臺灣博碩士論文加值系統

研發以βIII-tubulin為定向分化策略來增加幹細胞分化神經元
摘要
神經幹細胞提供很好移植治療退化性神經疾病的細胞來源，許多研究顯示神經幹細胞不僅能夠在體內或體外分化成神經元細胞，而且具有神經生理的特性和功能。但現今所有分化成神經幹細胞的分化條件下，都有混合了的多種細胞的不純細胞群體、或無法大量量產星狀細胞或神經元細胞等共同的問題，深深影響在臨床上利用幹細胞治療退化性神經疾病的普遍性。本研究主要透過結合細胞譜系篩選法和迫使分化法等兩種方法來研發以βIII-tubulin為定向分化策略來增加幹細胞分化神經元細胞。研究主軸分為三方向：第一，以βIII-tubulin啟動子為主的細胞譜系篩選法、第二，利用calbindin-1神經元分化誘導因子迫使幹細胞分化法、及第三，結合細胞譜系篩選法和迫使分化法等兩種方法來達到更有效率的神經元分化。本研究結果顯示：第一，在神經分化的過程，βIII-tubulin啟動子不僅能將報告基因啟動及表現，而且應用該啟動子為主的細胞譜系篩選法成功在神經幹細胞(MEB5)分化成可被MAP2抗體辨識之神經元。另外，我們也成功透過dsRNA的方法及siRNA的方法來干擾NRSF等策略來達到增加約30%神經元分化的細胞群體。第二，神經元分化誘導因子迫使幹細胞分化法中，我們大量表達calbindin-1基因在神經幹細胞中，比對照組有增加超過40%的幹細胞被迫使分化成可被MAP2抗體辨識之神經元分化，且這些神經元擁有相對較長和多的神經突、以及抑制了幹細胞往星狀細胞的分化。最後，我們利用
結合calbindin-1基因作為神經元分化誘導因子及神經保護因子之迫使幹細胞分化法與βIII-tubulin啟動子之細胞譜系篩選法有效增加迫使幹細胞分化成神經元。此法不僅增加分化成神經元的專一性，更有誘導因子與啟動子間之類似「正回饋」互動來增加分化成神經元的效率。

ABSTRACT
ENRICHMENT OF STEM CELL DERIVED NEURON BY βIII-TUBULIN TARGETED LINEAGE SELECTION

Neural stem cells are the prime candidate for studying the neuro-genesis and as a donor sources for the cell replacement therapy in several neurological diseases. Study indicated that neural stem cell has the potential to proliferate in vitro and differentiate into neuronal-like cells with biochemical and electro-physiological properties. Although the neural stem cell provided an unlimited source of neuron, the current differentiation protocol could be difficult to achieve the sufficient amount of homogenous neurons for the therapeutic cell transplantation. Therefore, we employed a hybrid approach of “lineage selection” and “forced differentiation” for enriching the stem cell derived neuron with the lineage restricted genes and promoters. To accomplish our goal, we studied on the possible use of the three difference approaches: (1) βIII-tubulin as a promoter based lineage selection, (2) Calbindin-1 as a neuronal inducer in forced differentiation and (3) βIII-tubulin and calbindin-1 as a hybrid approach for neuronal differentiation. Our results shown: first, the characterized the minimal promoter of βIII-tubulin was sufficient for up-regulating the reporter gene in the differentiated cells and was applied in the MEB5 differentiation and the GFP expressed cells were always recognized by the MAP2 antibody. Furthermore, we demonstrate the effectiveness of the both NRSE dsRNA and NRSF siRNA strategies in mediating the neuronal differentiation. Second, we employed the over-expression of calbindin-1 for promoting the neuronal differentiation of neural stem cell through tetracycline and βIII-tubulin regulated system. The exogenous overexpressed calbindin-1 was significantly increased the neuronal differentiation in MEB5 with more than 70% of transfected cells had been identified by MAP2 in compared with 30% in control group. These were more primary neuritic outgrowth and longer neuritic length in the calbindin-1 expressed cells, whereas it greatly reduced the glial differentiation from 30% to 9%. Finally, we employed the conditional inducible calbindin-1-GFP expression by βIII-tubulin promoter during neuronal differentiation; this novel approach was not only using calbindin-1 as a neuronal regulator and protector, but also using βIII-tubulin as neuronal specific lineage selection for enrichment of the neuronal differentiation. Our data shown that this approach was enriched the specificity of the neuronal differentiation, and a proposed positive feedback facilitates the neuronal differentiation.

TABLE OF CONTENTS

ACKNOWLEDGEMENT iv
ABSTRACT v
LIST OF ABBREVIATION ix
LIST OF FIGURES xiii
LIST OF TABLES xvi
1 INTRODUCTOIN 1
2 SPECIFIC AIMS 8
3 RESEARCH DESIGN 9
4 MATERIALS AND METHODS 15
4.1 Cell culture 15
4.2 Construction of expression vectors 16
4.3 Transfection and GFP reporter assay 19
4.4 Dual luciferase reporter assay 20
4.5 Reverse transcription chain reaction analysis 21
4.6 Immuno-cytochemistry assay 22
4.7 Western blot analysis 23
4.8 Measurement of neurite outgrowth and process 24
5 RESULTS 25
5.1 Characterization of neural stem cell from NSC E14.5 and MEB5 cells 25
5.2 Enrichment of stem cell derived neuron by βIII-tubulin promoter targeted lineage selection 26
5.3 Improved stem cell derived neuron by altering the NRSF 28
5.4 Enriched stem cell derived neuron by over-expression of tetracycline regulated calbindin-1 28
5.5 βIII-tubulin and Calbindin-1 as a hybrid approach for neuronal differentiation 30
6 DISCUSSION 31
7 CONCLUSION 41
REFERENCES 42

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