中心粒在牙齒生長發育及牙髓幹細胞的神經引導中所扮演的角色

Abstract

牙齒的生長和神經發育在再生醫學中被視為極為重要的議題。牙齒的生長取決於琺瑯質和牙本質之間的相互作用，進而形塑牙齒的結構。而神經發育則會影響許多涉及牙齒損傷及修復的議題。在早期的牙齒胚胎階段，神經與牙齒的發育過程會透過不同的訊號路徑相互連接，例如Wnt和Hedgehog等訊號路徑。而細胞內的胞器---中心粒，參與了許多細胞分化和影響細胞方向移動的過程。當中心粒相關基因突變，導致中心粒發生異常時，會產生神經性的系統疾病像是亨丁頓舞蹈症（Huntington''s Disease）和原發性小頭畸形症（Primary microcephaly），也可能有會口腔結構異常像是第一型口腔顏面手指症候群（Oral-facial-digital syndrome type1）、缺牙、牙釉質發育不全等情形發生。我們的假設是牙齒的生經生長和牙髓幹細胞在發育過程中受神經的吸引而遷移，中心粒在此過程中影響細胞，另外在牙齒的生長發育過程中牙釉質的形成也和中心粒有關。因此我們的研究將分成兩個部分來討論，第一個部分探討中心粒對牙齒神經生長和遷移的影響，第二個部分則著重在牙齒發育的過程中，中心粒在造釉母細胞（ameloblast)的作用和變化。 本次研究透過小鼠神經組織和人類牙髓幹細胞的共培養來觀察幹細胞受神經吸引的影響造成細胞遷移的過程，將中心粒在免疫螢光染色後，利用共聚焦顯微鏡觀察它們的位置。同時為瞭解去除中心粒對細胞的影響，故而添加中心粒抑制劑（Centrinone）來觀察細胞活性的變化。另外為了研究中心粒在牙齒生長過程的變化，將C57BL/6的健康小鼠臼齒進行組織切片染色和免疫螢光染色，觀察出生後1、3、5、7、9天的組織和細胞內的中心粒變化。其中特別比較了第九天健康小鼠臼齒和中心粒相關蛋白缺失的小鼠其組織和細胞內的中心粒變化。 從研究結果可以發現，隨著神經組織和牙髓幹細胞之間距離的減少，牙髓幹細胞的中心粒靠近神經組織的比例也會越來越多。而與神經組織共培養的距離越近，也會增加牙髓幹細胞向神經組織的遷移速度。相較之下，單獨培養或和纖維母細胞共同培養的牙髓幹細胞則會表現出較弱的遷移能力，顯示神經組織是會對牙髓幹細胞有吸引的影響。另外添加中心粒抑制劑也會影響細胞的遷移和中心粒的聚合過程，不過一旦沖洗掉中心粒抑制劑後，中心粒的聚合和細胞遷移的能力都會恢復正常。而在觀察不同天數的小鼠臼齒組織中，造釉母細胞和琺瑯質（enamel）在出生後一到九天發生了顯著變化。造釉母細胞中的中心粒表現出動態的位移變化，從細胞核靠向琺瑯質的邊緣。同時也發現牙釉質的厚度和中心粒的數量有正向的關聯性。而在正常小鼠和中心粒異常的小鼠比較中可以發現，中心粒相關蛋白缺失的老鼠其牙釉質的厚度、造釉母細胞的長度以及中心粒的分佈存在明顯差異，顯示CPAP缺陷對牙齒發育的影響。 中心粒在影響細胞運動和極化有兩種可能的情況，一種是中心粒位於細胞中心，但是細胞核卻是向後移動，像是NIH3T3細胞。另一種則是細胞和中心粒都會向前移動，像是牙髓幹細胞。因此牙髓幹細胞對神經組織的吸引力可能有助於牙胚對神經的引導生長，從而幫助牙髓幹細胞分化為神經。但具體機制還需要進一步的體內和體外研究來證實。而有關牙齒的生長發育過程，從研究中證實了成釉細胞內的中心粒對分泌期的琺瑯質生成有正面的影響。反之，中心粒相關蛋白缺失的時候會造成中心粒的破壞，進而影響造釉母細胞的形態和功能，這樣的發展與在原發性小頭畸形患者中觀察到的牙釉質發育不全相類似。不過仍須進一步的研究來闡明分子機制以及與造牙本質細胞（odontoblast）中心粒的潛在相互作用。總括而言，從中心粒的角度來了解牙齒和神經的生長發育對於臨床醫療工作，尤其是修復牙齒損傷和促進神經再生，具有重要的啟示。期望未來的研究能繼續深入探討中心粒的分子機制，為更有效臨床治療方法的開發奠定基礎。

