奈米鑽石在胚胎癌幹細胞之神經分化作用的生物相容性及標定應用

Abstract

奈米鑽石是一種相當具有潛力的碳奈米材料，目前已被開發作為生物醫學的應用，例如藥物運送及生物標定。在本論文中，我們發現螢光奈米鑽石是一種具有生物相容性及可被檢測的奈米材料，可應用作為胚胎癌幹細胞之神經分化作用的標定。螢光奈米鑽石不會影響老鼠P19及人類NT2/D1兩種胚胎癌幹細胞株的細胞型態、存活率、生長能力及細胞凋亡。胚胎癌幹細胞在處理0.1至50 μg/ml的螢光奈米鑽石24小時後，細胞內的螢光強度會隨著螢光奈米鑽石之濃度增加而增高。螢光奈米鑽石會進入這些細胞中，螢光奈米鑽石會存在胚胎癌幹細胞之細胞質中，並與SSEA-1蛋白（幹細胞標誌）位於相同位置。然而，螢光奈米鑽石並不會顯著改變胚胎癌幹細胞中的SSEA-1蛋白表現。胚胎癌幹細胞可利用處理視黃酸而誘導細胞進行神經分化的作用。但在分化過程中的神經細胞，螢光奈米鑽石的存在並會不影響神經細胞的型態及TUJ1（神經細胞標誌）的基因與蛋白表現。此外，螢光奈米鑽石不會誘發神經分化過程中的細胞毒性與細胞凋亡。螢光奈米鑽石也不會誘發神經細胞細胞毒性及細胞凋亡。再者，螢光奈米鑽石可利用共軛焦顯微鏡與流式細胞儀進行這些分化的神經細胞的檢測及分析。綜合以上結果，我們的研究提供螢光奈米鑽石是一種具有生物相容性及生物影像的標定劑，可應用於幹細胞之神經分化過程的追蹤。

Nanodiamond (ND) is a promising carbon nanomaterial that has been developed for biomedical applications such as drug delivery and bio-labeling. In this study, we show fluorescent nanodiamond (FND) that is a biocompatible and detectable nanomaterial, which can be used for the labeling of neuronal differentiation in embryonic carcinoma stem (ECS) cells. FND did not alter cell morphology, viability, growth ability and apoptotic levels in both the mouse P19 and human NT2/D1 ECS cells. The fluorescence intensities were increased via a concentration-dependent manner by treatment with FND (0.1-50 μg/ml for 24 h) in these ECS cells. FND particles were taken into cells. FND was co-localized with SSEA-1 protein (stem cell marker) in the cytoplasm of ECS cells. However, FND treatment did not significantly alter the SSEA-1 protein levels in ECS cells. The neuronal differentiation of ECS cells was induced by retinoic acid (RA) treatment. The existence of FND in cells did not alter the neuronal cell morphology and the gene and protein expressions of TUJ1 (neuron marker). Besides, FND did not induce cytotoxicity and apoptosis during the process of neuronal differentiation. Also, FND did not induce cytotoxicity and apoptosis in neuronal cells. Furthermore, these differentiated neuronal cells carry FND particles, which can be detected by confocal microscope and flow cytometry. Together, our findings demonstrate that FND is a biocompatible and bio-imaging agent for the labeling and tracking in the neuronal differentiation of stem cells.