| 摘要: | 間葉幹細胞在特別的條件下可以分化為神經幹細胞。然而對於確切的調控機制，尚未明朗。對於胚胎發育與成熟個體的組織恆定，Wnt一直扮演重要角色。先前報告指出，Wnt有能力調控神經幹細胞及前驅細胞的身經分化如軸突樹突的生長與突觸形成。此外，Tenascin家族中的Tenascin-C與R也能控制多種神經前驅細胞的分化、移動、存活。因此我們假定Wnt與Tenascin將參與在特定的微環境中調控間葉幹細胞的最終神經分化。在Wnt的研究中，Neurotrophins所引起的初步神經化會增加Wnt7a的表現。進一步發現，Wnt7a與Wnt訊號刺激劑─鋰鹽會誘導突觸蛋白Synapsin-1的表現。而此由Wnt所引起的Synapsin-1表現，也會被Wnt抑制劑所抑制。再進一步觀察Wnt7a的效果，發現Wnt7a所引起的神經特化卻不被鋰鹽所刺激，展現出Wnt7a有引起兩種訊號路徑的能力。在Tenascin的研究中，我們將幹細胞培養於兩種主要環境，一是將Tenascin溶於培養基，另一種是將Tenascin先附著於培養盤上再行培養。及時定量PCR分析顯示，Tenascin-C與R同時溶於培養基中能促進神經分化，但是並無法刺激突觸指標的表現。而有趣的是，以Tenascin-C附著的培養盤，能刺激軸突生長與突觸形成。再將幹細胞培養於Tenascin-R與C一起附著的盤上，除了突觸指標增加外，也幫助神經與寡突細胞分化並抑制神經膠細胞分化。此外，功能阻礙實驗也顯示，Integrin α7與α9β1是Tenascin產生功能的主要受器。這些試驗顯示了Wnt7a與Tenascin在間葉幹細胞神經分化的成熟上有關鍵作用。這些資訊也拓展了中胚層幹細胞療法於神經再生醫學上的應用。
Mesenchymal stem cells (MSCs) can differentiate into neural stem cells (NSCs) under specific conditions. Wnt was reported to be crucial during embryonic development and adult tissue homeostasis. Wnts regulate neurogenesis, such as neurite and synapse formation, of the neural stem or progenitor cells. In addition to Wnts, tenascin (Tn) family members, TnC and TnR, have been demonstrated to regulate differentiation and migration, as well as neurite outgrowth and survival in numerous types of neurons and progenitor cells. Thus, we hypothesize that Wnt signaling and different forms of Tn regulate the terminal neuronal differentiation in neurotrophin-induced hMSCs. Wnt was also reported, to stimulate hMSC with neurotrophins containing medium (retinoic acid, nerve growth factor, and brain-derived neurotrophic factor; NBR) resulting in the expression of Wnt7a, which enhanced the hMSCs to express neuronal markers in our subsequent analysis, Synapsin-1 (SYN) was induced by Wnt7a and lithium, a glycogen synthase kinase (GSK)-3βinhibitor, in the NBR-induced hMSCs, but was further inhibited by the Wnt inhibitors. Furthermore, hrWnt7a triggered the formation of cholinergic, dopaminergic, GABAergic, and serotonergic neurons. hMSCs were then further cultured in media incorporated with soluble Tn, or on pre-coated Tn. In a qualitative PCR analysis, adding a soluble TnC and TnR mixture to the medium significantly enhanced the expression of neuronal and glial markers, whereas no synaptic markers were expressed. Conversely, in the groups of cells treated with coated TnC, hMSCs showed neurite outgrowth and synaptic marker expression. A combination of TnC and TnR significantly promoted hMSC differentiation in neurons or oligodendrocytes, induced neurite and synapse formation, and inhibited differentiation into astrocytes. In a functional blocking study, integrin α7 and α9β1 blocking antibodies inhibited, respectively, 80% and 20% of the mRNA expression by the hMSCs in the coated Tn mixture. In summary, our results demonstrated novel mechanisms and functions of Wnt7a and Tn for regulating neural differentiation. Data can be broadly employed in the use of MSC to treat neurodegenerative disease. |
