| 摘要: | 外泌體經研究證實在未來再生醫學治療中是具備可信且極具影響力的，在細胞間的訊息因子傳遞中有效發揮作用，從而有助於發現癌症發病機制和過程，使其在疾病診斷與治療研究發展中逐漸受到矚目。現今已有愈來愈多實驗開始進行外泌體相關的研究，然而，以外泌體為基礎的研究快速擴展但是卻對於萃取外泌體的方法缺乏共識及標準量化，因此現今找出適合萃取出外泌體的方法至關重要。目前，外泌體樣品的萃取已有了幾種方法，超高速離心法是最常用的技術，但反覆地進行超高速離心會破壞外泌體的結構和生物學完整性。我們評估了使用超高速離心法和超微薄膜過濾合併粒徑篩析法從牙髓間葉幹細胞中萃取出的外泌體。首先，我們直接比較了兩種方法萃取外泌體的能力，以整合性且有系統性的方式比較與分析外泌體的特性、進行標準量化、特性分析與蛋白質體分析。我們經由超高速離心和超微薄膜過濾合併粒徑篩析法皆成功萃取出了牙髓間葉幹細胞外泌體，然而，超高速離心法在萃取外泌體的產量方面超過了超微薄膜過濾合併粒徑篩析法，但超微薄膜過濾合併粒徑篩析法之樣本顆粒尺寸分布更為平均。接著我們更進一步評估與比較兩種不同萃取方法之牙髓間葉幹細胞外泌體對於牙周韌帶幹細胞(PDLSCs) 的增殖能力、細胞遷移和骨分化能力的影響，隨著外泌體劑量的增加，效果也更加顯著。為了提高在臨床的應用價值，我們仍需要更進一步對外泌體膜蛋白的種類及功能、基因表達，以及可能的信息傳遞途徑進行更深入的研究。
Exosomes have been proven to be credible and influential in future regenerative medicine treatment selection.It plays an effective role in the transmission of information factors between cells,contributing to the discovery of cancer pathogenesis and processes so that it has gradually attracted attention in the development of disease diagnosis and treatment research.At present, more and more experiments have begun to conduct exosome-related research.However,exosome-based research has expanded rapidly,but there is a lack of consensus and standards for the method of isolating exosomes.Therefore,it is crucial to find a suitable method for the isolation of exosomes.There are several methods for the isolation of exosome samples now.Ultracentrifugation is the most used technique,but repeated ultracentrifugation can destroy exosome structure and biological integrity.We evaluated the isolation of exosomes from dental pulp mesenchymal stem cells using ultracentrifugation and ultrafiltration combined with size exclusion chromatography.We directly compared the ability of the two methods to isolate exosomes,comparing and analyzing their exosome characterization,standard quantification,characterization,and proteomic analysis in an integrated and systematic manner.Both ultracentrifugation and ultrafiltration combined with size exclusion chromatography were successful in isolating exosomes.Ultracentrifugation isolates exosome yields higher,while the sample distribution particles of ultrafiltration combined with size exclusion chromatography have more average size. Next, we evaluated and compared the proliferation ability,migration,and osteogenesis of periodontal ligament stem cells (PDLSCs) when co-cultured with dental pulp mesenchymal stem cells exosomes (DPSCs-exos) obtained through two different isolation methods.Moreover,as the dosage increased,the effects became more significant.To enhance the clinical utility,we will further evaluate and compare the two different isolation methods of dental pulp mesenchymal stem cells exosomes (DPSCs-exos)-the types and functions of membrane proteins,various gene expression,and possible discussion of message transmission pathways. |
