殼聚醣是常用的支架材料，具有促進癌症細胞群聚成球狀並提升幹細胞的特性。為了可以模擬體內的微環境，因此我們利用電紡絲技術製備出殼聚醣奈米纖維絲，藉由奈米纖維的疊加形成3D孔洞結構。此外，為了進一步提升癌症幹細胞的選殖效果，我們將殼聚醣電紡絲以細胞外基質玻尿酸進行改質，並與殼聚醣膜做為對照。由生長曲線結果發現，玻尿酸改質的電紡絲因其近似腫瘤微環境(niche)環境，會使細胞生長數率略微減緩，但是相較於膜上的細胞，絲上的細胞生長速率有大幅的提升。而在抗藥性實驗結果證實，玻尿酸改質所篩選的細胞其化療的耐受度有大幅的提升。藉由即時聚合酶反應(qPCR)發現從改質的電紡絲上取下的細胞具有的細胞幹性、上皮間質轉換、基質降解能力、血管新生與抗藥性等基因表現均有提升。而這些結果也可以透過侵入性與細胞遷移實驗中得到驗證。最後我們利用流式細胞儀分析，發現與所篩選的細胞其癌症幹細胞相關的CD24及CD44表面標誌比例有大幅提升，證實我們所發展的殼聚醣奈米纖維接枝玻尿酸的確能提升癌症幹細胞的比例。此基材將所擴增篩選的細胞將有助於將來在抗癌藥物的開發和癌症治療的研究。;Chitosan is a frequently used material for scaffold fabrication because it can promote cancer cells to form spheroid and enhance stem cell-relative characteristics. To mimic in vivo microenvironment, chitosan nanofibers were prepared using electrospinning technology, and their superposition can form a 3D porous structure. To effectively promote cancer stem cell adhesion, hyaluronic acid (HA) was grafted to electrospun chitosan fibers, and HA grafted chitosan films were applied as the control groups. The growth curve analysis demonstrated that HA modification reduced proliferation rates of surface cells because of its similarity to the cancer stem cell niche. On the other hand, cells grew on the fibers were faster than those on the films. The results of drug resistance examinations indicated that the chemotherapy tolerance of the cells collected from HA modified fibers was greatly improved. The levels of gene transcription determined by quantitative polymerase chain reaction (qPCR) indicated that the cells collected from HA modified fibers demonstrated high stemness, epithelial–mesenchymal transition, matrix degradation, angiogenesis, and drug resistance abilities. These results were consistent to their superior performances in colony formation, migration, and matrix invasion. Finally, flow cytometry analysis was applied to identify cell makers. The results indicated that the screened cells highly expressed the cancer stem cell-related makers CD24 and CD44, suggesting that the use of HA grafted chitosan fibers indeed increased the ratio of cancer stem cells. We expect these selected cells should be applicable to the development of anticancer drugs and the research of cancer therapy.
