發展ㄧ個研究中樞神經細胞軸突的細胞培養裝置

Development of compartmentalized device for studying physiology of central axons

何星樺(Ho, Hsing Hua)

指導教授 ： 張兗君

國立清華大學/生命科學院/系統神經科學研究所/碩士(2015年)

https://doi.org/10.6843/NTHU.2015.00303

摘要

摘要
神經元(neuron)為高度特化的細胞，除了細胞本體(soma、cell body)，還有神經次細胞結構樹突(dendrites)及軸突(axon)。軸突在神經系統中扮演重要角色，軸突為細長結構的神經纖維(process)，從細胞本體延伸至目標細胞，進行訊息的傳遞。目前因缺乏相關研究，導致對於中樞神經系統軸突其受損及再生的機制並尚未了解。因此，本論文發展一個裝置，可將大鼠(SD rats)的海馬迴神經細胞 (hippocampal cells)進行體外培養 (in vitro)。本裝置利用雙層膜的構造來進行細胞本體及軸突的分離，第一層膜為孔徑1 μm PCTE (polycarbonate) 膜，第二層膜為具數千孔洞(孔洞直徑大小為150μm)且厚度約為500μm的 PDMS (polydimethylsiloxane)膜。將神經細胞養在coating PLL(poly-L-lysine)的PCTE膜上，因細胞本體的直徑約為10 μm，因此會被侷限在PCTE膜上，只有神經纖維(樹突及軸突)可以穿過PCTE膜往PDMS膜生長。PDMS膜除了coating PLL (poly-L-lysine)之外，另有ECM (Extracellular matrix)及laminin充滿孔洞，誘導神經纖維往PDMS膜生長。成熟的神經細胞其樹突只能長達300 μm，軸突可生長超過500 μm，利用厚度約500 μm的PDMS膜，因此只有軸突可長至PDMS膜的下方，藉此區分樹突及軸突。此裝置可利用螢光染色的方法觀察活體軸突的生長及收集單純的軸突，亦可將神經細胞與軸突培養在兩個不同的環境中進行研究，可應用於軸突與其他細胞的共同培養(co-culture)或是藥物試驗的實驗，將有助於未來研究軸突與目標細胞的突觸形成過程或是軸突的生長及再生的分子機制。

關鍵字

軸突

並列摘要

Abstract
A typical neuron consists of a cell body (soma), dendrites, and an axon. Axon is a long process extending from soma of a neuron and plays important roles in establishing the neuron-target connection and signal transmission. On the other hand, due to their limited capability of regeneration, injury to the axons of central neurons frequently leads to incurable neural disorders, such as spinal injury. Here, we have developed a compartmentalized device by using a composite membrane consisting of a PCTE (polycarbonate) and PDMS (polydimethylsiloxane) membrane and various protein components. The PCTE membrane is ~8 µm in thickness and contains pores of an average diameter of 1μm. Placed underneath the PCTE membrane is a layer of PDMS of 500 μm in thickness and contains holes of 100 µm in diameter that open to the two surfaces. The PCTE membrane is coated with poly-L-lysine, the the holes of PDMS membrane are filled with ECM (Extracellular matrix) plus laminin. This composite membrane is placed between two chambers. Cells dissociated from rat embryonic hippocampi are then plated onto the PCTE membrane. After ~ 2 weeks in vitro, the cell bodies and glial cells remain on the surface of PCTE membrane, and only axons can penetrate through the composite membrane to the opposite side. The advantages of this device include (1) the diffusion of small molecules across this composite membrane is limited, (2) the axons on the PDMS side of the composite membrane could be observed continuously under microscopy, and (3) a second population of cells can be grown on the PDMS membrane surface. This device could be used for the high-throughput screening for drugs affecting (1) synaptogensis, (2) axonal growth and (3) axon regeneration. This device could also be used to study the molecular basis of synaptogenesis, synaptic regulation and trafficking in axons.

並列關鍵字

axon

參考文獻

Burridge, K., C. E. Turner and L. H. Romer (1992). "Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly." J Cell Biol 119: 893-903.

Campenot, R. B. (1977). "Local control of neurite development by nerve growth factor." Proc Natl Acad Sci U S A 74: 4516-4519.

Cox, L. J., U. Hengst, N. G. Gurskaya, K. A. Lukyanov and S. R. Jaffrey (2008). "Intra-axonal translation and retrograde trafficking of CREB promotes neuronal survival." Nat Cell Biol 10: 149-159.

Daniloff, J. K., G. Levi, M. Grumet, F. Rieger and G. M. Edelman (1986). "Altered expression of neuronal cell adhesion molecules induced by nerve injury and repair." J Cell Biol 103: 929-945.

Iozzo, R. V. (1998). "Matrix proteoglycans: from molecular design to cellular function." Annu Rev Biochem 67: 609-652.