利用转基因斑马鱼研究小胶质细胞在视网膜感光细胞损伤及再生中的作用

Retinal injuries and photoreceptor dystrophies are significant causes of blindness in humans. The zebrafish retina is an important model for nervous tissue development and regeneration. In contrast to mammals, any insult that significantly depletes retinal neurons stimulates robust neuronal regeneration and recovery of function. Microglia are cells from non-neuronal lineages that reside in the central nervous system. In zebrafish, macrophages migrate from the yolk sac to the brain and retina at 26-30 hours post fertilization (hpf) and transform into microglia at 55-60 hpf. .Previous work in our lab shows that the targeted knockdown of csf-1r with morpholino oligonucleotides delays migration of macrophages from the yolk sac to the retina, and this delay results in microphthalmia, delay in cell cycle withdrawal among retinal progenitors and the absence of neuronal differentiation. Another previous study shows that clodronate-liposome injected into vitreous chamber depletes microglia partially and diminishes injury induced proliferation. However, gaps still remain regarding the role of microglia during photoreceptor regeneration. .Our aim is to test the hypothesis that microglia play a fundamental, neurogenic role in stem cell-based prhtoreceptor regeneration. Two sub aims will be undertaken: (1) transgenic fish Tg (pgrna:nfsB::EGFP) will be generate to provide a model for selective death of microglia and (2) photoreceptor regeneration will be studied in which microglia have been depleted. Firstly, fish transgenic for the pgrna-nfsB-EGFP construct will be generated by the Tol2 system. The bacterial nitroreductase (NTR)/metronidazole cell ablation system will be used to specifically induce death of microglia. Secondly, adult transgenic zebrafish will receive a photolytic lesion by a mercury arc lamp to induce the selective death of photoreceptors. After the light exposure, these procedures will be followed: at 1-2 days survival, photoreceptor apoptosis will be quantified using TUNEL assay; at 2-5 days survival, cell proliferation will be quantified using immunocytochemistry; at 5-10 days survival, cone and rod regeneration will be observed by in situ hybridization; at 1-10 days survival, the images of retina will be collected by SDOCT scanning. .The studies proposed here will contribute to the comprehensive understanding or photoreceptor regeneration in the human retina and will help develop therapies for stem cell-based treatment of retinal injury and disease in humans.

感光细胞功能丧失是人类致盲的重要原因，目前尚无有效治疗。斑马鱼的视网膜结构与人类高度相似，感光细胞损伤后可以再生，是研究感光细胞再生的理想模型。前期工作中发现，斑马鱼发育早期缺乏小胶质细胞导致视网膜发育明显延迟；成体鱼小胶质细胞部分受抑制能显著减缓细胞增殖过程。然而，现有方法不能彻底、稳定清除小胶质细胞，需要构建一种新模型对其调控作用进行深入研究。本项目将构建转基因斑马鱼Tg（pgrna: nfsB:: EGFP），小胶质细胞不仅被绿色荧光蛋白特异性标记，还携带了硝基还原酶，利用甲硝唑可以特异性导致硝基还原酶阳性细胞死亡的特点，达到在中枢神经系统稳定、彻底清除小胶质细胞的目的。以转基因斑马鱼为模型，利用免疫组化、原位杂交和影像学等方法，从细胞凋亡及增殖、感光细胞分化基因表达、细胞再生等角度研究小胶质细胞调控感光细胞再生的机制，为开发干细胞移植治疗人类视网膜感光细胞损伤提供理论依据。

斑马鱼的视网膜结构与人类高度相似，并且神经生成作用持续终生，是研究感光细胞损伤与再生机制的理想动物模型。本项目的主要研究内容为构建转基因斑马鱼Tg（pgrna: nfsB:: EGFP），以绿色荧光蛋白特异性标记小胶质细胞，再利用甲硝唑只导致硝基还原酶阳性细胞死亡的作用，实现小胶质细胞的稳定清除。我们的工作分为两部分：（1）观察感光细胞损伤后的细胞增殖与再生以及小胶质细胞在早期损伤中的作用，并对视网膜中央区损伤后的行为学变化进行了测定，是本项目的基础数据，我们将在此基础上以构建的转基因斑马鱼为模型，再研究小胶质细胞的调控作用。（2）通过Tol2系统构建转基因斑马鱼Tg（pgrna:nfsB::EGFP），进行了筛选与鉴定，这是本项目研究的动物模型。在一年资助期内，项目负责人累计发表论文和综述4篇，其中SCI收录期刊2篇，中文核心期刊1篇，中文在线期刊1篇。