构建载有mTOR-siRNA人工晶状体对晶状体上皮细胞生物学行为影响的研究

Posterior capsular opacification is one of the most common complication after cataract surgery which also lead to blinding once again. However, there is currently no effective treatment method.The main cause of posterior capsular opacification is the proliferation, migration and epithelial mesenchymal transformation of residual lens epithelial cells after cataract surgery. One focus of preventing posterior capsular opacification is inhibiting lens epithelial cell biological behavior through artificial lens modification. The aim of this issue is to design, synthesize and choose the excellent rapamycin target protein mTOR small interfering RNA(mTOR-siRNA).Then,constructing multilayer controlled releasing membrane with chitosan/mTOR-siRNA on acrylate intraocular lens surface through layer-by-Layer assembly technique.Thephysical,chemical,optical characteristics, biocompatibility hydrophobic properties and releasing degradation efficiency in vitro of the new artificial lens will be researched.By imitating the capsule membrane environment model in vivo,the effect of new type artificial lens on cell biology behavior will be observed in vitro. We will also observe the cell biology behavior after mTOR-siRNA transfection into cells and its influence on PI3K/Akt/mTOR signaling pathways. Our advantage is that we combine gene therapy with materials science to construct the new type artificial lens, which inhibit lens epithelial cells' proliferation efficiently. At the same time, the pathogenesis of posterior capsular opacification will be further studied . In a word, our study will lay the foundation for the gene therapy of treating posterior capsular opacification in vivo.

后发性白内障是白内障手术后最常见并发症之一，是术后再次盲的主要原因，目前尚无有效治疗方法。残留的晶状体上皮细胞增殖、移行、上皮间质转化是其根本原因。通过人工晶状体途径改变晶状体上皮细胞行为是抑制后发性白内障热点之一。本课题体外合成优良干扰效能的雷帕霉素靶蛋白小干扰RNA（mTOR-siRNA），通过层层自组装技术在丙烯酸酯人工晶状体表面构建载有壳聚糖/mTOR-siRNA的多层控释膜。观察新型人工晶状体物理、化学、光学特性、生物相容性、疏水性及体外降解释放效能；模拟体内囊膜微环境模型体外观察新型人工晶状体对细胞生物学行为影响；观察mTOR-siRNA转染至晶状体上皮细胞后对细胞生物学行为及对PI3K/Akt/mTOR信号通路的影响。意义在于结合基因治疗及材料学优势构建出高效抑制晶状体上皮细胞增殖的新型人工晶状体，同时进一步明确后发性白内障的发病机制，为在体实现基因治疗后发性白内障奠定基础。

项目按照预期计划正常完成课题任务。后发性白内障仍然是现代白内障手术无法克服的并发症之一，严重影响患者术后的视觉质量。后发性白内障的发生主要与晶状体上皮细胞的生物学行为有关，包括其术后的迁移、增殖、上皮间质转化等过程，PI3K/AKT/mTOR信号通路参与了术后的细胞异常代谢反应。mTOR基因是参与此过程的重要信号基因。本项目观察了应用RNAi技术干扰mTOR表达对晶体上皮细胞生物学行为及PI3K/AKT/mTOR信号通路的影响；将浸泡过mTOR-siRNA核酸溶液的IOL或张力环植入兔晶状体囊袋内，离体观察对囊袋内晶状体上皮细增殖、迁移及囊袋皱缩影响情况。体外构建的mTOR-siRNA成功转染HLEB3后明显抑制胞mTOR蛋白及mRNA的表达。mTOR-siRNA转染细胞后明显抑制细胞的增殖，并且呈时间依赖性。在有或无TGF-β诱导条件下，转染mTOR-siRNA组迁移率显著降低。TGF-β可诱导磷酸化AKT的增加及EMT标志物α-SMA的表达，但转染mTOR-siRNA后，α-SMA蛋白表达受到抑制。mTOR siRNA转染晶状细胞后，mTOR及其下游效应分子P70S6K 、p-P70S6K、p-AKT的 mRNA及蛋白表达水平明显下降。证实在HLEB3内存在PI3K/AKT/mTOR信号通路，且可能是通过抑制p70S6K 和 AKT表达来抑制HLEB3增殖、迁移、上皮间质转化过程。成功构建模拟体外微环境的囊袋培养模型，将浸泡过mTOR-siRNA核酸溶液的人工晶状体或张力环植入囊袋内，发现在白内障摘除术后24小时内晶状体上皮细胞开始从赤道部生长并向中央爬行，96h后爬满晶状体后囊，并且出现细胞纤维化、囊袋皱缩；而经过浸泡mTOR-siRNA核酸溶液的人工晶状体或张力环组则表现较少的细胞密度和迁移距离。总之，应用RNAi技术特定基因沉默mTOR，能够抑制mTOR信号通路及下游蛋白及核酸的表达，调控晶状体上皮细胞的生长及分化，进而能够实现减轻或推迟PCO发生，为PCO防治新手段提供了可能。