人神经嵴干细胞分化的分子机制及其生物学功能的研究

Embryonic NCSCs are pluripotent and NCSCs have been shown to persist in the adult in at least two differentiated tissues, the enteric nervous system of the gut and the skin and hair follicles. NCSCs have been well characterized in chick and murine systems. Despite the importance of neural crest cell biology in development and disease, the isolation of NCSCs of human origin has been elusive. We have recently identified a stem cell population in human hair follicles. These cells are distinctively different from epithelial or melanocyte stem cells. They are clonogenic, capable of self-renewal and pluripotent. We propose to characterize the bulge hNCSCs in human hair follicles, study the physiological function of hNCSCs ,and determine the signals for hNCSCs to differentiate to melanocytes. The proposed studies will establish a novel model to study human NCSCs in the somatic tissues. This model can be used to study various signalling pathways that are essential for hNCSC self-renewal and lineage determination, the physiological functions of hNCSCs in adult tissue, and the potential use of these cells for regenerative therapy. Also, because many diseases, including some cancers, are known to involve neural crest cells, the models developed in these studies may be useful in testing the etiologies of these diseases.

胚源性的神经嵴干细胞（NCSCs）具有多分化潜能，它至少存在于成人的两个分化组织中：肠道组织的神经系统以及皮肤毛囊组织。NCSCs在个体发育和疾病发生中有重要意义，包括某些癌症在内的许多疾病被证实与神经嵴细胞病变有关。由于人源NCSCs的分离技术尚不成熟，现有的对NCSCs的了解多来自小鸡和鼠类的研究。最近，我们在毛囊结构的隆突区鉴定出一个干细胞群。这些细胞与上皮干细胞或黑色素干细胞具有明显的差别，他们能形成克隆,能够自我更新并具有多潜能性，我们初步鉴定了人毛囊隆突区hNCSCs的生理功能和诱导hNCSCs分化为黑色素细胞的信号。本研究将建立一个新的模型，用于研究hNCSC自我更新、控制hNCSC谱系遗传、成体中hNCSCs的生理作用等重要生物学功能的关键信号转导通路，及探讨将这些细胞用于再生疗法的可能性。同时,该模型将有助于阐明一些与神经嵴细胞有关的疾病的病因。

我们已在课题研究上取得显著进展。其中一个主要问题是皮肤源性神经嵴干细胞（NCSC）的长期的培养，包括它的体外扩增培养和其他相关的研究。目前没有确切的方法直接分离出这些原代干细胞。为了克服这个障碍，我们最近发现一种新方法，直接从毛囊中分离出人神经嵴干细胞（hNCSCs）。我们将头皮组织切碎并用消化酶孵育分散过夜。这些毛囊被分离出来，再充分洗涤。加入胰酶将其与制成单细胞悬液。我们采用FACS分析检测表面标记CD271和HNK1。我们发现，hNCSCs分化过程中，miR-200c表达的增加，谱系特异性标志物的表达相应增加，而 BMI-1表达相应减少，BMI-1是一种干细胞标志物，参与干细胞的自我更新。我们将hNCSCs在含Forskolin和Heregulin-1 的分化培养基中培养3-6周。这些分化后的hNCSCs表现出显著的形态变化。hNCSCs呈现更加修长和梭形的细胞形态。此外，这些细胞也表达了雪旺细胞特异性标记物GFAP和S-100，并减少SMA的表达。