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博碩士論文 etd-0718116-090423 詳細資訊
Title page for etd-0718116-090423
論文名稱
Title
移植胚胎脊髓組織重建中段頸部損傷脊髓之作用
Reconstruction of mid-cervical injured spinal cord following transplantation of fetal spinal cord tissue
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
66
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2016-07-15
繳交日期
Date of Submission
2016-08-18
關鍵字
Keywords
移植、呼吸、神經追蹤劑、胚胎脊髓組織、脊髓損傷
fetal spinal cord, neural tracer, spinal injury, transplantation, respiration
統計
Statistics
本論文已被瀏覽 5662 次,被下載 22
The thesis/dissertation has been browsed 5662 times, has been downloaded 22 times.
中文摘要
頸部脊髓損傷使得調控呼吸神經路徑受損,也使運動神經元損傷,進而導致呼吸功能及運動功能癱瘓。細胞移植療法對於脊髓損傷是具有潛力的療法,可替代因脊髓損傷而失去的神經元並重建神經元之間的連結。本論文主要研究移植胚胎脊髓組織的分化情形,以及是否能重建受損的頸部脊髓神經網路,並整合宿主脊髓進一步調控呼吸以及運動功能。實驗組分成三組,移植組(脊髓損傷手術後移植胚胎脊髓組織)、假移植組(脊髓損傷手術後注入Hank’s平衡鹽溶液)以及假損傷組(僅移除頸部背側脊椎骨)。大鼠於頸部第4節脊髓左側半切損傷後1週移植14天的大鼠胚胎脊髓片段,大鼠於移植前1天、移植後1週、2週、4週以及8週透過全體腔呼吸測量(whole body plethysmography; WBP)以及曠野實驗(open field test)的方式,觀察呼吸以及運動行為。組織學結果證實胚胎脊髓組織可於移植手術後8週存活,並且可分化成神經元、星狀細胞、微膠細胞以及少量寡突膠細胞,且於移植組織內部發現血清素、多巴胺β水解酶、γ-胺基丁酸、囊泡谷氨酸轉運蛋白第2型以及乙醯膽鹼轉移酶表現,說明移植組織確實可受到神經纖維支配,也有可能透過神經元調控神經傳遞物質影響移植組織本身。於移植8週後,藉由逆向單突觸(cholera toxin B; CT-B)或逆向跨突觸(wheat germ agglutinin; WGA)神經追蹤劑塗抹於橫膈肌,觀察移植組織是否與橫膈運動神經系統連結,結果發現移植組織內有少量神經元被逆向跨突觸神經追蹤劑標記,說明移植組織可能可透過多突觸的方式來影響橫膈。但生理學數據顯示呼吸以及運動行為於移植組以及假移植組別之間並無明顯差異。這些證據顯示移植的胚胎脊髓組織可存活並可分化成神經細胞並且重建部分宿主呼吸運動神經網路。
Abstract
Cervical spinal cord injury usually leads to respiratory impairment and locomotor paralysis due to the lesion of the bulbospinal pathway and damage of spinal motorneurons. Cellular replacement therapy has a potential to replace damaged spinal cells and restore neural connectivity; therefore, transplantation of neural progenitors may be a promising therapeutic strategy for spinal cord repair. This study was designed to examine whether a delayed transplantation of fetal spinal cord(FSC)tissue can reconstruct cervical spinal neural circuit and modulate respiratory and locomotor function. Embryonic day 14 spinal cord tissue expressing green fluorescence protein(GFP)or hank’s balanced salt solution(HBSS)were transplanted in the C4 spinal cord hemi-section(C4Hx)cavity of adult Sprague Dawley rat at 1 week post-injury. Respiratory patterns and locomotor behaviors were evaluated before and at 1, 2, 4 and 8 weeks post-transplantation by whole body plethysmography and open-field tracking system, respectively. Histological data showed that a robust GFP expression was observed in the lesion side in most transplanted animals. Immunofluorescence staining revealed that neural cell markers [e.g., neuronal nuclei(NeuN)for neuron; glial fibrillary acidic protein(GFAP)for astrocyte; ionized calcium binding adapter molecule 1(Iba1)for microglia; receptor interacting protein(RIP)for oligodendrocyte] were detected in GFP-positive tissue. The graft-derived neuron can differentiate into specific neuron [e.g., serotonergic, 5-hydroxytryptamine(5-HT); noradrenergic, dopamine beta hydroxylase(DBH); cholinergic, choline acetyltransferase(ChAT)]. FSC-derived graft received a robust innervation of GABAergic(GAD65/67 positive)and glutamatergic(vGluT2 positive)fibers. Physiological data demonstrated that respiratory patterns and locomotor behaviors were similar between C4Hx animals received FSC or HBSS transplantation. Retrograde monosynaptic tracer (Cholera toxin B)or transsynaptic tracer(wheat germ agglutinin; WGA)were painted on the diaphragm at 8 week post-transplantation to examine whether transplanted FSC-derived graft can innervate the phrenic motor system. The result showed that there were some WGA-positive cells detected within the graft region. These data suggest that fetal spinal cord tissue can survive and differentiate into neural cells in the injured host spinal cord and reconstruct the spinal cord respiratory circuit; however, this approach is not sufficient to significantly improve the respiratory motor function.
目次 Table of Contents
論文審定書i
中文摘要ii
Abstractiii
圖次iv
表次v
縮寫表vi
目錄vii
第一章 前言1
1.1基本脊髓介紹1
1.2脊髓內的細胞類型1
1.3台灣脊髓損傷案例2
1.4脊髓損傷對於呼吸運動調控的影響2
1.5脊髓損傷後細胞移植的治療策略4
1.6胚胎脊髓的特性4
1.7移植胚胎脊髓組織對於損傷脊髓的影響5
1.8目的7
第二章 材料與方法8
2.1實驗動物分組8
2.2頸部脊髓損傷手術8
2.3螢光鼠移植胚胎脊髓組織9
2.4胚胎脊髓組織移植9
2.5全體腔呼吸測量系統10
2.6運動行為評估10
2.7神經追蹤劑以及免疫化學組織染色10
2.8神經元逆行追蹤劑11
2.9數據分析13
第三章 結果15
3.1胚胎脊髓組織於損傷脊髓的存活狀況15
3.2移植組織細胞種類於宿主組織灰白質區域的差異15
3.3移植組織內神經元種類16
3.4移植組織與宿主橫膈肌肉間的連結能力16
3.5移植胚胎脊髓組織對脊髓損傷動物呼吸的影響16
3.6移植胚胎脊髓組織對脊髓損傷動物運動的影響17
3.7GFP大鼠移植正常胚胎脊髓組織18
第四章 討論19
4.1胚胎脊髓組織於損傷脊髓存活情形19
4.2胚胎脊髓組織於損傷脊髓分化情形20
4.3胚胎脊髓移植組織與宿主組織連結情況21
4.4胚胎脊髓組織對於呼吸功能影響21
第五章 圖與說明 23
第六章 表與說明 45
第七章 參考文獻 47
第八章 附錄51
8.1歷年發表摘要與壁報51
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