初代背根神經節神經細胞於植物化學素及奈米鑽石研究之應用

Abstract

初代培養分離性神經細胞 (primary dissociated neuron culture) 是一個已經被廣為使用且強而有力的研究工具。雖然有很多其他較易取得的系統可供使用，且這些系統也都可以長出神經突狀的突出 (neurite-like protrusion)；例如像是神經母細胞瘤 (Neuro-2a)、誘導性嗜鉻細胞瘤神經 (PC12) 與胚胎瘤細胞分化的神經 (P19)，但是因為這些系統所表現的特性都無法完全涵蓋真正神經細胞的特性，因此建立一個初代神經培養系統是必要的。而我們選擇了初代背根神經節神經細胞 (primary dorsal root ganglion neuron culture) 作為此研究的主軸。初代背根神經節神經細胞擁有神經突生長快速、屬於周邊神經系統與神經突可再生等特性。在此研究中，我們專注於研究初代背根神經節細胞於植物化學素 (phytochemical) 及奈米鑽石 (nanodiamond) 之應用。 薑黃素是一種植物化學素，目前已知具有對抗多種疾病的潛力，例如：阿茲海默症與帕金森氏症。先前的研究顯示以神經生長因子誘導的PC12神經 (NGF-induced PC12 neurons) 為實驗材料，薑黃素會造成劑量相關性的神經突伸長。但是在促神經基因Ascl1 誘導的P19 神經 (proneural gene Ascl1-induced P19 neurons) 中卻造成神經突有劑量相關性的縮短。我們認為PC12與P19這兩種細胞產生的神經突對於薑黃素有相反的表現型，可能源自於周邊神經系統與中樞神經系統本質上的差異。在本實驗中，我們發現薑黃素對於初代背根神經節神經細胞的神經突生長以及細胞存活率沒有明顯的影響，但是對於初代皮層神經細胞 (primary cortical neurons) 的神經突生長及細胞存活率有劇烈的抑制效果。這些結果顯示薑黃素確實在不同神經系統具有不同的作用，但是確切原因需待進一步的查證。 近年來，螢光奈米鑽石 (fluorescent nanodiamond) 是一個非常受到矚目的奈米材料，因為其具有奈米級大小及所發出的螢光不會有光漂白現象，因此非常有潛力能夠發展成為生物探針。在本研究中，我們使用初代背根神經節神經細胞當作素材，進行奈米螢光鑽石的毒性測試，發現並不會對神經細胞的存活率造成影響，但是神經突的生長受到抑制。在活細胞影像實驗中，可以觀察到神經突的生長錐 (growth cone) 在碰觸到奈米螢光鑽石顆粒的時候，有退縮或轉向的現象，因此我們推論，神經突的縮短是因為奈米螢光鑽石的空間障礙所造成。

Primary neuron cultures have been widely used as a powerful tool to answer questions in various fields. Even though there are several models that could be obtain more easily, for example neuroblastoma cells (Neuro-2a), induced-pheochromocytoma neurons (PC12) and embryonal carcinoma differentiated neurons (P19), although these models have capability to form neurite-like formation that performing some properties of real neurons, however, all of these models are distinct to real neurons by lacking particular cellular or molecular features. Owing to these reasons, primary dissociated neurons culture has been chosen for this research. Primary dissociated dorsal root ganglion (DRG) neurons culture has characteristics as rapid neurite growth, derived from peripheral nervous system (PNS), and the ability to regenerate after injury. In this study, we established the dissociated DRG neuron culture and focused on studying the function of phytochemical and nanodiamond on neurite outgrowth and neuronal toxicity. Curcumin has been shown to exhibit a plethora of anti-disease potentials, such as anti-Alzheimer’s disease and anti-Parkinson’s disease potentials. In nerve growth factor (NGF)-induced PC12 cells, curcumin application resulted in longer neurites. Interestingly, this is different from the phenotype we observed in proneural gene Ascl1-induced P19 neurons. Curcumin application resulted in a dosage-dependent reduction in neurite length. We hypothesized that the opposite phenotypes is caused by the intrinsic difference between neurons from the CNS and from the PNS. This is because we also observed a reduction of neurite length in curcumin-treated cortical neurons (CNS), but an increase in DRG neurons (PNS). In recent years, fluorescent nanodiamond (FND) has become a promising new type of nanomaterial for biomedical applications. Nanodiamonds are fluorescent and do not photobleach or photoblink, these properties made them useful as biosensors. In this research, FND application showed no neuronal toxicity but caused a dosage-dependent neurite length reduction in primary dissociated DRG neurons culture. We hypothesize that the neurite reduction of DRG neurons was caused by the spatial obstacle of FND, and this was supported by the time-lapse live cell imaging.