利用無標記之多變數曲線解析法所組成之拉曼光譜和影像研究大腸癌細胞於細胞分裂時之細胞組成和分布

Abstract

細胞分裂為細胞繁殖過程中最重要的一環，一旦調控機制出問題，便有機會轉為癌細胞。對於細胞分裂的過程，分子層級的研究不只可以使我們更了解生命延續的機制，更有機會發展出預防及治療癌症的方法。目前，許多生物分析方法已經可將胞器或細胞組成顯影於細胞之中。然而，許多的方發皆須破壞細胞或額外加入染劑，因此會使正常的細胞功能受損，或是破壞細胞本身。拉曼光譜學是一項極具影響力的技術，可以在不添加染劑的狀況下研究細胞的組成及分布。在此實驗中，我們利用激發波長為632.8 nm之共軛焦拉曼光譜研究HCT116大腸癌細胞，並將細胞內成分如蛋白質和脂質的分布顯影於細胞中。我們取得細胞間期、細胞分裂期以及細胞群集之細胞空間解析光譜，相較於傳統的光譜分析方式，利用多變數曲線解析法 (multivariate curve resolution, MCR)，成功地分析出不具背景訊號且高解析度的蛋白質及脂質光譜，所組成的拉曼影像也具有較好的影像對比度。從拉曼影像中，我們發現蛋白質在所有細胞中大致上為均勻分布，但脂質在細胞中卻集中於正在分裂的細胞之分裂溝。經此結果可推測，在細胞分裂時，某些脂質滴或是富含脂質的胞器會集中於分裂溝處，在細胞分裂的過程中扮演重要的角色。此外，我們也發現兩種不同的自體螢光(F1和F2)，F1的最大放射波長為667 nm，並出現於細胞的周圍；然而，F2的最大放射波長為704 nm，並且出現於分裂細胞之分裂溝處，和上述所提之脂質的分布極為相似。由分布位置不同可推斷出此兩種自體螢光應源自於不同分子，且F2可能在細胞分裂時於分裂溝才會增加。

Mitotic cell division is a crucial biological process related to the reproduction of living cells. Dysregulation of this event may lead to tumor formation. Detailed molecular-level investigation of cell division will not only deepen our understanding of the fundamental question of how life continues, but it could also open up new possibilities of diagnosis/prognosis of cancer cells. Many biological techniques have been used to study the distributions of organelles and cellular components during cell division. However, the conventional methods usually require destructive procedures or external labeling and hence are prone to alter normal cell functions or cause sample damage. Raman spectroscopy is a powerful alternative for investigating cellular components and their distributions in vivo and in a label-free manner. In this work, we used 632.8-nm excited confocal Raman microspectroscopy to study colon cancer cells (HCT-116) and visualize the distributions of intracellular components such as proteins and lipids. We obtained space-resolved Raman spectra of interphase and M-phase cells, and cell clusters in which many cells are adherent to each other, and constructed Raman images by employing a multivariate data analysis method known as multivariate curve resolution (MCR). MCR analysis of the Raman hyperspectral data has successfully yielded background-free intrinsic spectra of proteins and lipids and their spatial distributions with much higher image contrast than the conventional univariate approach can provide. The MCR Raman images show that the distribution of proteins looks nearly homogeneous in all types of cells, whereas lipids are localized at the cleavage furrow of dividing cells. The present results indicate that some phospholipids or phospholipid-rich organelles are recruited and accumulated at the cleavage furrow during cell division, playing an important role in regulating cytokinesis. Furthermore, we found two different autofluorescence components (denoted F1 and F2). F1 with an emission maximum at 667 nm appears at the peripheral part of all cells, whereas F2 (at 704 nm) is localized in the cleavage furrow of M-phase cells in a very similar manner to the lipid component. This difference in distribution suggests that the two autofluorescence components arise from different chemical species in the colon cancer cell, and F2 may increase when cells are in the process of dividing.