楊西苑臺灣大學:分子與細胞生物學研究所王東晟Wang, Tung-ChengTung-ChengWang2010-06-022018-07-062010-06-022018-07-062008U0001-0701200823232600http://ntur.lib.ntu.edu.tw//handle/246246/184667在老鼠食道中，其外肌肉層(muscularis externa)在早期發育期間是由平滑肌所組成，但稍後被骨骼肌由頭往尾端的發育過程中被漸進式地取代。然而對於食道骨骼肌細胞的發育來源卻尚未完全了解。過去十年間，關於食道骨骼肌細胞發育來源有以下兩種假說被提出：一者是由已分化的食道平滑肌轉化為骨骼肌 (transdifferentiation)，另一者是由獨特的骨骼肌先驅細胞 (skeletal muscle cell precursors) 分化而來。此議題目前之所以仍處於兩派學說的爭議中，其歸咎於骨骼肌先驅細胞的存在還未被完全證實。在這個研究當中，利用免疫螢光染色法 (immunostaining)，使用巢蛋白 (nestin) 做為肌肉幹細胞的標示 (myogenic cell marker)，藉此辨識出食道骨骼肌先驅細胞的存在。此項研究中發現巢蛋白不只出現在骨骼肌先驅細胞，同時也出現在平滑肌先驅細胞。然而，巢蛋白表現在平滑與骨骼肌細胞的時序是不同的。巢蛋白在平滑肌發育中會較早消失於已分化的細胞，相反地，巢蛋白稍後在骨骼肌發育中會持續表現，並與兩個骨骼肌分化蛋白(MyoD和skeletal muscle myosin heavy chain,)共同表現於不同分化階段的細胞中。此外，吾人利用巢蛋白與平滑肌專一性分化蛋白(smooth muscle α-actin)及前述兩個骨骼肌分化蛋白，標示出位於各個不同分化階段的平滑與骨骼肌細胞之免疫螢光類型 (immunotypes)。並且發現平滑與骨骼肌肉的專一性分化蛋白並不如平滑肌轉化為骨骼肌的機制中所預測地會共同表達在同一細胞中。最後，在利用5-溴脫氧尿嘧啶核苷 (BrdU) 來標定增生細胞的實驗中，其結果也顯示出巢蛋白所標示的食道骨骼肌先驅細胞確實處於高度增生狀態並為食道骨骼肌細胞的來源。總結，吾人利用巢蛋白做為肌肉幹細胞的標示，成功地找尋出食道骨骼肌先驅細胞，並且對於食道平滑肌與骨骼肌分別走向不同的發育途徑也提供更完整的證明。During the esophageal myogenesis in rodent, the muscularis externa is initially composed of smooth muscle cells which are later replaced by the skeletal muscle in a craniocaudal progression. However, the ontology of the esophageal skeletal muscle has not been clearly understood. For the past decade, the origin of esophageal skeletal muscle has been proposed as two mechanisms: the smooth-to-skeletal muscle cell transdifferentiation and the cell differentiation of distinct skeletal muscle precursors. This issue remains controversial with respect to the existence of esophageal skeletal muscle precursors. In the present study, we used the immunostaining of nestin, a marker for myogenic progenitors, to identify the presence of esophageal myogenic precursors. The results showed that nestin was not only expressed in skeletal muscle precursors but in smooth muscle precursors as well. However, the temporal expression patterns are different in that the nestin expression of smooth muscle lineage disappeared early in differentiated cells, whereas the nestin of skeletal muscle lineage was expressed throughout the late fetal development and coincidently labeled with two markers for skeletal muscle cell differentiation, MyoD and skeletal muscle myosin heavy chain. In addition, the cell phenotypes of different developing stages in both smooth and skeletal muscle lineages were determined by immunotyping approach with nestin, smooth muscle α-actin, and the two skeletal muscle markers. Moreover, no mixed phenotype of cells expressing both smooth and skeletal muscle-specific proteins was detected, as would be expected in transdifferentiation mechanism. Finally, BrdU incorporation analysis revealed that the nestin-immunoreactive skeletal muscle precursors had high proliferation potential and suggested that they were the source of developing esophageal skeletal muscle cells. Taken together, we identified the esophageal skeletal muscle precursor cells by immunoreactivity of nestin and provided evidence for the distinct differentiation pathways for each muscle type in rat esophageal myogenesis.Cover page……..………………………………………………………………………..i謝..................................................................................................................................ii要……………………………………………………………………………………..iiibstract……………………………………………………………………………….....ivontent………………………………………………………………………………......1ntroduction………………………………………………………………………….......3he general structures of adult esophagus………………………………………......3sophageal myogenesis: early development of visceral smooth muscle cells...........3sophageal myogenesis: the formation of smooth muscle musculature....................4sophageal myogenesis: the replacement of esophageal smooth muscle by skeletal muscle.........................................................................................................................5he origin of esophageal skeletal muscle cells: transdifferentiation of smooth muscle cells................................................................................................................6he origin of esophageal skeletal muscle cells: distinct skeletal muscle differentiation.............................................................................................................7he unknown esophageal skeletal muscle precursors................................................8he introduction of intermediate filament protein nestin...........................................9dentification of esophageal skeletal muscle precursor cells by nestin immunoreactivity.......................................................................................................10aterials and Methods....................................................................................................12nimals......................................................................................................................12ntibodies..................................................................................................................12mmunofluorescence microscopy..............................................................................13esults.............................................................................................................................15tage of the onset of smooth muscle cell differentiation................................................15tage of smooth muscle cell differentiation and keletal muscle cell determination.............................................................................16tage of skeletal muscle cell differentiation..............................................................17tage of esophageal muscle type conversion: expansion of skeletal muscle cells and degeneration of smooth muscle cells.........................................................................18dentification of distinct nestin-IR cells as skeletal muscle precursors.....................19roliferation potential of nestin-IR esophageal skeletal muscle precursors..............20iscussion.......................................................................................................................22pecial features of esophageal skeletal muscle.........................................................22he origin of esophageal skeletal muscle cells.........................................................22mmunotyping of esophageal smooth muscle lineage..............................................24mmunotyping of esophageal skeletal muscle lineage..............................................25estin identifies two distinct origins of esophageal smooth and keletal muscle cells..................................................................................................26ransdifferentiation in esophageal myogenesis........................................................27roliferation capacity of nestin-IR skeletal muscle precursors.................................28mbryonic origin of esophageal skeletal muscle precursors....................................29ate of esophageal smooth muscle............................................................................30ossible role of esophageal smooth muscle...............................................................31onclusion.......................................................................................................................33eferences.......................................................................................................................34ables...............................................................................................................................40igures and illustrations...................................................................................................41application/pdf1200533 bytesapplication/pdfen-US巢蛋白免疫表現型肌肉先驅細胞食道壁肌肉發育分化性轉型nestinimmunotypingmuscle precursorsesophageal myogenesistransdifferentationhttp://ntur.lib.ntu.edu.tw/bitstream/246246/184667/1/ntu-97-R93b43006-1.pdf