Mi zou yuan xing shen jing ji xi bao, shen jing xian wei he chang nei wei huan jing dui xiao shu di yuan xing shen jing ji xi bao qian yi de zuo yong

人類先天性巨結腸症（HSCR）主要表現為結腸末端的神經節缺失或稀少。 結腸末端的神經節來源於迷走源性神經脊細胞和骶源性神經脊細胞。迷走源性神經脊細胞是腸神經系統的主要來源，已被廣泛研究，而關於哺乳類包括人類的骶源性神經脊細胞的研究卻相當稀少。小鼠胚胎的骶源性神經脊細胞遷移途徑近期已被闡釋，其由神經管背側遷出並向腹側移動，於後腸附近聚集成旁神經節然後進入腸內。本研究運用一系列實驗鑒定了對小鼠骶源性神經脊細胞由旁神經節遷移至腸內過程有影響作用的因素。研究發現骶源性神經脊細胞是沿著神經纖維並向口端方向遷移進腸內，同時由於迷走源性神經脊細胞於腸內向尾端遷移，它們在後腸末端相遇並相互作用，我們首先研究了這種相互作用以瞭解迷走源性神經脊細胞如何影響骶源性神經脊細胞的遷移。利用帶綠色螢光的小鼠骶源性神經脊細胞和可變螢光的小鼠迷走源性神經脊細胞（鐳射激發下由綠色變為紅色），以及鐳射共聚焦顯微鏡活細胞成像術，我們觀察了這兩種細胞在腸內相遇時的行為。當骶源性神經脊細胞和迷走源性神經脊細胞在神經纖維上相遇是，它們都停止了移動，不能前行。培養3天以後，骶源性神經脊細胞和迷走源性神經脊細胞在後腸中共同形成了神經細胞網路，其中骶源性神經脊細胞相對只占了小部分細胞。由於骶源性神經脊細胞沿著由旁神經節發出的神經纖維遷移至腸內並且當在神經纖維上遇到迷走性源性神經脊細胞時便停止移動，我們進而研究了神經纖維在骶源性神經脊細胞遷移中的地位。活細胞成像術和免疫組化實驗表明旁神經節發出的神經纖維對骶源性神經脊細胞的遷移是非常重要的，它有助於細胞遷移同時，但當細胞到達纖維末端時它便也限制了細胞的遷移。儘管如此，體外實驗表明在一定培養條件下，骶源性神經脊細胞的遷移並不需要這些神經纖維。由於有報導表明腸內微環境能影響腸神經脊細胞的遷移，我們利用2D電泳和質譜檢測了12.5天（骶源性神經脊細胞進入後腸之前）和13.5天（骶源性神經脊細胞進入後腸）胎鼠後腸中的蛋白表達情況。大多數鑒定到有差異表達的蛋白都與蛋白折疊、細胞生長和細胞骨架組織有關。我們選取了與細胞粘附和肌肉收縮有關的鈣離子依賴膜結合蛋白Anxa6，並結合腸內的平滑肌發育進行了進一步的研究。結果顯示在13.5天胎鼠中，旁神經節的口端方向有一段約600微米的腸的腹側的平滑肌還沒有發育，可能與腸神經脊細胞的遷移有關。但腸內平滑肌發育是否及如何影響腸神經脊細胞的遷移還需要進一步的研究。綜上所述，骶源性神經脊細胞的遷移是一個複雜的過程，迷走源性神經脊細胞，神經纖維和腸內微環境都參與並能影響這個遷移過程。Hirschsprung’s disease (HSCR) in humans is characterized by the absence or reduction of enteric ganglia in the distal part of the colon. It is known that all enteric ganglia in the distal colon originate from neural crest cells (NCCs) at both vagal and sacral levels during embryonic development. Vagal NCCs have been well characterized as the main cellular source of the enteric nervous system (ENS), but however, information on the mammalian, including human, sacral NCCs is still scarce. Sacral NCCs in mouse embryos have been recently identified to be able to migrate from the dorsal neural tube to the mesenchyme, aggregate as pelvic ganglia adjacent to the hindgut and then enter the distal hindgut. In the present study, a series of experiments were performed to determine the factors that were involved in modulating their entry to the hindgut and their migration within the distal hindgut, using mouse embryos.Having entered the hindgut, sacral NCCs migrated along nerve fibers in a caudal-to-rostral direction while vagal NCCs were colonizing the hindgut in a reverse, rostral-to-caudal direction. The migratory behaviors of the vagal and sacral NCCs were examined at the time when these two populations of NCCs met each other with live cell confocal imaging in the distal hindgut using GFP-expressing sacral NCCs (green fluorescent) and Ednrb-Kikume labeled vagal NCCs (red fluorescent) from transgenic mice. The rostral migration of sacral NCCs was observed to be temporarily affected when they met vagal NCCs on the nerve fiber. However, after 3 days in organotypic culture, sacral and vagal NCCs were found to intermingle with each other to form an interconnected cellular network in the hindgut with much greater cellular contribution from vagal NCCs than sacral NCCs. Hence, vagal NCCs were able to affect the migration and thus the final location of sacral NCCs within the hindgut.Since sacral NCCs have been observed to enter the hindgut by migrating on the nerve fibers extending from pelvic ganglia, the role the nerve fibers in migration was then examined. Results obtained from time-lapse