胞外基质刚度调控由PTB/nPTB敲低介导的人源神经元转分化效率的研究

将人源成年真皮成纤维细胞（HAFs）在体外直接转化为诱导神经元（iNs），为神经退行性疾病的细胞治疗提供了一种新策略。有研究表明细胞外基质（ECM）刚度等力学因素可以改善由过表达转录因子介导的神经元重编程，但是对于连续敲除成纤维细胞中RNA结合蛋白PTB和nPTB介导的人源神经元体外转分化体系，三维ECM刚度对其的调控机制我们还知之甚少。根据前期数据，我们发现在神经元直接转化过程中，细胞对力学刺激反应途径中的一些重要基因被上调，并且在后期采用刚度更低的Matrigel能使神经元生成效率更高。在本项目中，我们计划将人源星形胶质细胞脱细胞制成三维类人脑细胞外基质（hBLECM），并建立ECM刚度与细胞转分化之间的数学力学模型，深入研究ECM刚度在神经元转分化起始期和成熟期不同的力学-生物学调控机制，以期引入hBLECM优化现有转分化体系，获得更多的功能神经元用于基础研究和临床转化。

Direct conversion from human adult fibroblasts (HAFs) to induced neurons (iNs) in vitro implicates a promising strategy for cell therapy in neurodegenerative diseases. It is known that mechanical cues such as extracellular matrix (ECM) stiffness can improve the direct neuron reprogramming by overexpressing a combination of neuronal transcriptional factors, but the effects of three-dimensional ECM on transdifferentiation of iNs via sequential knockdown of RNA-binding proteins PTB and nPTB in HAFs remain unexplored. According to our preliminary data, we found some genes from pathway of cellular response to mechanical stimulus were upregulated during direct iNs conversion, and generation efficiency of iNs was higher with commercial Matrigel coating with lower stiffness at late stage. In this proposal, we plan to prepare a three-dimensional human brain-like extracellular matrix (hBLECM) from decellularized human astrocytes with supplements of related scaffold proteins and neuronal growth factors, which could mimic human brain microenvironments for development and growth of neurons. In addition, we also plan to construct a mathmatical-mechanical modulation between ECM stiffness and transdifferentiation efficiency. Using this self-designed hBLECM, we next study the mechanical-biological mechanism of ECM stiffness to facilitate neuronal transdifferentiation at early or late stage, separately. And based on these key mechanical properties of hBLECM, we aim to establish an advanced system from current in vitro HAFs-iNs protocol to obtain more functional neurons for both research and clinical purposes.