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研究生:魏志安
論文名稱:促進成年海馬迴神經前驅細胞增殖的藥物篩選
論文名稱(外文):Promoting proliferation of adult hippocampal neural progenitor cells in vitro
指導教授:賴桂珍
學位類別:碩士
校院名稱:國立政治大學
系所名稱:神經科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
畢業學年度:102
語文別:中文
論文頁數:106
中文關鍵詞:海馬迴成年神經新生神經球神經前驅細胞
外文關鍵詞:hippocampusNeural progenitor cellsneurospheresAdult neurogenesis
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在成年的哺乳類動物大腦中有兩個區域,可以不斷的有新的神經細胞生成,一個位於大腦側腦室旁內側(Subventricular zone of anterior lateral ventricle ;SVZ),另一個位於海馬迴(hippocampus)內的齒狀迴(Subgran-
ular zone of dentate gyrus ;SGZ) ,其中海馬迴是本論文主要探討的腦區。
神經前驅細胞(Neural progenitor cells :NPC)因具有自我更新(self
-renewal)、增殖(proliferative)、多能(multipotent)的能力以及遷移性(Migration),所以可利用海馬迴內生性的神經前驅細胞(NPC),促進其增殖以替代因損傷、老化或疾病而損失的神經細胞。神經前驅細胞經由細胞體外培養過程會形成神經球(Neurospheres),神經球和神經前驅細胞同樣具有自我更新以及可以分化成其他神經細胞的能力。
本研究觀察到,對成年神經新生進行體外藥物的篩選中,化合物Chemical-X,有明顯的促進神經新生的能力。實驗中取健康成年雄性大鼠為實驗動物,分離出成年大鼠之海馬迴神經前驅細胞。用Chemical-X處理後,觀察神經球自我更新能力,以及再把新生成的神經球利用免疫螢光染色處理,瞭解神經前驅細胞經藥物處理後所新生成的細胞,是否仍維持在神經前驅細胞的狀態。進而評估藥物能否達到促進神經新生的目的。

中文摘要………………………………………………………………Ⅰ
英文摘要………………………………………………………………..Ⅱ
目錄……………………………………………………………………Ⅳ
圖次……………………………………………………………………Ⅷ
表次……………………………………………………………………ⅩV
縮寫對照表……………………………………………………………….…ⅩⅣ
第一章、緒論………………………………………………………… 1
第一節 成年神經新生(Adult neurogenesis)…………………… 1
一、成年神經新生(Adult neurogenesis)的區域…..………… 1
第二節 海馬迴(Hippocampus)構造………………………… 3
一、海馬迴的學習記憶功能………………………………………. 4
二、神經前軀細胞(Neural progenitor cells, NPC)特性………... 5
三、成年神經新生發育過程的細胞標記(Cell maker)……….….. 6
第三節 影響成年神經新生的因素………………………………… 7
一、腦內微環境 (niche)…………………………………………. 7
二、環境(Environmental).……………………………………..8
三、荷爾蒙(hormones)….………………….…………………9
四、運動(Exercise).…..…….……………………………....11
五、學習 (Learning)….………………………………………….11
六、老化 (Aging)……………..………………………………….12
七、藥物 (Drug)…………..……………………………………13
第四節 天然化合物…………………………..…………………………..15
一、厚朴的介紹….…………………………...………………....15
二、和厚朴酚藥理作用…….……..….…………….…….…......16
第五節 研究目的與策略…………….……………………………………17
第二章、實驗材料與研究方法..……………………………………………...18
第一節 實驗材料…………………………………………………………...18
一、細胞培養材料………….…………………..…………..……18
二、化學藥品……………………………………….……………18
三、抗體..………………..………..…..……………….………...20
四、實驗儀器.…………………………………………………….20
五、 軟體…………………………………..…………….……….21
六、實驗藥品配製…………………...…………………………..21
七、實驗動物..……………………………………..…………….22
第二節 成年海馬迴神經前驅細胞(neural progenitor cells)分離
培養………………………………………………….............… 23
一、海馬迴神經前驅細胞的分離………………………………...23
二、細胞計數…………...…………………………………………24
三、神經球(neurosphere)的計算……………………………….24
四、細胞培養皿塗層……………………………………….……24
五、培養基的配置……….………….. ……………….………... 25
六、酵素的製備(Enzyme cocktail)…..…….…………………...25
七、Percoll的製備…..……………………………….…….……26
八、phosphate buffer saline(PBS)的製備……….……………26
九、DPBS-glucose平衡鹽溶液的製備……………………..…26
十、Harvest medium(HM)的製備……………………………...26
第三節 免疫螢光染色(Immunofluorescence)................….…….…….…27
一、細胞免疫螢光染色 .…..…….……..………………………27
二、IdU, CldU 細胞免疫螢光染色…..….………………..……28
三、封片膠的製備………..……….…..……..…………….……28
第四節 細胞螢光強度分………………………………….….……………..28
一、Nestin/GFAP螢光強度分析………….……………………29
二、IdU/CldU螢光強度分析…….…...………………....……29
第五節 實驗數據分析…………………………………...…………………30
第三章 結果…………………………………………...………………………31
第一節 分離培養的神經前驅細胞(NPC)組成及分期……..……….....…31
第二節 Microglia的分離與神經前驅細胞(NPC)純度的提高………..…36
第三節 和厚朴酚對體外神經前驅細胞(NPC)的影響……………………39
第四節 Sonic hedgehog對體外神經前驅細胞(NPC)的影響………......52
第五節 Chemical-X對體外神經前驅細胞(NPC)的增殖分析…………..59
第六節Chemical-X藥物對體外神經前驅細胞(NPC)的增殖後分析.…..73
第四章 討論……………………………………………………………..……77
第五章 結論……..……………………………………………………………80
參考資料 …………………………………………………………………....81





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