Bhlhb5对小鼠视网膜无长突细胞和双极细胞亚型命运决定的作用研究

视网膜变性疾病是目前主要致盲眼病，缺乏有效的治疗方法，干细胞移植是该类疾病新的研究热点。神经细胞的细胞谱系和命运决定研究是探索干细胞定向分化的关键，也是干细胞移植治疗的基础。申请人前期通过Bhlhb5基因敲除和细胞谱系研究，发现Bhlhb5为GABA能无长突细胞和II型OFF视锥双极细胞的生成所必需，并作用于视网膜干细胞的分化方向选择过程。Bhlhb5表达缺失造成上述两种神经细胞数量显著减少和分化方向改变，提示Bhlhb5可能作用于此两种细胞的命运选择过程。为了验证Bhlhb5对此两种视网膜无长突和双极细胞亚型的命运决定作用，本项目将在利用前期转基因、基因敲除小鼠模型的基础上，结合腺相关病毒载体分别在小鼠胚胎期和成体视网膜中过表达Bhlhb5基因，观察Bhlhb5基因过表达对视网膜Bhlhb5细胞谱系无长突和双极细胞亚型的影响，验证Bhlhb5诱导视网膜干细胞定向分化的潜力。

Retinal degeneration (RD) diseases are the main cause of blindness in clinical Ophthalmology. Now there is no effective treatment for RD, stem cell transplantation provides a promsing therapy for RD. Cell lineage and fate determination research are the basis of retinal stem cell directional differentiation, and play crucial roles in the stem cell transplantation therapy. Our preliminary study showed Bhlhb5, a basic helix-loop-helix transcription factor, is key regulator in the generation of retinal amacrine and bipolar subtypes. By tracing the cell lineage of Bhlhb5, we revealed that Bhlhb5 is required for the generation of GABAergic amacrine and Type 2 OFF-cone bipolar cells. Deletion of Bhlhb5 resulted in the loss of these two interneuron subtypes, accompany by the cell fate conversion of retinal amacrine subtypes. Our data suggest Bhlhb5 may be the key factor for cell fate determination of GABAergic amacrine and Type 2 OFF-cone bipolar cells, and may be a effective candidate for induced directional differentiation of retinal stem cells. In order to confirm our hypethesis, we will over-express Bhlhb5 in both embyronic and adult mouse retina by generating the transgenic and knockout models and AAV vector that continuous expressing Bhlhb5. Our project will verify the role of Bhlhb5 in the determination of mouse retinal amacrine and bipolar subtypes, as well as the potential application of Bhlhb5 in the directional differentiation of retinal stem cells.