許旺細胞培養液誘導乳牙幹細胞神經分化之動態培養研究

Schwann Cells Cultured Medium Induce Neuronal Differentiation of SHED Cells under Dynamic Culture

潘俞靜(Yu-Jing Pan)

指導教授 ： 蘇文達

國立臺北科技大學/工程學院/生物科技研究所/碩士(2014年)

https://doi.org/10.6841/NTUT.2014.00269

摘要

人類乳牙幹細胞(Stem cells from human exfoliated deciduous teeth, SHED)可作為組織工程使用細胞之替代來源，因為它具有分化成多種類型細胞的能力。許旺細胞在周邊神經系統中扮演著重大的角色，為周邊神經主要的結構及功能性的細胞，並且會釋放神經營養物質幫助神經再生。本研究探討利用老鼠許旺細胞(Rat Schwann cells, RSCs)所培養過的培養液誘導SHEDs神經分化的效益。
本實驗將SHEDs及RSCs單獨培養在聚二甲基矽氧烷(poly dimethyl siloxanes, PDMS )材料中，靜態培養是利用培養過RSCs的培養液來每2天更換一次SHEDs培養所需的培養液；動態是將每個PDMS載體以矽膠管串聯50mL培養液，利用蠕動幫浦進行灌流式培養。實驗以qPCR檢測神經細胞相關基因(Nestin、β-III tubulin、GFAP、γ-enolase)的表現，並利用共軛焦螢光顯微鏡觀察其細胞形態變化與特定神經分化相關蛋白質(Nestin、β-III tubulin、GFAP、γ-enolase)的表現。
SHEDs 靜態誘導培養7天時，出現類似神經球(neurospheres)的細胞聚集；動態培養則可提前在第3天出現細胞聚集。之後將神經球與RSCs培養液再培養，發現神經球可以進一步分化成類神經元及神經膠質細胞。後續利用qPCR以及免疫螢光染色分析，在靜態與動態培養時，神經特定的基因與蛋白質的表現量都比平面培養的SHEDs高。從本實驗的結果表示，RSCs培養所分泌在培養液中的神經營養物質，不論在靜態或動態串聯誘導培養，都能有效的幫助乳牙幹細胞走神經的分化，未來可運用在神經損傷的修復。

關鍵字

乳牙幹細胞 ； 許旺細胞 ； 聚二甲基矽氧烷 ； 動態培養 ； 神經分化

並列摘要

Stem cells from human exfoliated deciduous teeth (SHEDs) have been considered as alternative sources of tissue engineering because of their multi-potential to differentiate into multiple cell lineages. Schwann cells (SCs) are primary structural and functional cells in peripheral nervous system and play a crucial role in peripheral nerve regeneration by releasing of neurotrophic factors. In this study, we have evaluated the neural differentiation potential effects of SHEDs by Rat Schwann cells (RSCs) cultured medium.
In our study, SHEDs and RSCs were cultured on polydimethylsiloxanes (PDMS) matrix individually, and were divided into static and dynamic culture. Static culture was changed every two days by aspirating off cultured RSCs medium; Dynamic culture was series connected under perfusion culture. We measured neural differentiation capacity of SHEDs by qPCR, and detected neuronal differentiation associated gene (Nestin, β-III tubulin, GFAP, γ-enolase). The morphology of SHED cells on PDMSs are observed by using confocal microscopy as well as the measure the immunocytochemistry of neural protein for Nestin, β-III tubulin, GFAP, and γ-enolase.
Based the results, SHEDs cells grown on RSCs cultured medium in static culture formed neurospheres after 7 days; Dynamic culture could early form neurospheres after 3 days culture. After incubation with RSCs cultured medium, these neurospheres further differentiated into a cell population that contained neuron or neuroglial. Immunefluorescent staining and qPCR measuring showed the nestin, β-III Tubulin, GFAP, and γ-enolase of neural marker on the differentiated cells.These results showed higher expression than those cells cultured planar dish and indicate that RSCs cultured medium under both culture methods can induce the neural differentiation of SHEDs cells, and show a new therapeutic possibility for nerve damage repair.

並列關鍵字

Stem Cells from Human Exfoliated Deciduous Teeth ； Rat Schwann cell ； Polydimethylsiloxanes ； Dynamic culture ； Neural differentiation

參考文獻

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