| 摘要: | 根據流行病學的研究發現，牙周病是一種慢性發炎疾病且在世界各地皆有超過百分之九十的成年人都患有不同嚴重程度的流行比例。牙周病的發生主要是由食物殘渣、脫落的口腔上皮細胞、唾液和口腔細菌所構成的生物薄膜(牙菌斑)所致，且在口腔內微生物與宿主的免疫反應交互作用下，導致齒槽骨的吸收與牙齦發炎，引發牙周組織的破壞及牙周囊袋的形成，最終因齒槽骨不斷的流失而造成牙齒的鬆動及脫落。小球藻 (Chlorella sp.)，又稱綠球藻，含有豐富的蛋白質、胜?、胺基酸、醣類、脂質、維生素和礦物質等，近??小球藻的各種萃取物已開始在許多疾病的預防與治療等體內/外試驗中被廣泛研究。本論文作者以粗萃後的普通小球藻 (Chlorella vulgaris) 蛋白經中空纖維管柱(Hollow Fiber Filter Module)分離純化後，分別得到五種組別的胜?(Short-Chain Peptides)，包括 <1 kDa、1-3 kDa、3-10 kDa、10-30 kDa、>30 kDa等小球藻胜?組 (CVPs)，以探討小球藻不同分子量功能性胜?，對於抗牙周病致病菌及抑制牙齦炎之體內/外藥理活性評估與分子作用機制。研究結果顯示，分子量 3-10 kDa 與 10-30 kDa (10 mg/mL)的小球藻胜?分別能夠抑制轉醣鏈球菌 (Streptococcus mutans) 、血鏈球菌 (Streptococcus sanguis)及牙齦??單胞菌 (Porphyromonas gingivalis) 的生長進而達到抗菌作用。同時也可以透過降低牙周病菌生物膜的形成與其對細胞的貼附能力，提供對口腔上皮細胞(SCC-4)的保護作用。在細胞試驗中，我們也證實了分子量1-3 kDa與3-10 kDa (0.01 和 0.03 mg/mL)的小球藻胜?不但提高了LPS誘導下受損的口腔上皮細胞及巨噬細胞 (RAW264.7)之存活率，也能達到抑制TLR4的表現量，並分別降低了細胞所產生的相關發炎因子如 IL-1β, TNF-α及IL-6。此外，在細胞劃痕試驗中觀察到小球藻胜? 3-10 kDa對口腔上皮細胞具有傷口癒合的作用，同時也具有抑制破骨細胞生成的功效。綜觀上述，特定分子量的小球藻功能性胜?對於改善牙周病初期與後期的可能作用機制與其抗菌、抗發炎、細胞修護、抗破骨細胞及調節因LPS所誘發的TLR4的表現量有著正關聯性。其次，本研究選用上述數據中抗菌效能最佳之小球藻胜?3-10 kDa進行第二類醫療器材(J.5090)之口腔抗菌凝膠雛形設計與開發。首先，以口腔黏膜易吸附之各類賦形劑、增稠劑、黏著劑、保濕劑及2% 與5% 添加比例之小球藻胜?3-10 kDa等製定抗菌凝膠配方(10 g/管)，並針對雛形凝膠進行酸鹼值測定，膠體機械強度，溶解速率，生物黏合度測試，以及抗菌、細胞毒性與動物實驗。其中，小球藻胜?3-10 kDa 抗菌凝膠表現了理想的基礎特性，包含平均值約6.2之偏中性酸鹼度，膠體機械強度>2000 cP (黏性單位)，24小時內>50%的殘留度，生物黏合度>0.02 N (拉力單位)，且分別能夠在24小時之內抑制大腸桿菌 (Escherichia coli)及金黃色葡萄球菌 (Staphylococcus aureus) >90% 的生長率，同時也在ISO 10993-5的測試中達到0-1級>80% 的細胞存活率。另外，動物實驗顯示，小球藻胜?3-10 kDa抗菌凝膠可有效降低由LPS加上S. sanguis及P. gingivalis所誘發的口腔病菌生成，與血清中單核細胞趨化蛋白-1 (MCP-1)的產生，值得作者更進一步的探討與研究，希冀未來能夠成為牙周病的潛在治療與替代方案。最後，本研究採用了小球藻胜?<30 kDa的綜合胜?組別(內含抗發炎作用為佳之小球藻胜? 1-3 kDa與最具抗菌作用之小球藻胜? 3-10 kDa)，開發出具抗牙周病菌附著功能之口腔保健口含錠(400 mg/錠)。從研究數據中得知，小球藻胜?<30 kDa的綜合胜?40 mg/mL具有抗菌以及抗生物膜的形成。同時，該綜合胜?組合物也可降低口腔病菌貼附於細胞的作用，並具有清除自由基DPPH+與ABTS+之抗氧化的能力。在此，作者希望透過上述實驗結果與所延伸設計、開發之雛型與產品，能夠對牙周病患者帶來更多的解決方案及創造出更多口腔健康的福音與福祉。
According to epidemiological research, periodontal disease (PD) is a chronic inflammatory condition affecting over 90% of adults worldwide to varying degrees. The disease is primarily caused by a biofilm (dental plaque) composed of food residues, sloughed oral epithelial cells, saliva, and oral bacteria. Interaction between oral microbiota and host immune responses leads to inflammation of the gingiva and absorption of alveolar bone, resulting in destruction of periodontal tissues and formation of periodontal pockets. Ultimately, continuous bone loss leads to tooth loosen and tooth loss. Chlorella sp., also known as green algae, is rich in proteins, peptides, amino acids, carbohydrates, lipids, vitamins, and minerals. In recent years, various extracts of Chlorella have been widely studied in vitro and in vivo for prevention and treatment in various diseases. In this study, the authors purified peptides from Chlorella vulgaris using Hollow Fiber Filter Module, resulting in five