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研究生:黃佩宇
研究生(外文):Pei-Yu Huang
論文名稱:人類臍帶間質幹細胞移植對顳葉癲癇動物模式的影響
論文名稱(外文):Functional roles of human umbilical mesenchymal stem cell transplantation in adult epileptic rats
指導教授:林永煬林永煬引用關係
指導教授(外文):Yung-Yang Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:62
中文關鍵詞:人類臍帶間質幹細胞顳葉癲癇癲癇重積狀態毛果芸香鹼
外文關鍵詞:Human umbilical mesenchymal stem cellTemporal lobe epilepsyStatus epilepticusPilocarpine
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顳葉癲癇 (temporal lobe epilepsy) 為常見的神經疾病,特徵上包含自發性反覆癲癇發作 (spontaneous recurrent motor seizure,SRMS) 以及學習與記憶相關之損傷。然而,多數抗癲癇藥物 (antiepileptic drugs) 針對癲癇症狀提供治療時,卻無法緩和癲癇形成過程 (epileptogenesis) 。其中的神經病理機制也尚未完全了解。本實驗將探討人類臍帶間質幹細胞 (human umbilical mesenchymal stem cell,簡稱HUMSC) 移植到以毛果芸香鹼 (pilocarpine) 誘發之大鼠中產生的影響。主要探討目標為 (1) 量測海馬迴 (hippocampus) 中的分子與細胞變化程度、(2) 觀察SRMS的行為表現。實驗將雄性 Sprague-Dawley rats (200-300 g) 分成四組:(1) 非癲癇鼠、(2) 接受人類臍帶間質幹細胞移植之非癲癇鼠、(3) 癲癇鼠、 (4) 接受人類臍帶間質幹細胞移植之癲癇鼠。自腦室內 (intracerebroventricular) 注射pilocarpine (1.2 mg/μl; 2-3 μl) 誘發癲癇重積狀態 (status epilepticus),並在誘發1.5個小時後注射diazepam以抑制發作。於癲癇誘發後24小時,移植人類臍帶間質幹細胞 (105; 10 μl) 至左側海馬迴CA3區域,而於誘發癲癇重積狀態後第28天犧牲動物。利用人類臍帶間質幹細胞表面標誌物CD105,判別人類臍帶間質幹細胞之存活與分布情形。分別利用結晶紫染色法 (Nissl stain)、Timm染色法 (Timm’s stain) 及免疫組織染色法分析海馬迴神經細胞損失、苔狀纖維異常增生 (mossy fiber sprouting) 、神經細胞特異性核蛋白 (neuron-specific nuclear protein,NeuN) 及神經胜肽Y (Neuropeptide Y,NPY) 的表現量。在癲癇大鼠接受人類臍帶間質幹細胞移植組中,海馬迴CA3、CA1區域的神經細胞損失情形減少且NPY的表現量上升。同時,此組中有一半的實驗大鼠並未觀察到苔狀纖維異常增生的現象。於誘發癲癇重積狀態後之2至11週,癲癇鼠與接受幹細胞移植鼠在行為觀測上均無SRMS。我們觀察到僅癲癇鼠於第12週開始發生SRMS。本實驗結果顯示移植人類臍帶間質幹細胞至pilocarpine誘發之大鼠中,可能增進其細胞存活、調控其突觸重建 (synaptic reorganization) 以及延長首次SRMS發生的時間。
Temporal lobe epilepsy (TLE) is a common neurological disease characterized by spontaneous recurrent motor seizure (SRMS) and impairment of learning and memory. However, most antiepileptic drugs provide symptom treatment without alleviating the process of epileptogenesis. In addition, the exact neuropathological mechanisms still remain unclear. We plan to investigate the effects of human umbilical mesenchymal stem cell (HUMSC) transplantation in rats with pilocarpine-induced epilepsy model. Specifically, the present study aimed to (1) examine the molecular and cellular changes of hippocampal cytoarchitecture, and (2) observe the behavioral manifestation of SRMS. Male Sprague-Dawley rats (200-300 g) were divided into four groups: (1) control rats, (2) control rats