神經幹細胞在不同高分子薄膜上行為之研究

Abstract

在研究中，神經幹細胞從14-15天大的Wistar大鼠胚胎大腦皮質被分離出來，並培養於無血清、含有20 ng /ml bFGF生長因子的培養基，使其增生成神經細胞團。另外，利用相轉換法中的乾式法，將幾丁聚醣、聚甲基丙烯酸甲酯和聚偏二氟乙烯製備成無孔洞的緻密型薄膜。經過6天的增生培養，將神經幹細胞團培養在幾丁聚醣、聚甲基丙烯酸甲酯和聚偏二氟乙烯三種高分子基材上，分別培養在無血清、含有20 ng /ml bFGF生長因子的培養基或是有含10﹪胎牛血清、不含bFGF生長因子的培養基中。 將神經幹細胞團培養在三種高分子基材五天中，利用光學顯微鏡觀察細胞的行為變化，並進行一連串的實驗。從光學照片中，可以觀察到神經幹細胞團在無血清、含有20 ng /ml bFGF生長因子的培養基的條件下，會隨著培養基材的不同，而有行為及型態上的差異。然而，在有含10﹪胎牛血清、不含bFGF生長因子的培養基中，神經幹細胞團培養於不同基材上卻都表現出相同的行為：細胞團中的細胞會往外遷移。而從細胞免疫螢光染色結果可以看出，無論在有無血清的培養基中，神經幹細胞在三種高分子基材上都可以分化出神經細胞、星狀細胞以及寡樹突細胞，且血清對於神經幹細胞團在三種基材上分化出來的細胞類型並沒有顯著的影響。 從結果中可以推論，或許是因為血清中的蛋白質沈澱在基材上，使不同的基材都被同樣的蛋白質覆蓋住，因此基材對神經幹細胞的行為影響幾乎不見，血清有助於神經幹細胞團的貼附，且有些細胞會從細胞團中遷移到有覆蓋蛋白質的基材上。

In this study, 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 20 ng /ml bFGF for 6 days. Then chitosan, poly (methyl methacrylate) (PMMA), and poly (vinylidene fluoride) (PVDF) were used as substrates to culture neurospheres under serum-free medium in the presence of 20 ng /ml bFGF or under medium with 10﹪FBS. The substrates were prepared by dry process of phase inversion method and the resulting structure of all substrates was nonporous and dense. After culturing the neurospheres on the substrates for 5 days, the neurospheres were observed by an optical electron microscope and underwent a series of experiments. From phase contrast pictures, it was found that under serum-free condition, the neurospheres adapted different morphologies and behaviors on different substrates. However, under medium with 10﹪FBS, the neurospheres exhibited the same behaviors on different substrates: the cells in the neurospheres migrated outward. Further, in the result of indirect immunocytochemical staining we found that the neurospheres could differentiate into neurons, astrocytes, and oligodendrocytes on all of the three substrates with or without the serum. Moreover, the presence or the absence of the serum in the medium did not influence the differentiation pathway of cells in the neurosphere. From the experimental results, it was concluded that same set of proteins in the FBS precipitated and covered the surfaces of each of the substrates; thus the influences of different substrates on the neurospheres almost disappeared. Further, the cells in the neurosphere exhibited the tendency to migrate outward to get in touch with the proteins that covered the substrates.