丙戊酸 (VPA) 是一種在臨床上被用來抗驚厥和穩定情緒的藥物。除了治療神經功能紊亂的功效以外, 最近的研究發現VPA可以導致癌細胞的凋亡及分化，增加胚胎幹細胞的分化，並在提升由成體細胞誘導成多功能幹細胞的效率。至今，VPA在幹細胞發育的作用並不十分清楚。在這項研究中，我們使用體外培養體系，來研究VPA在小鼠神經幹細胞，神經前體細胞和胚胎幹細胞的生物學效應。 / 實驗發現VPA可以抑制神經前體細胞株C17.2和神經幹細胞的增生。另一方面，VPA可以增加這兩類細胞分化形成神經元，但同時並沒有增加神經膠質細胞和少突膠質細胞的分化。另一方面，VPA抑制胚胎幹細胞向神經類細胞分化。實驗顯示這種抑制作用源自於VPA對胚胎幹細胞早期譜系分化的抑制。 / 胚胎幹細胞分化實驗顯示，VPA可以抑制胚胎幹細胞向神經外胚層譜系分化，並促進對中胚層和內胚層譜系的分化。經VPA處理後的胚體不能夠產生β-微管蛋白III陽性樹突狀突起。我們還發現，在含血清的培養液中分化的胚體會出現跳動的細胞群。此類細胞群在VPA組裡面的比例較高。因此可以推斷VPA有區增強心肌分化的功能。免疫熒光染色也顯示，VPA處理組中較多細胞為內胚層蛋白標記 FOXA2和甲胎蛋白(AFP)陽性。經VPA處理六天後的胚體胚層標誌基因表達量顯示，神經外胚層標記 Pax6和Noggin在VPA處理組中表達量降低。VPA處理組的中胚層標記brachyury和Mef2c以及內胚層標記GATA4和FOXA2胚層標記的表達量均有上調。 / 在神經幹細胞, 神經前體細胞以及胚胎幹細胞的分化中，可以從VPA處理組觀察到經典 wnt信號通路上調。定量逆轉錄聚合酶鏈反應檢測顯示在VPA處理組裡面, 神經幹細胞, 神經前體細胞以胚體的 wnt信號分子的表達水平的上調。免疫墨點法也檢測到VPA處理組中活化β-catenin較對照組為高。 / Wnt信號通路抑製劑 IWP-2可以破壞棕櫚酰化wnt信號，從而抑制信號分子的成熟和分泌。IWP-2抵銷了VPA 在pNSC中促進神經元的分化的作用。另一方面，抑制wnt信號通路增加了VPA處理組胚體中β-微管蛋白III陽性樹突狀突起的生成, 並且減少跳動細胞群以及內胚層標記蛋白FOXA2和甲胎蛋白陽性的細胞比例。定量逆轉錄聚合酶鏈反應檢測顯示，IWP-2處理組的胚體和對照組相比，神經外胚層標記Pax6和Noggin被上調，中胚層標記 brachyury和Mef2c和內胚層標記 GATA4和FOXA2均被下調。IWP-2 VPA雙重處理組和VPA處理組相比也呈現相同趨勢。這些數據表明，VPA對於神經幹細胞的神經元分化和胚胎幹細胞的胚層譜系分化的影響是通過wnt信號通路介導的。 / 體內研究可以觀察到VPA處理會導致小鼠胚胎中出現的一系列畸形，其中出現有關神經管閉合，異常出血和小腸生長畸形的症狀。胚層標誌分析指出，從VPA注射組孕鼠體內取出的早期階段胚胎具有和在胚胎幹細胞實驗中類似的胚層標記基因表達模式。中胚層和內胚層標記上調而外胚層標記下調。另一方面，VPA在全腦缺血小鼠的治療方面有幫助改善認知功能的作用。 / 這項研究為說明 VPA的致畸作用提供了一個潛在的機制。同時也能夠顯示VPA對全腦缺血患者的潛在治療效果。 / Valproic acid (VPA) is commonly used as a mood stabilizer and anticonvulsant. Despite the clinical relevance to neural disorders, recent studies of VPA revealed the apoptotic and differentiating effects on cancer cells, enhancement of lineage commitments of embryonic stem cells and augmentation of the efficiency in the induction of somatic cells into pluripotent stem cells. Up to date the roles of this small molecule in stem cell development are not well understood. In this study, in vitro culture system was used to elucidate the biological effects of VPA on mouse primary neural stem cells (pNSC), neural precursor cells C17.2 and embryonic stem cells, D3 and E14Tg2a. / VPA was found to inhibit the proliferation of NPC C17.2 and pNSC. On the other hand, it enhanced the differentiation into neurons but not astrocytes and oligodendrocytes. The differentiation of ESC revealed that VPA treatment inhibited the differentiation of ESC into the neuro-ectodermal lineage but promoted the commitment towards the mesodermal and endodermal lineages. / Embryoid bodies (EB) derived from VPA-treated ESC displayed less cell foci with β-tubulin III+ protrusions, but an increase of beating cell clusters, suggesting that VPA enhanced cardio differentiation. Immunofluorescence staining demonstrated that a higher portion of the cells in the VPA-treated group were positive for endodermal markers, FOXA2 and α-fetoprotein. Quantitative RT-PCR (q-PCR) for dermal markers in EB differentiated and treated with VPA for six days showed that neural-ectodermal markers, Pax6 and Noggin, were down-regulated, whereas mesodermal markers, brachyury and Mef2c, and endodermal markers, GATA4 and FOXA2, were all up-regulated. / In studies of the molecular signalling mediated by VPA, quantitative RT-PCR revealed an up-regulation in the gene expression level of wnt molecules in VPA-treated pNSC, NPC and EB. Western blotting also envisaged a higher level of activated β-catenin proteins in VPA treated cells. / The wnt pathway inhibitor IWP-2 was employed to disrupt the palmitoylation of wnt and block the maturation and secretion of the signalling molecule. It was noted that the application of IWP-2 negated the VPA-enhanced neuronal differentiation