神經幹細胞在組織培養皿和聚乙烯醇基材上不同行為之探討

Abstract

本研究為探討大鼠胚胎之大腦皮層神經幹細胞在組織培養皿(TCPS)和聚乙烯醇(PVA)基材上不同行為之研究。文獻中顯示神經幹細胞在聚乙烯醇基材上無法存活，且細胞增生有受到抑制的現象，另一方面，神經幹細胞可存活於一般組織培養皿上，且會持續增生，我們想了解培養在上述兩種基材中的神經幹細胞，同樣是懸浮狀態，為何會面臨不同命運，聚乙烯醇基材如何影響細胞造成細胞死亡。 針對本實驗目的我們做了以下四種假設，首先，我們觀察到培養在聚乙烯醇基材上的神經幹細胞幾乎沒有增生的現象，因而假設當細胞增生能力受到抑制後，細胞會走向分化，由實驗結果顯示細胞在死亡之前沒有經歷分化的路徑。其二，一般而言，神經幹細胞培養在組織培養皿上再移到多聚賴氨酸(PDL)基材上會貼附並且分化，然而我們發現培養在聚乙烯醇上的細胞再移到聚賴氨酸基材上卻呈現懸浮狀態，因而假設細胞無法貼附的原因是因為細胞膜表面負電減少，經由細胞電泳結果顯示，培養在聚乙烯醇上的細胞其表面電性確實減少，我們推測可能與細胞膜表面的唾液酸(sialic acids)有關。其三，我們進一步假設細胞無法貼附的原因和細胞凋亡(apoptosis)早期，磷脂絲胺酸(PS)會由膜內外翻至膜外有關，因此使用Annexin V和TUNEL方法檢測細胞凋亡的情形，結果顯示聚乙烯醇造成細胞死亡是經由細胞凋亡的路徑，但細胞的路徑和機制尚未明瞭。另一方面，我們加了細胞凋亡抑制劑，希望減少細胞死亡的數目，但實驗結果不如預期。其四，我們假設加了抗細胞凋亡的藥物沒有效果是由於聚乙烯醇釋放到培養液中，將細胞包覆，造成細胞的凋亡。我們將培養液(medium)預先泡在聚乙烯醇基材上一天後，拿此培養液去進行細胞培養，發現細胞也會死亡，表示其高分子會些微釋放到培養皿中，將細胞包覆，造成細胞死亡。

In this study, we explored the different behavior patterns of embryonic rat cortical neural stem cells on two biomedical polymer substrates¬¬—tissue culture polystyrene (TCPS) and polyvinyl alcohol (PVA). A previous study had found that neural stem cells could not survive on PVA and the material might inhibit their proliferation. By contrast, neural stem cells can continuously proliferate and survive on TCPS. Hence, the purpose of this study was to investigate what were the factors that determined the different behaviors of neural stem cells between TCPS and PVA given that they are both suspended in culture. Based on the purpose of this study, four hypotheses were generated. First, since the proliferation of neural stem cells was not observed on PVA, it was reasonable to assume that neurospheres could undergo differentiate as proliferation was inhibited. According to our assessment, neural stem cells did not differentiate on PVA before cell death. Second, neurospheres in general would attach to poly-D-lysine (PDL) substrate, where they are transferred to after being pre-cultured on TCPS. However, it was found that neurospheres failed to attach to PDL as they were pre-cultured on PVA before being transferred to PDL. Therefore, a proposed hypothesis was that the surface charge of neurospheres reduced probably. Apparently, the decrease of surface charge of neurospheres was measured by cell electrophoresis. It maybe related to negateively charged sialic acids of NCAM on cell membrane. Third, to confirm the translocation of phosphatidylserine (PS) from the inner to outer membrane, which represented to cell apoptosis, annexin V and TUNEL assays were applied. The results had verified that apoptosis pathway was the way through which cell death was induced when being cultured on PVA. Further results showed that additional anti-apoptotic agents had no significant effect on the number of damaged cells when they were cultured on PVA. Therefore, the mechanism of the apoptotic pathway on neural stem cells remained unexplored. Lastly, we hypothesized that such a cellular apoptosis might the result of PVA slightly releasing to medium. Cell death also occurred when they were cultured in medium pre-treated with PVA substrate. It indicated that neural stem cells were wrapped around by the release of PVA, which caused cell death.