receiving HUMSC grafts, (3) epileptic rats receiving HUMSC grafts, and (4) epileptic rats receiving sham grafts. Status epilepticus (SE) was induced by pilocarpine (1.2 mg/μl; 2-3 μl) via intracerebroventricular administration, and was suppressed with diazepam at 1.5 hours later. HUMSCs (105; 10 μl) were injected into left hippocampal CA3 region at the first day after SE. Animals were sacrificed at the 28th day after SE. The survival and distribution of HUMSC in the hippocampal were confirmed by the detection of its surface marker (CD105). Hippocampal cell loss, aberrant mossy fiber sprouting (MFS), neuron-specific nuclear protein (NeuN) and neuropeptide Y (NPY) expression, were assessed by Nissl stain, Timm’s stain, and immunohistochemistry, respectively. The neuron loss in CA1 and CA3 regions were decreased and NPY expression was increased in the epileptic rats receiving HUMSC grafts. Additionally, in this group, half of rats did not reveal MFS. The behavioral performance of SRMS were not observed in epileptic rats receiving sham grafts and epileptic rats receiving HUMSC grafts from 2 to 11 weeks post SE, whereas we observed that only the epileptic rats receiving sham grafts began to demonstrate SRMS at 12th week. In conclusion, HUMSC transplantation may promote cell survival, modulate synaptic reorganization, and prolong the occurrence of SRMS in pilocarpine-induced rats.
目錄 I
中文摘要 IV
Abstract VI
第一章 緒論 1
第一節 癲癇 1
1. 癲癇 1
2. 顳葉癲癇 2
3. 顳葉癲癇形成原因 2
4. 顳葉癲癇病理特徵 4
5. 顳葉癲癇與新生神經細胞 5
第二節 顳葉癲癇動物模式 6
1. 癲癇動物模式種類 6
2. 毛果芸香鹼 (pilocarpine) 7
第三節 癲癇治療方法與原理 8
1. 癲癇治療 8
2. 癲癇病灶切除術 9
3. 內生性抗癲癇物質 9
4. 抗癲癇藥物 11
5. 基因療法 12
第四節 人類臍帶間質幹細胞與幹細胞療法 13
1. 人類臍帶間質幹細胞 13
2. 幹細胞療法 14
3. 癲癇的幹細胞療法 15
4. 神經滋養因子與幹細胞療法 16
第二章 實驗目的 18
第三章 實驗材料與方法 19
第一節 實驗藥品 19
1. 人類臍帶間質細胞培養 19
2. 動物實驗 19
3. 組織染色實驗 20
4. 抗體 20
第二節 儀器設備與相關實驗材料 21
第三節 實驗方法 22
1. 人類臍帶間質細胞培養 22
2. 實驗動物及組別 22
3. 顳葉癲癇動物模式 23
4. 人類臍帶間質幹細胞移植流程 24
5. 組織型態研究之製備 24
6. 免疫組織染色法 25
7. 結晶紫染色法 26
8. Timm染色法 26
9. 自發性反覆癲癇發作之行為分析 27
10. 統計方法 27
第四章 結果 28
第一節 癲癇動物模式行為變化 28
第二節 癲癇動物模式對海馬迴結構變化之影響 28
第三節 人類臍帶幹細胞療法對顳葉癲癇動物模式之影響 30
第四節 人類臍帶間質幹細胞移植存活及分布情形 30
第五節 人類臍帶間質幹細胞移植對MFS之影響 31
第六節 人類臍帶間質幹細胞移植對NPY表現細胞之影響 31
第七節 人類臍帶間質幹細胞移植對SRMS之影響 32
第五章 討論 33
第一節 顳葉癲癇動物模式 33
第二節 癲癇所引起的神經損傷與幹細胞療法之關係 34
第三節 NPY與癲癇療法之關係 35
第四節 MFS與癲癇療法之關係 36
第五節 幹細胞療法抑制癲癇的可能治療機制 37
第六章 結論 39
第七章 參考文獻 40
第八章 圖表 50
第九章 附錄 62
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