The growth and neural development of teeth are regarded as crucial topics in regenerative medicine. Tooth growth is influenced by the interaction between enamel and dentin, shaping the overall tooth structure. Concurrently, neural development plays a significant role in addressing issues related to tooth damage and repair. During the early stages of tooth embryogenesis, the processes of neural and tooth development are intricately connected through various signaling pathways, such as Wnt and Hedgehog. The organelle within cells, known as the centriole, participates in numerous cellular differentiations and processes that influence cell directional movement. Mutations in centriole-associated genes leading to abnormal centriole function are implicated in neurological disorders such as Huntington''s Disease and Primary Microcephaly, as well as oral structural abnormalities like Oral-facial-digital syndrome type 1, missing teeth, and enamel hypoplasia. Our hypothesis is that innervation of tooth is correlated with the neural attraction of dental pulp stem cells and centrioles play important role during the cell migration process. In addition, the development of enamel during tooth growth is also correlated with centrioles. This study is divided into two main parts to investigate these phenomena. The first part explores the impact of centrioles on neural growth and migration of dental pulp stem cells (DPSCs). The second part focuses on the role and changes of centrioles in ameloblasts during tooth development. Through co-culturing mouse neural tissue and human dental pulp stem cells, we observed the cellular migration process during neural atteaction. Immunofluorescence staining of centrioles, followed by observation with confocal microscopy, allowed us to understand their positions. To assess the influence of centrioles'' removal, a centriole inhibitor (Centrinone) was added, and changes in cell activity were monitored. Additionally, examining the tissue sections of healthy C57BL/6 mouse molars at various postnatal days (1, 3, 5, 7, 9) provided insights into centriole changes during tooth growth. A comparative analysis of healthy and CPAP/p53 double knockout mice on the ninth day highlighted differences in enamel thickness, ameloblast elongation, and centriole distribution, illustrating the impact of CPAP deficiency on tooth development. Results revealed that as the distance between neural tissue and DPSCs decreased, more DPSCs exhibited centrioles near neural tissue, correlating with increased migration towards neural tissue. Co-culturing with neural tissue enhanced DPSCs'' migration, while DPSCs cultured alone or with fibroblasts showed weaker migration. The addition of Centrinone affected cell migration and centriole aggregation, with recovery upon Centrinone removal. Observing tissue changes in mouse molars at different postnatal days indicated significant alterations in ameloblasts and enamel. Dynamic shifts in centriole localization within ameloblasts, moving from the cell nuclei towards enamel, were observed. Positive correlations between enamel thickness and centriole quantity were noted. Comparative analysis of normal and CPAP/p53 double knockout mice highlighted substantial differences in enamel thickness, ameloblast elongation, and centriole distribution, emphasizing the impact of CPAP deficiency on tooth development. In conclusion, gaining insights into the growth and neural development of teeth from the perspective of centrioles holds significant implications for clinical healthcare, particularly in repairing tooth damage and promoting neural regeneration. Further research is needed to delve into the molecular mechanisms of centrioles, laying the foundation for the development of more effective clinical treatment methods.