仿聚離胺酸材料對神經細胞培養的研究

Abstract

聚離胺酸(poly-D-lysine)在一般細胞培養上是很常被使用的材料，尤其在神經培養上更是經常當成不同材料間的比較的對照組。由於細胞表面整體是偏負電性，而poly-D-lysine在pH=7.4的培養環境下，側鏈末端的NH3+所帶的正電有助神經細胞貼附。 本實驗是選用幾種結構相近於聚離胺酸(poly-D-lysine)的高分子，以及利用體外神經培養的方法，來判斷各種材料的不同官能基對神經表現的差異。目的是要發展新的材料能取代舊有的poly-D-lysine。另外也想藉此瞭解poly-D-lysine的結構是在哪一個部分有助於神經培養，最後也想藉以製造比poly-D-lysine更便宜更有效的材料。 神經細胞的表現會由MTT和LDH進行初步分析，而形態會使用位相差和免疫螢光法分析；細胞的功能性蛋白質表現則使用immunoblot確認量的變化；至於活體傳導功能表現則以glutamate release和phospho-synapsin I(Ser603)的定量方式檢驗。 數種材料中還是以類似於poly-D-lysine的側鏈4個碳會有最高的小鼠顆粒型神經細胞貼附量，但調整塗佈(coating)的量後，大部分材料形態上都會有類似表現。 同時我們也發現，當降低塗佈的量時，神經會微量聚集但尚可形成完整的大格子網路結構。如果把功能表現量除掉細胞總量，其單位細胞功能表現量並不會比傳統的單顆貼附形態的效果差。由於體內神經元並非都以分散貼附的方式生長，因此這種形態也可嘗試成為新的培養模式。

Poly-D-lysine is a material that usually used in cell culture, especially to be deal as control material for other materials test in neural culture. The NH3+ group on poly-D-lysine would be positive charge at pH=7.4 which is a common conditions in cell culture, and it will be helpful for neuron adherence on material due to the overall negative charge on the cell surface. The study is to determine the different behaviors of neuron cultured on several materials which have similar structures to the poly-D-lysine by using in vitro neuron culture. We wish to find new materials that can be substitutes for poly-D-lysine. Besides, we want to know which part of structure of poly-D-lysine is useful for neural culture, and finally to create new materials that cheaper and more effective than poly-D-lysine on neural culture. The behaviors of neurons are determined by LDH and MTT first, and the morphology is checked by using microscope including phase contract and fluorescence. The expression of functional proteins is checked by immunobloting. And finally, the ability of transmission is determined by measurement of the glutamate release and phosphor-synapsin I(Ser603). In this study, the structure have four carbon on the residue which is similar to poly-D-lysine is the best one in the numbers of cerebellar granule cells attached on material. But if we adjust the concentration of coating, neurons can have similar expression on almost all materials. And we also found that the low concentration of coating will made neurons have a little slide to form a big network without neurites broken. Besides, if we divide the among of functional expression with cell number, the average behaviors of big-network forms are not worse than single-adherent forms. Although neurons grow in vivo are not always at single-attachment forms, the big-network forms caused by low-concentration-coating might be a new model for neuron culture.