探討雷射光刺激對神經母細胞瘤造成軸突回縮之效應

Neurite Retraction Caused by Blue Light Stimulation on Neuroblastoma

劉婕婷(Chieh-Ting Liu)

指導教授 ： 李超煌 ； 林俊元

國立中正大學/理學院/物理學系/碩士(2014年)

摘要

細胞運動對於研究細胞行為是非常重要的主題。目前在細胞運動的研究領域中，大多數研究團隊是對細胞施以化學藥物並觀察細胞對此刺激之反應，然而物理性刺激引發的細胞運動就比較不為人所了解。一個有趣的研究主題是神經細胞軸突(neurite)之伸長或收縮運動對物理性刺激的反應，有助於科學家開發新的神經修復或再生(neurogenesis)技術。

在本論文中，我們利用波長473 nm的藍光光點刺激老鼠神經母細胞瘤之軸突，觀察到細胞軸突會朝向胞體(soma)回縮，或者是胞體朝向光照位置移動而縮短軸突長度。利用本實驗室發展之非干涉式廣視野光學測繪術對細胞膜的表面形貌進行測繪，我們同時測量了神經母細胞瘤的細胞膜粗糙度。我們將藍光光點引發的軸突回縮現象與細胞膜粗糙度互相對照，發現在細胞被藍光照射引發軸突縮短時，胞體上的細胞膜粗糙度有上升的趨勢，可能代表胞體的硬度降低以促進軸突縮短。而在對細胞分別以blebbistatin及monastrol進行前處理後，被藍光照射之細胞軸突將不再回縮，部分軸突會斷裂並殘留於基板上。由此可知藍光刺激所引發的軸突回縮現象由第二型肌動蛋白(myosin II)及第五型驅動蛋白(kinesin V)協同主導，且若其中一項蛋白被抑制後，軸突將失去運動能力，無法進行軸突回縮。

相較於原子力顯微鏡，非干涉式廣視野光學測繪術使用光作為探測工具，屬於非侵入式的量測方式。此方式可量測到細胞膜細微的表面高度變化，對外在刺激具有極為敏銳的感測度，因此提供除了細胞的外在形貌、螢光表現、物理性質之外，另一種細胞診斷參數。我們也利用此技術量測神經母細胞瘤受類澱粉樣蛋白纖維(amyloid-beta fibril)刺激後之細胞膜粗糙度，發現細胞膜粗糙度會下降。受類澱粉樣蛋白纖維處理後的細胞存活率稍低，細胞內活性氧物質含量較高。然而，實驗組與控制組細胞在存活率上並無顯著差異，而細胞膜粗糙度卻有顯著差異，代表在類澱粉樣蛋白刺激下，細胞膜粗糙度具有較高的敏感度。這是以細胞膜粗糙度作為細胞診斷參數的另一個實例。

關鍵字

細胞膜粗糙度 ； 非干涉式廣視野光學測繪術 ； 藍光刺激 ； 軸突回縮

並列摘要

The motion of cells is among the most important cellular behaviors. Most researchers investigate the cell motion under chemical stimulations. In contrast, the cell motion induced by physical stimulations has not been fully understood. A very interesting cell motion related to physical stimulations is the growth or retraction of neurites from a neuronal cell. Sufficient knowledge about the neurite motion under physical stimulations could help scientists to develop new techniques for neurogenesis.

In this thesis, we used a focal spot of 473 nm blue light to illuminate the neurite of a mouse neuroblastoma, and observed that either the neurite retracted to the soma, or the soma moved toward the illuminated site, hence the length of neurite was shortened. We also measured the membrane roughness of neuroblastoma under the blue light stimulation by using the non-interferometric wide-field optical profilometry (NIWOP) technique developed in our lab. We found the membrane roughness increased while the neurite was retracting under the illumination of blue light. The increase in membrane roughness could result from the decrease of cell stiffness on the soma, which facilitates the retraction of the neurite. In addition, we observed that the light-induced neurite retraction was partially impeded by either blebbistatin or monastrol treatment. We thus conjecture that the blue light-induced neurite retraction is driven by both myosin II and kinesin V. Inhibiting one of the two motor proteins can largely reduce the retraction ability of neurites.

In comparison with atomic force microscopy, the NIWOP technique is non- intrusive measurement because it uses light to probe the sample. NIWOP can detect the small changes of cell membranes, which could be very sensitive to external stimulations. Membrane roughness can thus be a cellular diagnostic parameter in addition to cell morphology, intensity of fluorescently labeled proteins, and other physical properties. We also measured the membrane roughness of neuroblastoma under the treatment of amyloid-beta fibril. The amyloid-beta fibril reduced membrane roughness, decreased the cell viability, and increased the intracellular reactive oxygen species. However, the difference in cell viability is insignificant compared with the change in membrane roughness. This is an example of using membrane roughness as a diagnostic parameter for quantifying the effects of amyloid-beta fibril.

並列關鍵字

blue light stimulation ； membrane roughness ； non-interferometric wide-field optical profilometry ； neurite retraction

參考文獻

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