Sirtuin1调控转录因子Pax6对视网膜前体细胞分化的作用

Retinal progenitor cells (RPCs) have low differentiation efficiency related to the decrease of Pax 6. Reports show that retinal differentiation of human embyonic stem cell (hESC) is partialy due to the dramatic reduce of deacetylase SirT1, which lead to the selective high expression of retina gene (PAX6, DLL4 and TBX3). The findings indicate that SirT1 may play an important role in determine the retinal progenitor fate. In this propsal, we are planing to modulate SirT1 to dynamically regulate Pax6 so that to induce RPCs differienate into diverse neuroretinal cells. We will introduce the hESC line and human PDL stem cell into retina determination condition. To detect and the location of SirT1 and Pax6 by immunocytochemistry staining. To analysis the relationship of SirT1 and retinal differentiation signaling pathway by detecting SirT1 activity and translocation and by real time PCR and western blot. To confirm the relationship between SirT1 and retinal progenitor cells differtiation by modulate SirT1. Identifiy the qualification of the induce retinal progenitor cells to analysis the modulate effect of SirT1 on differentiation effect. To elusiate the molecular mechanism of SirT1 and Pax6 involved retinal progenitor cells differtiation. Expect to increase induce efficiency by modulate SirT1.

视网膜前体细胞（Retinal progenitor cells,RPCs）的分化效率低与Pax6下降导致的多潜能性下降有关，研究发现干细胞分化时，去乙酰化酶Sirtuin1（SirT1）明显降低，直接引起Pax6的表达，促进向视网膜分化。本研究提出假设，利用商品化的SirT1调节剂调控RPCs的Pax6，以保持其多潜能分化性。因此本研究拟利用人胚胎干细胞和人牙周膜干细胞进行视网膜诱导为RPCs，在不同时段利用定量PCR和Western blot检测SirT1与Pax6及视网膜基因，观察SirT1的酶活性和亚细胞定位的动态变化，分析SirT1与Pax6的关系；通过对SirT1和Pax6的调控，对RPCs进行标记物及功能鉴定，分析RPCs分化为成熟神经视网膜细胞的成分和比例，明确SirT1与Pax6的调控关系及对分化效率的影响。以期通过调控SirT1提高RPCs多潜能性并阐明相关机制。

目前对视神经神经退行性疾病缺乏有效的治疗手段，主要是由于中枢神经细胞的不可再生性，而干细胞替代治疗可能是治疗这类疾病的有效方法。本研究主要对两种人成体干细胞是否能诱导成视神经细胞的可行性进行了论证，研究发现人牙周膜干细胞通过诱导能分化成具有视网膜前体细胞（retinal progenitor cells, RPCs）特征的细胞，分别从形态学，细胞标记物和功能进行鉴定证明其可靠性并且具有继续分化为神经细胞的潜在分化能力；此外，又利于另一种来源更加广泛的人脂肪提取干细胞进行诱导分化，结果发现脂肪干细胞经过定向诱导，也能分化成为RPCs并且具有分化为光感受器细胞的分化潜能。这些结果都证明了人成体干细胞分化为视神经细胞的潜能，这为缺乏供体的视神经细胞提供了更方便有效的干细胞来源，为将来视神经疾病的临床应用提供了重要的依据。此外，通过大鼠的视神经两种损伤模型分析sirtuin7个成员在损伤后不同时间点mRNA的动态变化，结果发现7个成员的表达模式表达有差异，提示sirtuin成员在损伤及修复过程中参与程度不同。