confocal live cell imaging and immunohistochemical localization indicated that nerve fibers extending from pelvic ganglia were very important for sacral NCCs migration. It was found that these nerve fibers could both assist in sacral NCC migration and also restrain their migration once the cells reached the distal tip of the fibers. However, under specific in vitro conditions, sacral NCCs were still able to migrate without the presence of nerve fibers.The gut microenvironment surrounding the migrating NCCs has also been reported to affect NCCs migration. Therefore, protein molecules with differential expression levels prior to and after the entry of sacral NCCs to the distal hindgut between E12.5 to E13.5 were examined with 2-dimensional gel electrophoresis and mass spectrometry. The proteins identified with significant changes of expression (more than 1.5 folds) were grouped according to their predicted biological functions and involved in protein folding, cell growth and cytoskeletal organization. Among them, Anxa6, a calcium-dependent membrane binding protein related to cell adhesion and muscle contraction, was further examined for its relationship with the muscle development in the hindgut at E12.5 to E14.5. The results showed that a segment of the hindgut (about 600μm) rostral to pelvic ganglia exhibited an incomplete layer of smooth muscle at E13.5. Whether Anxa6 and the smooth muscle are involved in the sacral NCC migration is worth further investigations.In summary, sacral NCCs migration is a complex process regulated by their interactions with nerve fibers, vagal neural crest cells and possibly molecules in the hindgut microenvironment through which they migrate.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.Chen, Jielin.Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.Includes bibliographical references (leaves 201-214).Abstract also in Chinese.Abstract --- p.I摘要 --- p.IVAcknowledgements --- p.VITable of contents --- p.XIIAbbreviation --- p.XIIIChapter Chapter 1 --- General Introduction --- p.1Chapter 1.1 --- The enteric nervous system (ENS) --- p.1Chapter 1.1.1 --- Embryonic origin and development of the ENS --- p.2Chapter 1.1.2 --- Hirschsprung’s disease (HSCR) --- p.5Chapter 1.2 --- Enteric neural crest cells (ENCCs) --- p.7Chapter 1.2.1 --- Vagal neural crest cells (NCCs) --- p.8Chapter 1.2.2 --- Sacral neural crest cells (NCCs) and pelvic ganglia --- p.10Chapter 1.2.3 --- Interactions between neural crest cells (NCCs) --- p.14Chapter 1.3 --- Microenvironment within the gut --- p.16Chapter 1.3.1 --- Effect of molecules on ENCCs migration --- p.16Chapter 1.3.2 --- Effect of tissue age on ENCC migration --- p.19Chapter 1.3.3 --- Absence of ENCCs facilitates ENCC colonization --- p.20Chapter 1.4 --- Objectives of the present study --- p.22Chapter Figures and Legends --- p.25Chapter Chapter 2 --- Migratory behaviors of sacral and vagal neural crest cells in the distal hindgut --- p.32Chapter 2.1 --- Introduction --- p.32Chapter 2.2 --- Materials and methods --- p.36Chapter 2.2.1 --- Mouse strains --- p.36Chapter 2.2.2 --- Isolation of gut tubes and pelvic ganglia --- p.36Chapter 2.2.3 --- Photo-conversion of Ednrb-kikume labeled neural crest cells within the gut --- p.37Chapter 2.2.4 --- Preparation of general culture medium and organ culture agarose gel --- p.38Chapter 2.2.5 --- Organotypic culture --- p.39Chapter 2.2.6 --- Time-lapse live cell confocal microscopic imaging --- p.39Chapter 2.2.7 --- Whole mount gut preparations for immunohistochemical staining --- p.41Chapter 2.3 --- Results --- p.42Chapter 2.3.1 --- Conversion of green fluorescent, Ednrb-kikume labeled vagal NCCs into red fluorescent --- p.42Chapter 2.3.2 --- Ednrb-kikume labeled cells were Sox10 immunorecative --- p.42Chapter 2.3.3 --- No discernible photo-toxicity after photo-conversion --- p.43Chapter 2.3.4 --- Sacral NCCs migration was hindered by vagal NCCs when they met on the nerve fiber --- p.43Chapter 2.3.5 --- Vagal NCCs migrated toward each other to form an interconnected network --- p.45Chapter 2.3.6 --- Sacral NCCs contributed much fewer cells than vagal NCCs in the terminal hindgut --- p.46Chapter 2.4 --- Discussion --- p.47Chapter 2.4.1 --- Ednrb-kikume mouse is a potentially ideal animal model for studies of NCCs migratory behaviors --- p.47Chapter 2.4.2 --- Migration of sacral NCCs in the hindgut was affected by vagal NCCs --- p.49Chapter 2.4.3 --- Migratory behaviors of vagal NCCs --- p.52Chapter 2.4.4 --- Vagal NCCs potentially preferred to move on nerve fibers --- p.53Chapter 2.4.5 --- Sacral NCCs contributed much less to the cellular network than vagal NCCs --- p.53Chapter 2.5 --- Summary --- p.55Chapter Table 2-1 --- Primary and secondary antibodies used in the experiments --- p.56Chapter Figures and Legends --- p.57Chapter Chapter 3 --- The relationship between nerve fiber extension and sacral neural crest cell migration in vitro --- p.81Chapter 3.1 --- Introduction --- p.81Chapter 3.2 --- Materials and methods --- p.86Chapter 3.2.1 --- Mouse strains --- p.86Chapter 3.2.2 --- Preparation of fibronectin (FN) coated coverslips and confocal dishes --- p.86Chapter 3.2.3 --- Preparation of media --- p.87Chapter 3.2.4 --- Isolation of pelvic ganglia --- p.87Chapter 3.2.5 --- In vitro culture of pelvic ganglia in 4-well plates or confocal dishes --- p.88Chapter 3.2.6 --- Live cell imaging using Nikon live cell imaging system --- p.88Chapter 3.2.7 --- WGA treatments on the pelvic ganglia culture --- p.89Chapter 3.2.8 --- Effect of embryonic cell proliferation medium and stem cell proliferation medium on pelvic ganglia growth in vitro --- p.90Chapter 3.2.9 --- Immunohistochemical staining --- p.90Chapter 3.3 --- Results --- p.92Chapter 3.3.1 --- Sacral NCCs and nerve fibers from the pelvic ganglia were in close association in vitro --- p.92Chapter 3.3.2 --- Migratory behaviors of sacral NCCs on the nerve fiber in vitro --- p.92Chapter 3.3.3 --- WGA treatments affected the growth of nerve fibers and sacral NCCs migration in vitro --- p.93Chapter 3.3.4 --- Sacral NCCs migrated without nerve fibers when cultured in proliferation media --- p.95Chapter 3.4 --- Discussion --- p.97Chapter 3.4.1 --- In vitro culture of pelvic ganglion --- p.97Chapter 3.4.2 --- Migratory behaviors of sacral NCCs in vitro --- p.98Chapter 3.4.3 --- Sacral NCCs migration was affected by the extension of nerve fibers from pelvic ganglia in vitro --- p.101Chapter 3.4.4 --- Nerve fibers from the pelvic ganglia were not necessary for sacral NCCs migration in vitro --- p.103Chapter 3.5 --- Summary --- p.106Chapter Figures and Legends --- p.107Chapter Chapter 4 --- Differentially