peptide groups (CVPs): <1 kDa, 1-3 kDa, 3-10 kDa, 10-30 kDa, and >30 kDa. The study aimed to investigate the pharmacological activities and molecular mechanisms of these Chlorella peptides in combating periodontal pathogens and inhibiting gingival inflammation. The results showed that CVP 3-10 kDa and 10-30 kDa (at 10 mg/mL) inhibited the growth of Streptococcus mutans, Streptococcus sanguis, and Porphyromonas gingivalis, thereby exerting antimicrobial effects. These peptides also protected oral epithelial cells (SCC-4) by reducing biofilm formation and adherence to cells. Cell experiments demonstrated that CVP 1-3 kDa and 3-10 kDa (at 0.01 and 0.03 mg/mL) improved the survival rates of LPS-induced damaged oral epithelial cells and macrophages (RAW264.7), inhibited TLR4 expression, and reduced inflammation factors IL-1β, TNF-α, and IL-6. Moreover, scratch assays revealed that CVP 3-10 kDa promoted wound healing in oral epithelial cells and inhibited osteoclastogenesis activity in macrophages. Overall, specific molecular weight Chlorella peptides showed potential mechanisms in improving early and advanced stages of periodontal disease, correlating positively with anti-bacterial, anti-inflammatory, cell repair, anti-osteoclastogenic properties, and modulation of TLR4 expression induced by LPS. Furthermore, based on the optimal anti-bacterial efficacy data, the study developed an oral anti-bacterial gel prototype (J.5090) using CVP 3-10 kDa. The gel formulation (10 g/tube) included various excipients easily absorbed by oral mucosa, and was evaluated for pH, viscosity (>2000 cP), dissolution rate (>50% residual within 24 hours), biological adhesion (>0.02 N), anti-bacterial efficacy against Escherichia coli and Staphylococcus aureus (>90% growth inhibition within 24 hours), and cytotoxicity (>80% cell survival in ISO 10993-5 testing). Animal experiments demonstrated that CVP 3-10 kDa gel effectively reduced oral pathogen production induced by LPS with S. sanguis and P. gingivalis, as well as serum monocyte chemoattractant protein-1 (MCP-1) levels. These findings warrant further exploration and research, aiming to potentially provide an alternative therapeutic approach for periodontal disease. Lastly, a comprehensive mixture CVP <30 kDa, combining anti-inflammatory peptides (1-3 kDa) and highly anti-bacterial peptides (3-10 kDa), was used to develop oral health lozenges (400 mg/tablet). This mixture demonstrated anti-bacterial and anti-biofilm formation abilities, reduced oral pathogen adherence to cells, and exhibited antioxidant capabilities against free radicals DPPH+ and ABTS+. In conclusion, through these experimental results and the development of prototypes and products, the authors hope to offer more solutions for periodontal disease patients and contribute to oral health and well-being. |
