探討脂肪幹細胞以及BALB/c 3T3纖維母細胞在蠶絲布上的生長特性

Investigation of the properties of adipose-derived stem cells and BALB/c 3T3 fibroblasts grown on silk fabric-based scaffolds

林俐岑

疝氣 ； 蠶絲蛋白 ； 脂肪幹細胞 ； 纖維母細胞 ； 蠶絲布 ； 膠原蛋白 ； hernia ； fibroin ； adipose-derived stem cell ； fibroblast ； silk fabric ； collagen

清華大學分子醫學研究所學位論文

2017年

碩士

張晃猷

英文

疝氣是一種常見的疾病，為腹腔內的器官從正常的位置跑到不正常的位置被擠壓到腹腔外。而在疝氣修補手術中需植入修補網片，以重建及增強薄弱腹壁組織的強度，避免再次復發。大部分的修補網片是人工合成材料，這些材料可能會造成病患的異物感或引發免疫反應。蠶絲蛋白是一種天然聚合物，近年來在組織工程的應用非常廣泛。由於蠶絲有良好的力學特性以及生物相容性，利用蠶絲所編織成的布具有作為疝氣修補網片的潛力。本研究利用蠶絲布作為讓細胞生長的支架，並探討其免疫刺激性，以了解蠶絲布作為疝氣修補網片的可能性。首先，在溶解蠶絲蛋白或膠原蛋白處理或沒有處理過的蠶絲布上培養人類脂肪幹細胞以及BALB/c 3T3纖維母細胞，再利用 LIVE/DEAD 染劑觀察細胞在蠶絲布上的生長狀況。我們也利用 alamarBlue assay 進行細胞活性分析。結果顯示，經過溶解蠶絲蛋白處理過的蠶絲布細胞生長狀況最為良好。接著，我們測量脂肪幹細胞分泌的生長因子，如鹼性成纖維細胞生長因子以及肝細胞生長因子，結果顯示，跟細胞生長在培養皿中比較，在蠶絲布上生長的脂肪幹細胞會分泌較多的生長因子。我們也測試蠶絲布是否會誘導老鼠巨噬細胞RAW264.7分泌發炎因子TNF 及 IL-6，結果顯示，蠶絲布不會誘導巨噬細胞分泌 TNF 以及 IL-6。最後，我們測試蠶絲布的降解能力，將蠶絲布浸泡在細胞培養液中持續三週，觀察其重量變化，結果顯示，蠶絲布在細胞培養液中不容易被分解。綜合以上結果，蠶絲蛋白和膠原蛋白可作為良好基質提供細胞生長，而蠶絲布有良好的生物相容性，並不會引起發炎反應。未來，蠶絲布可作為良好的疝氣修補材料。

A hernia is a common disease occurs when an organ protrudes through a tissue holding it in the normal position. Surgeons use meshes to repair hernias. Most of the meshes are made of synthetic materials such as polyethylene, polypropylene, polyester and polytetrafluoroethylene that may easily lead to foreign body reactions. Silk fibroin from silkworm is a nature polymer applied widely in tissue engineering, possessing excellent mechanical property and biocompatibility. We proposed that silk fibroin has a potential to be used as a hernia repair mesh when knitted into a fabric. This study utilized silk fabric, obtained from a commercial source, as a scaffold to support cell growth. We investigated the characteristics of cells grown on the scaffold and determined whether the scaffold evokes an immune response. First, BALB/c 3T3 and adipose-derived stem cells (ADSCs) cultured on a silk fabric with or without dissolved fibroin or collagen coating were examined by LIVE/DEAD staining. Cell viability of BALB/c 3T3 and ADSCs was measured using alamarBlue assay. Results showed that silk fabric with fibroin coating improved BALB/c 3T3 and ADSCs growth. Furthermore, we found that basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) were produced in a higher level from ADSCs cultured on silk fabric with fibroin coating. Next, we tested TNF and IL-6 production from macrophage line RAW264.7 cultured on silk fabric. The result showed that silk fabric would not trigger TNF and IL-6 production from RAW264.7. Furthermore, silk fabric would not degrade in culture medium for several days. Therefore, silk fabric with fibroin or collagen coating can be developed into a novel hernia repair mesh in the future. To sum up, silk fibroin and collagen provide a suitable matrix for cell growth on silk fabric.

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