間質幹細胞分化成神經細胞過程之蛋白質表現量差異與型態變化

Changes in protein expression and morphology of mesenchymal stem cells in neuronal differentiation

蕭震宇(Chen-Yu Hsiao)

指導教授 ： 李文乾

國立中正大學/工學院/化學工程學系/碩士(2013年)

摘要

許多研究指出，從成人體內骨髓取出的間質幹細胞，具有體外分化成為神經細胞的能力。本實驗室過去利用Neurobasal medium做為基礎培養基，添加B27，音波狀蛋白(Sonic Hedgehog，簡稱SHH)、鹼性成纖維細胞生長因子(Basic fibroblast growh factor，簡稱bFGF)及成纖維細胞生長因子8(fibroblast growth factor 8，簡稱FGF8)，針對SWH代謝路徑剃除之間質幹細胞(簡稱me_SWH hMSCs)，分化成為神經細胞。本研究根據觀察，me_SWH hMSCs經過五天分化培養下，細胞長度較未分化前之細胞，平均增長了1.52±0.09倍。同時，增長倍數大於2倍以上的細胞比例，約為整體的13.3%。me_SWH hMSCs細胞在Neurobasal medium中，會隨者時間而凋亡，造成可分化成神經細胞之存活率不高。相對初始培養細胞數目，在未添加SHH、bFGF、FGF8等生長因子刺激分化下，培養至第六天之細胞存活率僅為19.6%。再添加生長因子之誘導培養基(Neuronal induction medium，簡稱NIM)中，培養第六天之細胞整體存活率則為59.5%。本實驗室在過去的研究中，針對me_SWH hMSCs分化至神經細胞時蛋白質體進行分析，發現到有3個蛋白發生明顯增減。此三個蛋白分別為uKHC(全名ubiquitous kenesin heavy chain，又稱Kinesin-1 heavy chain， 縮寫KIF5B)、PKC-ε(全名Protein kinase C epsilon type)、NME1(又稱Nm23-H1，全名NME/NM23 nucleoside diphsphate kinase 1)。經過細胞分化誘導成神經細胞後，可以發現到uKHC與PKC-ε兩者蛋白隨者時間增加。相較於未誘導的細胞，第五天誘導分化的神經細胞，uKHC和PKC-ε兩者蛋白質之表現增加率分別為154.7%和159.6%。然而，誘導分化五天後的細胞，NME1蛋白表現率卻下降至35.7%。根據文獻，uKHC為形成軸突之組成動力蛋白，而PKC-ε與太多訊號傳遞路徑有關。未來的研究包括利用磁性奈米顆粒接NME1的抗體，送入分化細胞中，來先研究NME1與何種蛋白結合，以探討NME1的作用機制。

關鍵字:SWH、間質幹細胞、神經細胞、分化、UKHC、NME1、PKC-ε

關鍵字

PKC-epsilon ； KIF5B ； 神經分化 ； 神經細胞 ； 間質幹細胞 ； NM23-H1

並列摘要

Many studies have proposed human mesenchymal stem cells (hMSCs) from bone marrow can differentiate into neuron cells in vitro. In this study, we used SWH-hypermethylated (me_SWH) hMSCs which were modified with targeting DNA hypermethylation of genes in Salvador/Warts/Hippo (SWH) pathway as the model. Previous studies indicated that me_SWH hMSCs can be induced for neuronal differentiation by using a neurobasal medium supplemented with B27, Sonic hedgehog (SHH), fibroblast growth factor 2 (bFGF) and fibroblast growth factor 8 (FGF8). After 5-day differentiation, the cell length of me_SWH hMSCs increased 1.52 ± 0.09 folds in comparison with undifferentiated cells. Approximately 13.3% of the differentiated cells became double or larger in cell length. However, me_SWH hMSCs cells were caused to apoptosis in the neuronal induction medium. The survival rate was 59.5% after induction for 6 days. If growth factors SHH, bFGF, and FGF8 were not included in the medium the survival rate was only 19.6%. Three proteins: ubiquitous kinesin heavy chain (uKHC or Kinesin-1 heavy chain, KIF5B), protein kinase C epsilon type (PKC-ε), and NME/NM23 nucleoside diphosphate kinase 1 (Nm23-H1 or NME1 in short), were found differentially expressed by neuronal induction. Results from western bolting indicate that both PKC-ε and KIF5B increased with time of induction. Compared to the undifferentiated cells, the expression levels of PKC-ε and KIF5B increased 154.72% and 159.6%, respectively, by the induction for neuronal differentiation for 5 days. However, after the differentiation for five days, the Nm23-H1 level was reduced to 35.7% of its original level. According to the literature, KIF5B is the composition of the axon dynein and PKC-ε participates in several major signal pathways. Future studies include the use of magnetic nanoparticles bound with NME1 antibody, and delivery of magnetic nanoparticle-antibody conjugates into differentiated cells to study the proteins interacted with NME1 and the mechanism of interactions.

Keywords: mesenchymal stem cells, SWH-hypermethylated MSCs, neuronal differentiation, UKHC, PKC-ε, Nm23-H1

並列關鍵字

neuronal differentiation ； MSC ； NM23-H1 ； PKC-epsilon ； KIF5B

參考文獻

Abeliovich, A.,and Hammond, R.(2007) Midbrain dopamine neuron differentiation: Factors and fates. Developmental Biology, (304), 447 – 454

Akita, Y.(2002) Protein Kinase C-E (PKC-ƒÃ): Its Unique Structure and Function, J Biochem, (132), 847-852

Arthur, A.,(2008) Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells, ( 26-7),1787-1795.

Bae, K. S., Park, J. B., Kim, H. S., Kim, D. S., Park, D. J., and Kang, S. J. (2011) Neuron-like differentiation of bone marrow-derived mesenchymal stem cells. Yonsei Med J, (52), 401-412.

Baghbaderani B.A., Behie L.A., Sen A., Mukhida K., Hong M, and Mendez I. (2008). Expansion of human neural precursor cells in large-scale bioreactors for the treatment of neurodegenerative disorders. Biotechnology Progress, (24), 859-870,