of pNSC. On the other hand, wnt inhibition increased the incidence of EB having β-tubulin III⁺ protrusions and reduced the numbers of beating EB, FOXA2⁺ and α-fetoprotein⁺ cells in EB-derived cultures of both controls and VPA-treated group. Besides, IWP-2 enhanced the gene expression of neural-ectodermal markers, Pax6 and Noggin, but repressed the gene expression of mesodermal markers, brachyury and Mef2c, and endodermal markers, GATA4 and FOXA2, in EB cultures treated with and without VPA. Data suggest that VPA modulated the neuronal differentiation of pNSC and the dermal commitment of ESC via the wnt pathway. / In vivo study demonstrated that VPA mediated malformations in mouse embryos including deficits in neural tube closure, abnormal bleeding and intestine outgrowth. Dermal marker analysis of VPA-treated embryos at the early stage of development displayed a similar pattern of gene expression noted in in vitro ESC study. Mesodermal and endodermal genes were up-regulated while the ectodermal genes were down-regulated. In global brain ischemic mice, VPA helped restore the cognitive impairment, suggesting the potential therapeutic effect of VPA in global brain ischemia. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lau, Shong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 182-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / List of publications --- p.ii / Abstract --- p.iii / 綜述 --- p.vi / Table of content --- p.viii / Lists of figures --- p.xii / List of tables --- p.xix / List of abbreviations --- p.xx / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Stem cells --- p.1 / Chapter 1.2 --- Embryonic stem cells --- p.2 / Chapter 1.3 --- Application of stem cells for therapeutic purpose --- p.2 / Chapter 1.3.1 --- Application of stem cells in neurological disorders --- p.3 / Chapter 1.3.2 --- Application of stem cells in cardiac repair --- p.6 / Chapter 1.3.3 --- Application of stem cells in diabetes --- p.8 / Chapter 1.3.4 --- Application of haematopoietic stem cells --- p.9 / Chapter 1.3.5 --- Application of messenchymal stem cells --- p.10 / Chapter 1.4 --- Reprogramming --- p.11 / Chapter 1.4.1 --- Somatic cell nucleus transfer (SCNT) --- p.11 / Chapter 1.4.2 --- Altered nucleus transfer --- p.11 / Chapter 1.4.3 --- Induced pluripotent stem cells --- p.12 / Chapter 1.5 --- Propagation of ESC --- p.15 / Chapter 1.6 --- Molecular mechanism of ESC pluripotency and self-renewal --- p.16 / Chapter 1.6.1 --- Oct3/4 and Sox2 --- p.16 / Chapter 1.6.2 --- Nanog --- p.17 / Chapter 1.6.3 --- LIF/JAK/STAT3 --- p.17 / Chapter 1.6.4 --- Wnt --- p.18 / Chapter 1.6.5 --- Basic fibroblast growth factor (bFGF) --- p.18 / Chapter 1.6.6 --- TGF-β/Activin/Nodal --- p.18 / Chapter 1.7 --- Wnt signalling pathway --- p.19 / Chapter 1.7.1 --- Canonical wnt pathway --- p.20 / Chapter 1.7.2 --- Planar cell polarity pathway --- p.21 / Chapter 1.7.3 --- Wnt/Ca²⁺ Pathway --- p.22 / Chapter 1.7.4 --- Signal specificity --- p.23 / Chapter 1.7.4.1 --- Wnt 1 --- p.24 / Chapter 1.7.4.2 --- Wnt 2 --- p.25 / Chapter 1.7.4.3 --- Wnt3/wnt3A --- p.25 / Chapter 1.7.4.4 --- Wnt4 --- p.27 / Chapter 1.7.4.5 --- Wnt5 --- p.28 / Chapter 1.7.4.6 --- Wnt7 --- p.29 / Chapter 1.7.4.7 --- Other wnts --- p.31 / Chapter 1.8 --- Valproic acid --- p.33 / Chapter 1.8.1 --- Effect of VPA on histone modification --- p.34 / Chapter 1.8.2 --- Interaction of VPA with extracellular signal-regulated kinase pathway --- p.35 / Chapter 1.8.3 --- Effect of VPA on PI3K pathway --- p.37 / Chapter 1.8.4 --- Effect of VPA on wnt pathway --- p.37 / Chapter 1.8.5 --- VPA