幾丁聚醣基材對於前十字韌帶細胞與滑膜類間葉幹細胞共培養系統影響之研究

Abstract

本研究分別從人類前十字韌帶及其滑膜分離出細胞，進一步由流式細胞儀與分化試驗證明從滑膜分離出來的細胞具有類似間葉幹細胞的特性，我們將其稱之為滑膜類間葉幹細胞。 幾丁聚醣是一個天然、生物可分解、生物可相容性、無生物毒性且經由美國食品藥品管理局認可的多醣類，本研究使用其作為培養細胞之基材。當前十字韌帶細胞與滑膜類間葉幹細胞分別種植於幾丁聚醣基材上時，細胞都會呈現懸浮且聚集的型態。而後我們將此兩種細胞在幾丁聚醣基材上且利用直接共培養系統，探討兩細胞之間的交互作用，並以TCPS作為實驗的對照組。 再者，我們先把兩種細胞分別用螢光標定後，以倒立式螢光顯微鏡去觀察兩種細胞在這兩種基材上共培養後的型態；更進一步，我們再利用曠時攝影去觀察兩種細胞的型態在基材上隨時間變化的情形。 最後，我們使用流式細胞分選儀將在基材上做共培養後的兩種細胞分選出來，並分別抽取RNA後，以即時定量聚合酶連鎖反應觀察兩種細胞在不同的基材上共培養後，各自基因的變化情形。

In this study, we isolated cells from human anterior cruciate ligament (ACL) and synovial membrane. Further we demonstrate that these cells from synovial membrane are similar with mesenchymal stem cells (MSCs) by flow cytomerty analysis and differentiation assay. So we can call the cell is synovium-derived stromal cells (SDMSCs). Chitosan is a natural, biodegradable, biocompatible, non-toxicity and the U.S. Food and Drug Administration (FDA) approval of polysaccharides. In this study, chitosan was used to as substrate. When anterior cruciate ligament fibroblasts (ACLFs) and SDMSCs were cultured on chitosan substrate, the cells morphology was suspension and accumulation. Then we culture ACLFs and SDMSCs on chitosan substrate at direct co-culture system to explore the interaction between two cells, and TCPS as the control group. Furthermore, we labeled the two cells by CellTrackerTM and used the inverted fluorescence microscope to observe the two cells pattern on substrate after direct co-cultured. Besides, we used time-lapse to observe the two cells pattern when the two cells direct co-culture on substrate dependent with time. Finally, we used the flow sorter system to separate the two cells on the substrate after direct co-culture. After RNA was extracted, we use real-time quantitative polymerase chain reaction (RT-PCR) to observe genes expression of ACLFs and SDMSCs on different substrates after direct co-culture.