褪黑激素與維他命C對於乳牙幹細胞幹性的影響

Abstract

乳牙幹細胞（SHEDs）是間質幹細胞（MSCs）的一種，乳牙幹細胞有自我更新的能力及分化成多種細胞的潛力，包括分化成成骨細胞、軟骨細胞和脂肪細胞等等。由於分化的潛力，乳牙幹細胞能促進組織再生，加上乳牙幹細胞易於取得及分離，他被認為是再生醫學和幹細胞療法的良好細胞來源。相較於其他間質幹細胞，例如牙髓幹細胞（DPSCs）、骨髓幹細胞（BMSCs）和臍帶幹細胞（UC-MSCs）等等，乳牙幹細胞可以在試管內實驗中存活較久的時間，且乳牙幹細胞比其他間質幹細胞能表現出更高的增值能力及分化的潛力。在幹細胞療法或幹細胞的研究當中，繼代培養是取得一定數量幹細胞的主要方法，然而，連續的繼代培養會導致幹細胞退化，進而使幹細胞的幹性下降，而幹性包括了幹細胞的增殖能力和分化潛力。這種幹性的退化將影響幹細胞的品質和有效性，並最終阻礙其在幹細胞治療中的實際應用。根據先前研究顯示，褪黑激素及維生素C可以在培養細胞時調節細胞的活性。因此，為了研究褪黑激素及維生素C在幹細胞的退化和幹性之間的有效性，我在乳牙幹細胞的培養中分別加入褪黑激素及維生素C並培養至第二代及第五代，接著利用3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) 技術檢測乳牙幹細胞的細胞存活率，進而比較在培養中有無加入褪黑激素及維生素C，對於幹細胞因為繼代培養而退化的情形。除了檢測細胞存活率之外，我也利用Alizarin Red S染色技術研究了乳牙幹細胞分化成成骨細胞的狀況，並分別比較了乳牙幹細胞繼代培養到第三代及第五代，在有無褪黑激素及維生素C的狀況下的分化情形。然而，根據先前的幹細胞研究顯示，乳牙幹細胞在培養時對環境很敏感，任何高濃度的外來因子都會影響他的狀態，而這會導致單獨加入褪黑激素或維生素C時受到濃度的限制。因此，我會進一步利用不同濃度比例將褪黑激素及維生素C合併來培養乳牙幹細胞，並研究乳牙幹細胞在合併培養後的細胞生長情形。而此研究結果顯示，在培養乳牙幹細胞時分別單獨加入褪黑激素及維生素C，可以延緩幹細胞的退化並提高幹細胞的增殖能力及成骨分化潛力，而在加入褪黑激素與加入維生素C之間的組別則無顯著差異。至於將褪黑激素及維生素C以不同濃度比例合併後，此研究結果顯示可提高乳牙幹細胞的的增殖能力。這些結果可能對進一步的幹細胞研究和未來治療的應用有用。

Stem cells from human exfoliated deciduous teeth (SHEDs) possess self-renewal capabilities and multi-lineage differentiation potential. Due to their differentiation potential, SHEDs promote tissue regeneration and are considered a valuable cell source in regenerative medicine and stem cell-based therapies. Compared to other mesenchymal stem cells (MSCs), SHEDs exhibit prolonged in vitro survival, a higher growth rate, and stronger differentiation potential. In stem cell research and therapies, in vitro serial subculture is the primary method for obtaining a sufficient quantity of stem cells. However, stem cells tend to deteriorate during serial subculture, resulting in diminished stemness, which includes their proliferative capacity and differentiation potential. This deterioration adversely affects the quality and efficacy of SHEDs, ultimately hindering their practical application in stem cell therapy. Previous studies have demonstrated that melatonin and vitamin C can modulate the functional activity of cells during culture. To assess the effects of melatonin and vitamin C on the stemness of SHEDs during deterioration, cell proliferation rates at passages 2 (P2) and 5 (P5) were compared using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Additionally, the osteogenic differentiation abilities at passages 3 (P3) and 5 (P5) were compared among the control, melatonin, and vitamin C groups. Studies have shown that SHEDs are sensitive to their culture environment, and high concentrations of exogenous factors can adversely affect their state. Consequently, the individual treatments of melatonin and vitamin C are limited in concentration. This study also investigates the combined treatment of melatonin and vitamin C at different concentration ratios. The combination treatment aims to address the limitations of individual treatments and enhance the stemness of SHEDs. The results indicated that separate treatments with melatonin and vitamin C significantly enhanced the proliferation rate and osteogenic differentiation ability of SHEDs compared to the control group, with no significant difference between the effects of melatonin and vitamin C. However, the combined treatment of melatonin and vitamin C significantly augmented the proliferation rate of SHEDs compared to the control group. These findings may be valuable for further stem cell research and future therapeutic applications.