纖維母細胞培養液誘導乳牙幹細胞分化成肝臟細胞的研究

Hepatic differentiation of SHED cells using fibroblasts cultured medium

陳曉薇(Xiao-Wei Chen)

指導教授 ： 蘇文達

國立臺北科技大學/工程學院/生化與生醫工程研究所/碩士(2014年)

https://doi.org/10.6841/NTUT.2014.00101

摘要

乳牙幹細胞可被用來取代成體幹細胞在組織工程上的應用，具有多功能分化的潛能，可以分化成多種細胞譜系，纖維母細胞NIH-3T3通常會與肝臟細胞進行共培養，已知可以改善培養環境，因為NIH-3T3會分泌細胞信號因子、ECM還可以促使肝細胞間產生相互作用力，最近文獻研究發現NIH-3T3與肝臟細胞共培養，因為增加細胞貼附力與間隙連接，使得細胞更穩定。本實驗中，我們將乳牙幹細胞以纖維母細胞NIH-3T3分泌的營養因子誘導細胞分化，評估乳牙幹細胞分化成肝細胞的潛力。
在我們的研究中，將乳牙幹細胞接種在含有三維結構的蠶絲蛋白多孔洞載體，每兩天更換3T3培養過的培養液到乳牙幹細胞中，持續28天。我們透過定量PCR測量所被誘導的幹細胞其肝細胞特定基因的表現(AFP & ALB)，並利用免疫螢光染色觀察肝細胞特有的蛋白(AFP & ALB)表現。利用酵素免疫分析法(ELISA)來測量白蛋白分泌活性和尿素合成量。結果指出 三維結構的蠶絲蛋白載體會比平面培養盤在肝細胞誘導能力更具效果，顯示出利用含有三維結構的蠶絲蛋白多孔洞載體更換3T3培養過的培養液來培養幹細胞，確實可以誘導乳牙幹細胞往肝細胞分化，提供了一個新款修復肝臟損傷治療的可行性。

關鍵字

乳牙幹細胞 ； NIH-3T3纖維母細胞 ； 分化 ； 肝細胞

並列摘要

Stem cells from human exfoliated deciduous teeth (SHEDs) have been considered as alternative sources of adult stem cells because of their multi-potential to differentiate into multiple cell lineages. It is known that co-culture improves culture environment, since it induces production of cell signaling factors and extracellular matrixes, and/or formation of heterotypic cell–cell interaction. In particular, recent studies of co-culturing hepatocytes with 3T3 cells revealed that the expression of hepatocyte functions is stabilized by an increase of direct heterotypic cell–cell contacts (cell adhesion and gap junction). In this study, we have evaluated the hepatic differentiation potential of SHEDs by NIH-3T3 cultured medium inducing.
In our study, SHEDs seeded in the 3D silk fibroin porous scaffold, and changed the cultivated medium every two days, for 28 days. We measured the hepatic gene expression (AFP and ALB) by qPCR and observed the protein expression of AFP and ALB by immune-fluorescent staining. The albumin secretion activity and urea synthesis were determined by performing an enzyme-linked immunosorbent assay (ELISA). These results showed higher expression than those cells cultured planar dish and indicate that NIH-3T3 cultured medium under 3D silk fibroin porous scaffold can induce the hepatic differentiation of SHEDs cells, and show a new therapeutic possibility for liver damage repair.

並列關鍵字

stem cells from human exfoliated deciduous teeth ； NIH-3T3 cells ； differentiation ； hepatocytes

參考文獻

6. 林峰輝, 組織工程之生醫材料. 台灣醫學, 2001.p677-680

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