幾丁聚醣對老化幹細胞之影響

Abstract

人類間質幹細胞是一種可自我新生且具有分化能力的細胞群，其可誘導分化成硬骨、軟骨及脂肪等細胞。再者，該類細胞可自成人體中分離出後再誘導成目標細胞群，所以在細胞治療領域上具有相當的優勢。但能自人體取出的間葉幹細胞數量有限，一般常見使用基因和病毒轉殖技術增進其體外增殖、演緩其分化和老化的性質。在先前研究中我們已發現若將邁入老化前的人類包皮纖維母細胞培養於幾丁聚醣基材上或於含有幾丁聚醣的環境中，皆能有效地使進入老化前的細胞延長複製代數、提升增殖能力和減少老化表徵。因此本研究進一步探討幾丁聚醣是否能調節人類間質幹細胞的增殖與老化相關功能表現。由數據結果發現將幾丁聚醣直接加入培養環境中，可使邁入老化前的細胞提升細胞活性以及增生的能力。進一步使用衰老相關的β-半乳糖苷酶染色法測定細胞的老化程度，可發現幾丁聚醣僅對細胞粒線體活性較低的細胞株有延緩老化及增加細活性的效果。另外，在老化相關蛋白的表現上，使用幾丁聚醣處理的細胞其p53與p21的蛋白質表現量較未處理的組別低，推測其具有延緩老化之效果。最後，以分化實驗驗證間葉幹細胞的分化特性，成骨分化實驗中發現有處理之組別較未處理的組別分化能力較差，而成脂分化能力則是50 µg/mL組別有顯著下降。但整體而言處理過的細胞經長期培養後總可複製代數與無處理組別無顯著差異。

Human bone mesenchymal stem cells (hBMSCs) are capable of self-renewal and differentiation to the osteogenic, chondrogenic, and adipogenic lineages. HBMSCs can be easily isolated from adults’ bone marrow or adipose tissue so they have potential to cell therapy. Our previous research has found that chitosan can enhance proliferation, delay aging and extend the number of replication times on pre-aging human foreskin fibroblasts effectively. Compared with the method of genetic engineering and virus transfection, adding chitosan into culturing medium directly has great potential to expand cells in vitro since it is easy to operate. In this study, we found that adding chitosan into medium can increase the cell viability and proliferation ability of the pre-aging hBMSCs. The expression of senescence-related proteins p53 and p21 of these cells is lower than the untreated group. Moreover, the treated cells had lower osteogenic differentiation ability than the untreated cells, and the adipogenic differentiation ability was significantly reduced in 50 µg/mL chitosan treated cells. However, through the senescence-associated β-galactosidase (SA-β-gal) assay, we found that chitosan only reduced the amount of SA-β-gal positive cells with low mitochondrial activity. There was also no significant difference in the total population doublings among treated and untreated groups after long-term cultivation.