c-Ski基因对青光眼性视神经损伤保护作用及机制研究

The pathological basis of glaucoma is retinal ganglion cells(RGCs) apoptosis. Besides lowering intraocular pressure(IOP), optic nerve damage and neuroprotection have become the current research focuses. However, there is still no effective clinical treatment for optic nerve injury so far. TGF-β2 mediated RGCs apoptosis is closely related to glaucomatous optic nerve damage. Recent researches showed that the changes occurred in cytoskeletal proteins of RGCs and extracellular matrix(ECM) may play a key role in the RGCs damage and apoptosis. Our previous studies showed that the c-Ski gene could degrade actin cytoskeleton of trabecular meshwork cells, remodel ECM, and thus reduce the resistance of aqueous outflowing as well as increase aqueous outflowing in rats. Based on our previous studies, we will transfer c-Ski gene, a repressor of TGF-β/Smad signaling pathway, into vitreous cavity of rat optic nerve damage model induced by excitatory amino acids, investigate the protective effects of c-Ski gene on RGCs, and clarify the exact mechanism of neuroprotection and possible side effects of transgene expression. This study will lay the foundation of gene therapy for glaucoma. We are striving for a breakthrough in the treatment of the second leading cause of irreversible blindness in the world.

青光眼是以视网膜神经节细胞(RGCs)凋亡为病理基础的疾病。除降眼压外，视神经损伤和保护已成青光眼研究热点，然而迄今为止，临床尚无针对青光眼视神经损伤的有效治疗手段。TGF-β2介导RGCs凋亡，与青光眼性视神经损伤密切相关。近期研究表明，RGCs细胞骨架和细胞外基质(ECM)改变可能在青光眼RGCs损伤和凋亡中起关键作用，我们前期的研究发现：c-Ski基因可降解小梁细胞肌动蛋白骨架和重塑大鼠小梁通道ECM，降低房水外流阻力，增加房水排出。据此，本课题在前期研究基础上拟用慢病毒载体将TGF-β/Smad信号通路辅阻遏因子c-Ski基因导入用兴奋性氨基酸诱导大鼠青光眼性视神经损伤模型玻璃体腔，探讨c-Ski基因表达对大鼠视网膜RGCs的保护作用，阐明其作用机制和转基因表达可能的副作用，为基因治疗青光眼的临床应用打下基础，争取在人类第二大致盲眼病青光眼的治疗上有所突破。

青光眼是以视网膜神经节细胞(RGCs)凋亡为病理基础的疾病。除降眼压外，视神经损伤和保护已成青光眼研究热点，然而迄今为止，临床尚无针对青光眼视神经损伤的有效治疗手段。TGF-β2介导RGCs凋亡，与青光眼性视神经损伤密切相关。本课题在前期研究基础上用腺病毒载体将TGF-β/Smad信号通路辅阻遏因子c-Ski基因导入用兴奋性氨基酸NMDA诱导大鼠青光眼性视神经损伤模型玻璃体腔，探讨c-Ski基因表达对大鼠视网膜RGCs的保护作用，阐明其作用机制和转基因表达可能的副作用。我们的研究证实：(1)成功构建c-Ski稳定过表达或干扰细胞株，明确c-Ski基因通过抑制 TGF-β2/Smad 信号通路调控 NMDA 诱导RGCs损伤细胞凋亡和细胞外基质表达。（2）建立了NMDA大鼠青光眼性视神经损伤模型，AAV-c-Ski/ AAV-shRNA-c-Ski转导大鼠眼，检测基因过表达/基因干扰及其对视网膜形态及功能影响，c-Ski基因可以抑制NMDA诱导视网膜神经节细胞复合体的变薄和RGCs的凋亡，改善视神经的功能，未见明显眼内副作用如炎症反应等；（3）免疫印迹方法检测c-Ski基因过表达/基因干扰通过TGF-β2/Smad信号通路调控大鼠青光眼模型ECM蛋白表达和RGCs凋亡，明确c-Ski对NMDA诱导的大鼠青光眼性视神经损伤模型的保护作用及机制。阐明基因治疗保护青光眼视神经损伤的机制,为基因治疗青光眼的临床应用打下基础。