expressed protein molecules in the distal hindgut before and after the entry of sacral neural crest cells --- p.128Chapter 4.1 --- Introduction --- p.128Chapter 4.2 --- Materials and methods --- p.132Chapter 4.2.1 --- Mouse strain --- p.132Chapter 4.2.2 --- Preparation of solutions for 2-dimensional (2D) gel electrophoresis --- p.132Chapter 4.2.3 --- Preparation of solutions for mass spectrometry --- p.133Chapter 4.2.4 --- Isolation of the distal hindgut and protein extraction --- p.133Chapter 4.2.5 --- Measurement of protein concentration --- p.134Chapter 4.2.6 --- 2D gel electrophoresis --- p.135Chapter 4.2.7 --- Mass spectrometry --- p.138Chapter 4.2.8 --- SDS-PAGE and Western blot --- p.139Chapter 4.2.9 --- Immunohistochemical staining of gut tubes and embryos --- p.140Chapter 4.2.10 --- Distal hindgut model reconstruction --- p.141Chapter 4.3 --- Results --- p.143Chapter 4.3.1 --- E13.5 was the critical stage at which sacral NCCs started to enter the hindgut --- p.143Chapter 4.3.2 --- Protein molecules identified by 2D electrophoresis and mass spectrometry --- p.144Chapter 4.3.3 --- Western blot analysis and immunostaining confirmed expression levels of Anxa6 --- p.145Chapter 4.3.4 --- Smooth muscle actin (SMA) and Anxa6 partially co-localized within the E13.5 hindgut --- p.146Chapter 4.3.5 --- Expression of SMA and Anxa6 before and after sacral NCC entry to the distal hindgut --- p.146Chapter 4.3.6 --- Reconstruction of distal hindgut images from serial sections with SMA and Anxa6 immunoreactivities --- p.147Chapter 4.4 --- Discussion --- p.149Chapter 4.4.1 --- Tissue age affected enteric NCC colonization --- p.149Chapter 4.4.2 --- Proteomics used in modern biological research --- p.150Chapter 4.4.3 --- Molecules differentially expressed in the distal hindgut at E12.5 and E13.5 --- p.151Chapter 4.4.4 --- Anxa6 and SMA expression in the distal hindgut --- p.153Chapter 4.4.5 --- The role of the smooth muscle development in sacral NCC entry into the hindgut --- p.155Chapter 4.5 --- Summary --- p.157Chapter Table 4-1 --- Identification of proteins by MALDI-TOF analysis and the MASCOT search program --- p.158Chapter Table 4-2 --- Differentially expressed proteins identified by 2-D electro-phoresis and MALDI-TOF/TOF and their predicted biological functions --- p.159Figures and Legends --- p.160Chapter Chapter 5 --- Conclusions and discussion --- p.182Chapter 5.1 --- Vagal NCCs hindered sacral NCC migration when they coalesced on the nerve fiber --- p.182Chapter 5.2 --- Nerve fibers from pelvic ganglia were important but not necessary for sacral NCCs migration in vitro --- p.186Chapter 5.3 --- Possible involvement of smooth muscle development in modulating sacral NCCs migration --- p.189Chapter 5.4 --- Future prospects --- p.191Chapter 5.4.1 --- Interactions of vagal and sacral NCCs within the hindgut of mouse embryos --- p.191Chapter 5.4.2 --- Role of nerve fibers for sacral NCCs migration ex vivo --- p.192Chapter 5.4.3 --- Role of Anxa6 in muscle development of the gut and NCCs migration --- p.193Chapter Appendix I --- Solutions used in 2-D electrophoresis --- p.195Chapter Appendix II --- Solutions for Colloidal Coomassie staining --- p.198Chapter Appendix III --- Procedures for embryo processing --- p.199Chapter Appendix IV --- Other solutions --- p.200References --- p.20