因治疗发现新的靶点。.