调控SOX9介导的Müller细胞过度活化对大鼠光损伤视网膜变性的保护作用及其机制研究

Currently, the mechanisms and treatment of retinal degenerative diseases are still major research concerns. The common pathological process of these diseases is retinal remodeling. As the principal glial cell, reactivation of Müller cells and glial scars formation play a critical role in the process of retinal remodeling. However, it’s still unknown how this process happens. The studies in central nervous system have indicated that SOX9-upreglutation contributed a lot to reactivation Müller cells and extracellular matrix deposition (ECM). SOX9 is only expressed in Müller cells in mature retina. Our research has shown that SOX9 is upregulated in retinal light damage of rats. And hence, we infer that SOX9 could play an important role in reactivation of Müller cells and ECM deposition. In this study, to explore the role for SOX9 in retinal remodeling, we focus on the relationship between SOX9 and reactivity of Müller cells. We evaluated the neuroprotective effect of reactive depression of Müller cell via down-regulating SOX9 in retinal light damage of rats and investigate the upstream and downstream molecules of SOX9. The results can provide a new clue to retinal degenerative diseases treatment.

视网膜变性疾病的病理机制及治疗是目前研究的难点。这些变性疾病的共同病理过程是视网膜重塑，作为视网膜主要的胶质细胞，Müller细胞过度活化及胶质瘢痕的形成是视网膜重塑的关键环节，但其活化的调控机制尚不清楚。中枢神经系统的研究提示SOX9基因在调控胶质细胞过度活化及细胞外基质（ECM）形成中扮演重要角色。SOX9在成体视网膜仅在Müller细胞表达，申请人前期的研究发现SOX9在光损伤诱导的视网膜变性大鼠中表达上调。故推测，SOX9可能是调控Müller细胞过度活化及ECM沉积的关键靶点。本研究拟采用光损伤诱导的视网膜变性大鼠模型，以SOX9与Müller细胞活化的关系为切入点，观察抑制SOX9表达后对大鼠视网膜光损伤的保护作用；对Müller细胞活性及ECM沉积的影响；探求调控SOX9的信号通路，阐明SOX9在视网膜重塑中的作用机制，为视网膜变性疾病的治疗提供新的思路。

视网膜变性疾病的病理机制及治疗是目前研究的难点。这些变性疾病的共同病理过程是视网膜重塑，作为视网膜主要的胶质细胞，Müller细胞过度活化及胶质瘢痕的形成是视网膜重塑的关键环节，但其活化的调控机制尚不清楚。中枢神经系统的研究已提示SOX9基因在调控胶质细胞过度活化及细胞外基质（ECM）形成中扮演重要角色。体外研究表明，CRISPR-Cas9介导的SOX9基因敲除不仅抑制Müller细胞GFAP的表达，也减弱其移行能力。以大鼠视网膜光损伤作为视网膜变性的动物模型。在光损伤前2周，将大鼠玻璃体腔内注入SOX9-shRNA病毒。光损伤3天、7天、14天后，用视网膜ERG评估视视网膜功能，用HE组织学染色评估视网膜外核层厚度。用Western Blot观察GFAP, vimentin, nestin和CSPGs的蛋白水平等，这些蛋白与Müller细胞胶质化和细胞外基质重塑相关。GFAP的表达情况进一步用免疫荧光确认。结果表明：光损伤3天、7天、14天后，SOX9-shRNA组和对照组相比，视网膜外核层厚度更厚，视网膜ERG的a、b波振幅更高。GFAP, vimentin, nestin和CSPG蛋白表达水平下降的更多。另外，在SOX9-shRNA组，每个时点GFAP的免疫荧光也更弱。这些结果提示SOX9基因可能是未来视网膜变性疾病的一个可能治疗靶点.