乙烯乙烯醇共聚物薄膜接枝短鏈胜肽於有血清狀態下培養神經幹細胞行為之研究

在此研究中，神經幹細胞從14~15天大的Wistar大鼠胚胎大腦皮層中分離出，培養於無血清、含有20 ng/ml bFGF生長因子的培養基，繼代培養1~2代後，使其增生成神經幹細胞團。此外研究一開始選用了6種基材在無血清狀態下培養神經幹細胞團，2種親疏水性不同的乙烯乙烯醇共聚物薄膜(EVAL44、EVAL27)、以化學方法接枝板素(Laminin)上的一段短胺基酸鏈段GYIGSR於兩種EVAL薄膜表面(ECY44、ECY27)、基材TCPS塗佈聚離氨酸(PDL)和Laminin。經由光學顯微鏡觀察，細胞團的細胞在無血清狀態培養下，不同基材上的爬行距離和生長型態會有所差異，其中以可促進細胞貼附和移動的Laminin最具顯著效果，不論是塗佈或接枝於基材上，細胞爬行距離最遠且樹突(dendrite)生長緻密，而PDL對於細胞爬行的效果最差，但突觸生長的細而長；另外使用免疫螢光染色法來分析神經幹細胞團分化情形，在無血清狀態下，細胞分化只受到基材的影響(低細胞密度下, d=75 spheres/cm2)，對於接枝YIGSR和塗佈Laminin，神經幹細胞分化為神經元(neuron)的比例較改質前的基材下降，另外有趣的是EVAL27可提高促進分化為neuron的比例，而每種基材上分化為astrocyte比例大略都相同。最後在基材間挑選其中兩種基材做進一步研究，爬行距離最遠的ECY44和最短的PDL。先將細胞團培養於基材ECY44和PDL上，在不同時間點下添加入胎牛血清進入培養基中來影響細胞分化和爬行，因為血清不但可提供養分促進細胞生存也可以增進細胞往外移動的能力，所以在不同時間點下添加血清，藉以觀察基材和血清兩者存在對於細胞分化和移動的影響。

關鍵字

大腦皮層神經幹細胞；乙烯乙烯醇共聚物；板素；胎牛血清；生醫材料；免疫螢光染色

並列摘要

In this research, at first the neural stem cells were isolated from the cerebral cortices of 14~15 days’ embryonic Wistar rats, and propagated under serum-free medium in the presence of bFGF at a concentration of 20 ng/ml. After subcultured first to twice times, neurospheres were cultured on 6 kinds of substrates under serum-free medium, hydrophobic and hydrophilic poly (vinyl alcohol-co-ethylene) membrane (EVAL44 and EVAL27); EVAL44 and EVAL27 membranes were modified by oligopeptide sequence, YIGSR, derived from laminin (ECY44 and ECY27); TCPS substrate coated with poly-D-lysine (PDL) and laminin. The neurospheres cultured on various kinds of substrates under serum-free medium, the behaviors and migration ability of the cells were quite different. From the phase contract pictures, no matter coating or crosslinking on the substrate, laminin or YIGSR had apparent effect to the migration ability of cells and the dendrites grew pre-pressingly. The PDL had the worst effects on migration ability and the dendrites of cells grew thin and long. Subsequently, in the result of indirect immunocytochemical staining, the differentiation of cells was only effected by the substrates under serum-free condition (at low concentration, d=75 spheres/cm2). And we found that the cells on modified polymer membranes, ECY44 and ECY27, or laminin-coated TCPS, the ratio of the neurospheres differentiated into neurons were less than unmodified. Among these substrates, EVAL27 could enhance the differentiation of neurons. And the ratio of the astrocytes was quite similar in these substrates. Finally, according to the migration distance of these substrates, we chosen EVAL44 (the farthest) and PDL-coated TCPS (the shortest) as the substrates to investigate the differentiation and migration of neurospheres under the presence of fetal bovine serum (FBS) into the medium. FBS not only can offer nutrients to promote cells to survive but also can promote the ability that the cells move outside. Therefore, at several of time, we added FBS into culture medium and observed the influence of cells by both of substrate and serum-soluble factor.

並列關鍵字

cortical neural stem cell ； poly (vinyl alcohol-co-ethylene) ； laminin ； fetal bovine serum ； biomaterial ； indirect immunocytochemical staining

參考文獻

1. Freshney, R.I., Culture of animal cells : a manual of basic technique. 2000.

2. Hung, C.H. and T.H. Young, Differences in the effect on neural stem cells of fetal bovine serum in substrate-coated and soluble form. Biomaterials, 2006. 27(35): p. 5901-8.

3. Song, H., C.F. Stevens, and F.H. Gage, Astroglia induce neurogenesis from adult neural stem cells. Nature, 2002. 417(6884): p. 39-44.

4. Reynolds, B.A. and S. Weiss, Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science, 1992. 255(5052): p. 1707-10.

5. Gage, F.H., et al., Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc Natl Acad Sci U S A, 1995. 92(25): p. 11879-83.

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乙烯乙烯醇共聚物薄膜接枝短鏈胜肽於有血清狀態下培養神經幹細胞行為之研究

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