幾丁聚醣對前十字韌帶纖維母細胞與滑膜細胞的老化與增生表現之影響

Abstract

本研究分別從人類膝關節中的滑膜組織及前十字韌帶分離出細胞，進一步將其放置在幾丁聚醣上進行培養，培養幾天後觀察細胞的增生及老化表現的改變。 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。當前十字韌帶與滑膜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。讓細胞在這個懸浮的狀態下持續三天後，再回種到TCPS (Tissue culture polystyrene) 上，並以沒有做任何處理的細胞作為對照組，探討細胞型態及功能的改變。 接著，我們從兩個層面來探討細胞功能，第一是增生能力，我們建立細胞生長曲線、運用MTT ([3-[4,5-dimethylthiahiazo-2-yl]-2,4-dipheny- tetrazolium bromide]) assay和BrdU (5-bromo-2’-deoxiuridine) 生長偵測組做ELISA定量，互相佐證其細胞增生能力的改變。第二則是老化表現，我們運用Senescence-associated --galactosidase (SA--gal) 染色的方法來觀察SA--gal活性是否有被抑制。 最後，我們蒐集各種不同年齡病人檢體所獲得的細胞，進而探討本細胞處理方法對於不同年齡病人間的效果。

In this study, we isolated primary cells form human synovial membrane and human anterior cruciate ligament. First, we seed the cells on the chitosan substrate and culture for a few days. After this treatment, we studied the change of cell function. Chitosan is a natural, biodegradable, biocompatible, non-toxic and U.S. Food and Drug Administration (FDA) approved polysaccharide. In this study, chitosan was used as the coating substrates. When synovial cells and anterior cruciate ligament fibroblasts were cultured on chitosan, all cells became suspended and aggregated into spheroids. After three days, we seeded the cell spheroids on TCPS (Tissue culture polystyrene), and studied change of cell proliferation and cell senescence expression. Furthermore, we established the population doubling curve, used MTT ([3-[4,5-dimethylthiahiazo-2-yl]-2,4-dipheny- tetrazolium bromide]) assay and BrdU (5-bromo-2’-deoxiuridine) assay, to study the cell proliferation change. We used the inverted microscope to observe the cell morphology and used senescence-associated-β-galactosidase (SA-β-gal) to study the cell senescence expression. Finally, we collected cells from various patients to study the relationship between the effect of chitosan treatment and patient’s age.