靶向Pim-1基因过表达修复受损视神经的作用及机制

Pim-1 may be a crucial protein in regulating intrinsic regeneration ability of RGC. Our previous study indicated that Pim-1 mRNA expression in the retina do not change after rat optic neve injury, but Lnc-Pim1 expression be transiently significantly up-regulated 7 days after rat optic nerve crush. We hypothesize that overexpression of Pim-1 in the RGC, as one being worth ten, will more effectively enhance the intrinsic regeneration ability of RGC after optic nerve crush..We will firstly display the changes of temporal expression patterns of Pim-1 and Lnc-Pim1 in the retina after crush of rat optic nerve in this project. Then, vitreal injection of Pim-1-AAV2, Lnc-Pim1-AAV2 or isLnc-Pim1will immediately be performed after optic nerve crush to increase Pim-1 or Lnc-Pim1 content in the retina. To explore effects of these gene therapies to repair the injured rat optic nerve and their mechanisms, retina culture in vitro, morphology, molecular biology, functional evaluation and so on will be used to search optic neve regeneration, survival, gene expressions, intracellular signal transduction in the RGC, and functional restoration. At the same time, the effects and mechanisms will be studied in the injured RGC in vitro. Proteins and gene binding sites targeted by Lnc-Pim1 will be identified with RACE, RNA pull down，protein mass spectrometry and CHIRP-Seq in the RGC. The results will provide a new treatment and its rationale for repairing human optic neve and central nervous system injury.

Pim-1可能是影响RGC内在再生能力的关键分子。预实验显示大鼠视神经损伤后视网膜Pim-1 mRNA表达变化不明显，Lnc-Pim1短暂上调。我们假设视神经损伤后过表达RGC中的Pim-1，将起到以一抵十的效果，能更有效地提高RGC的内在再生能力。本项目拟首先研究大鼠视神经压榨伤后视网膜中Pim-1、Lnc-Pim1表达变化；伤后玻璃体注射Pim-1-AAV2、Lnc-Pim1-AAV2、isLnc-Pim1，用视网膜体外培养、形态学、分子生物学和功能检测等方法，观察视神经再生、RGC存活、基因表达、细胞内信号转导和功能恢复等，结合对体外受损RGC的相应研究，并用RACE、RNA pull down、蛋白质谱、CHIRP-Seq等方法寻找RGC中Lnc-Pim1的靶蛋白和基因结合位点，研究靶向Pim-1基因过表达修复视神经伤的作用及机制，为临床治疗视神经及中枢神经伤提供新手段和理论基础。

Pim-1为许多细胞因子和生长因子细胞内信号通路的共同下游信号转导分子，对细胞增殖、分化、存活等至关重要。本项目聚焦于调节Pim-1表达，增强视网膜神经节细胞（RGC）内在再生能力，以治疗受损视神经并探讨相关作用机制。Pim-1蛋白表达于大鼠视网膜视网膜色素上皮细胞及80% RGC。大鼠视神经损伤后视网膜Pim-1 mRNA和蛋白先高表达，其后下调。用RACE法在大鼠原代RGC中获得Lnc-Pim-1序列全长，大鼠视网膜 Lnc-Pim1主要表达在RGC层及内核层，视神经损伤后RGC层Lnc-Pim1短暂高表达，之后低表达。玻璃体注射rAAV2-Pim-1感染大鼠视网膜，可激活视神经损伤后视网膜中Stat3、Akt1和Akt2通路，下调Cleaved caspase-3、Bad和Bax表达，上调Bcl-2、ßⅢ-tubulin、GAP-43表达，提高RGC存活并减少其凋亡，促进视神经再生及视觉功能恢复。在体外，神经营养因子CNTF、Nrn1上调受损神经元样细胞N-2a-N中Pim-1表达，结果Bcl-2、GAP-43表达上调，Bax、Cleaved caspase-3下调，减少了受损细胞凋亡，并促进其突起再生。以LV-Pim-1转染Neuro-2a细胞，增强了细胞增殖，并促进受损N-2a-N中GAP-43表达及突起再生。Pim-1 silencer敲低N-2a-N细胞，减弱了细胞活力、GAP-43表达及突起再生。用RACE法获得小鼠Neuro-2a细胞Lnc-Pim1序列全长，核质分离技术及FISH法均显示Lnc-Pim1大部分位于N-2a-N细胞质内。体外N-2a-N受损后Lnc-Pim1表达先低后高，之后再下调。LV-Lnc-Pim1转染Neuro-2a细胞，促进了细胞分化和受损N-2a-N突起再生，而Lnc-Pim1 silencer敲低Neuro-2a细胞，阻碍了正常N-2a-N突起生长。Neuro-2a细胞Lnc-Pim1过表达促进Pim-1表达，Pim-1过表达抑制Lnc-Pim1表达，二者敲低对彼此表达影响不明显。用CHIRP、RNA-seq及生物信息学方法初步探讨了N-2a-N细胞Lnc-Pim1作用的分子基础。总之，调节Pim-1表达可修复视神经损伤，调节Lnc-Pim1表达可修复受损神经元样细胞，为临床治疗视神经损伤提供了新的有力手段和理论依据。