on stem cells --- p.38 / Chapter 1.8.6 --- VPA on brain ischemia animal model --- p.39 / Chapter 1.9 --- Overall aim and design of the Study --- p.41 / Chapter 2.1 --- Cells /cell lines --- p.53 / Chapter 2.1.1 --- Neural precursor cell line C17.2 --- p.53 / Chapter 2.1.2 --- Primary neural stem cells --- p.53 / Chapter 2.1.3 --- Primary mouse embryonic fibroblast --- p.53 / Chapter 2.1.4 --- Embryonic stem cell line D3 --- p.54 / Chapter 2.1.5 --- Embryonic stem cell line E14TG2a --- p.54 / Chapter 2.2 --- Cell cultures and assays --- p.54 / Chapter 2.2.1 --- Medium and solutions --- p.54 / Chapter 2.2.2 --- Isolation of primary neural stem cells --- p.58 / Chapter 2.2.3 --- Maintenance and passaging of primary neural stem cells --- p.60 / Chapter 2.2.4 --- Cryopreservation and thawing of primary neural stem cells --- p.60 / Chapter 2.2.5 --- Coating of coverslips using ornithine and laminin (O&L) --- p.61 / Chapter 2.2.6 --- Differentiation of primary neural stem cells --- p.61 / Chapter 2.2.7 --- Isolation of mouse embryonic fibroblasts --- p.62 / Chapter 2.2.8 --- Maintenance and passaging of mouse embryonic fibroblasts --- p.63 / Chapter 2.2.9 --- Cryopreservation and thawing of mouse embryonic fibroblasts --- p.63 / Chapter 2.2.10 --- Preparation of gelatin-coated culture wares and cover slips --- p.64 / Chapter 2.2.11 --- Preparation of irradiated mouse embryonic fibroblast feeder layer --- p.64 / Chapter 2.2.12 --- Maintenance and passaging of neural precursor cell C17.2 --- p.64 / Chapter 2.2.13 --- Cryopreservation and thawing of neural precursor cell C17.2 --- p.64 / Chapter 2.2.14 --- Differentiation of neural precursor cell C17.2 --- p.65 / Chapter 2.2.15 --- Maintenance and passaging of mouse embryonic stem cell line --- p.65 / Chapter 2.2.16 --- Cryopreservation and thawing of mouse embryonic stem cell line --- p.65 / Chapter 2.2.17 --- Spontaneous differentiation of embryonic stem cells --- p.66 / Chapter 2.2.18 --- Neural differentiation of embryonic stem cells --- p.66 / Chapter 2.2.19 --- WST-1 proliferation assay --- p.67 / Chapter 2.2.20 --- Colony formation assay --- p.67 / Chapter 2.2.21 --- Two-stage neural differentiation assay --- p.68 / Chapter 2.3 --- Molecular analysis --- p.68 / Chapter 2.3.1 --- In-house prepared solutions and reaction mixes --- p.68 / Chapter 2.3.2 --- RNA extraction --- p.72 / Chapter 2.3.3 --- Synthesis of complementary DNA (cDNA) by reverse transcription --- p.73 / Chapter 2.3.4 --- Polymerase chain reaction (PCR) --- p.74 / Chapter 2.3.5 --- Quantitative polymerase chain reaction (qPCR) --- p.74 / Chapter 2.3.6 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting --- p.75 / Chapter 2.4 --- Microscopy and Immunofluorescence staining --- p.77 / Chapter 2.5 --- In vivo Study --- p.77 / Chapter 2.5.1 --- Embryo malformation study --- p.77 / Chapter 2.5.2 --- Establishment of a mouse global brain ischemia model --- p.78 / Chapter 2.5.3 --- Behaviour study --- p.79 / Chapter 2.5.4 --- Histological analysis --- p.79 / Chapter 2.5.4.1 --- Animal sacrifice for brain harvest --- p.79 / Chapter 2.5.4.2 --- Paraffin sectioning --- p.80 / Chapter 2.5.4.3 --- Haematoxylin and eosin staining --- p.80 / Chapter 2.5.4.4 --- Cresyl violet staining --- p.81 / Chapter 2.5.4.5 --- TUNEL assay --- p.81 / Chapter 2.6 --- Statistical analysis --- p.82 / Chapter 2.7 --- Equipments used in this study --- p.82 / Chapter Chapter 3 --- Effects of VPA on proliferation and differentiation of mouse NPC, pNSC and ESC --- p.88 / Chapter 3.1 --- Effects of VPA on mouse ESC proliferation --- p.88 / Chapter 3.1 --- Effects of VPA on mouse ESC proliferation --- p.88 / Chapter 3.2 --- Effects of VPA on differentiation and proliferation of neural lineage cells --- p.89 / Chapter 3.2.1 --- Effects of VPA on differentiation and proliferation of NPC C17.2 --- p.89 / Chapter 3.2.2 --- Effects of VPA on differentiation of pNSC --- p.89 / Chapter 3.2.3 --- Effects of VPA on proliferation and stemness of pNSC --- p.90 / Chapter 3.2.4 --- Effects of VPA on neural differentiation of mouse ESC --- p.91 / Chapter 3.2.5 --- Two-stage neural differentiation assay of mouse ESC --- p.91 / Chapter 3.3 --- VPA-mediated lineage commitment of mouse ESC --- p.92 / Chapter 3.3.1 --- Cardio differentiation of ESC --- p.92 / Chapter 3.3.2 --- Endodermal commitment of mouse ESC --- p.93 / Chapter 3.3.3 --- Germ layer marker expression in mouse ESC --- p.93 / Chapter 3.3.4 --- Up-regulation of histone acetylation in VPA-treated mouse ESC --- p.94 / Chapter 3.6 --- Summary --- p.94 / Chapter Chapter 4 --- Wnt signalling played as a mediator of VPA in the differentiation of ESC, NPC and pNSC differentiation. --- p.118 / Chapter 4.1 --- VPA treatment up-regulated wnt pathway in mouse ESC, NPC and pNSC --- p.118 / Chapter 4.2 --- Effect of wnt inhibitor IWP-2 on stem cells in differentiation cultures supplemented with VPA. --- p.119 / Chapter 4.2.1 --- Effects of wnt inhibitor IWP-2 on the neural differentiation of NPC C17.2 --- p.119 / Chapter 4.2.2 --- Effect of wnt inhibitor on differentiation of pNSC --- p.120 / Chapter 4.2.3 --- Effect of IWP-2 on the differentiation culture of EB --- p.121 / Chapter 4.2.4 --- Effect of wnt inhibitor on the lineage commitment of mouse ESC --- p.121 / Chapter 4.2.5 --- Effects of IWP-2 on VPA-induced lineage markers expressed by mouse ESC --- p.123 / Chapter 4.3 --- Effects of wnt inhibitor IWP-2 on ESC proliferation. --- p.124 / Chapter 4.4 --- Summary --- p.125 / Chapter Chapter 5 --- In vivo effects of VPA --- p.154 / Chapter 5.1 --- Effects of VPA on mouse embryo development --- p.154 / Chapter 5.1.1 --- Effects of VPA at the daily dose of 300mg/kg body weight --- p.154 / Chapter 5.1.2 --- Effects of VPA at the daily dose of 600 mg/kg body weight --- p.155 / Chapter 5.1.3 --- Effects of VPA at twice daily dose of 600 mg/kg body weight --- p.155 / Chapter 5.2 --- Effects of VPA on ischemic mice --- p.156 / Chapter 5.2.1 --- Behaviour test --- p.156 / Chapter 5.2.2 --- Histological and molecular assessment --- p.157 / Chapter 5.3 --- Summary --- p.157 / Chapter Chapter 6 --- Discussion --- p.171 / Chapter 6.1 --- Modulation of ESC fate at early developmental stage --- p.171 / Chapter 6.2 --- Neural differentiation of pNSC and NPC upon VPA treatment --- p.172 / Chapter 6.3 --- VPA-mediated wnt signaling on the differentiation of mouse ESC --- p.173 / Chapter 6.4 --- VPA-mediated wnt signalling in the neuronal differentiation of mouse neural stem/progenitor cells --- p.175 / Chapter 6.5 --- Effects of VPA on the proliferation of mouse ESC, pNSC and NPC --- p.176 / Chapter 6.6 --- Effects of VPA on mouse embryo --- p.177 / Chapter 6.7 --- Therapeutics of VPA in ischemic stroke --- p.178 / Chapter Chapter 7 --- Conclusion --- p.180 / References --- p.182
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328111 |
| Date | January 2012 |
| Contributors | Lau, Shong., Chinese University of Hong Kong Graduate School. Division of Anatomical and Cellular Pathology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xxi, 228 leaves) : ill. (chiefly col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
Page generated in 0.0148 seconds