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研究生:陳文傑
研究生(外文):Wen-Chieh Chen
論文名稱:吳郭魚腦神經原始細胞株(TB2)的特性
論文名稱(外文):Characterization of a Putative Neural Progenitor Cell Line Derived from Tilapia Brain
指導教授:溫秋明
學位類別:碩士
校院名稱:國立高雄大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:82
中文關鍵詞:A2B5星狀神經膠細胞GFAP寡突神經膠細胞神經前驅細胞硬骨魚類
外文關鍵詞:A2B5AstrocyteGFAPOligodendrocyteneural progenitor cellTeleost
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魚類和哺乳類動物中樞神經系統最大的不同,是魚類終其一生可以產生新的神經元,基於此項理由,魚類的中樞神經系統是研究中樞神經發育很好的材料。TB2是從吳郭魚腦組織分離出來的持續性細胞株,已經繼代超過100次。本實驗使用細胞免疫染色(immunocytochemistry),西方點墨法 (Western blot)及反轉錄聚合酶連鎖反應 (RT-PCR)等方法,鑑別TB2細胞是否具有神經細胞的特性。

實驗的結果,TB2細胞在免疫染色的實驗中可以表現神經元細胞的標記GABA,星狀神經膠細胞的標記glial fibrillary acidic protein (GFAP)、vimentin、S100,寡突神經膠細胞的標記galactocerebroside (GalC),寡突前驅細胞的標記A2B5,其中表現 GFAP (63%)、vimentin (71%)、S100 (61%)表現的細胞量較多,表現GABA (12%)、A2B5 (12%)、 GalC (14%)表現的細胞量較少。Western blot證實TB2所表現的tyrosine hydroxylase (TH)、GFAP、vimentin、glutamine synthetase (GS)、S100和myelin basic protein (MBP)的分子量大致和吳郭魚腦組織相同。在RT-PCR確認TB2之GFAP序列和吳郭魚腦組織相同,另外本研究也證實TB2細胞會表現cytokeratin 8 mRNA。

由於TB2細胞株表現神經元、星狀神經膠、寡突神經膠和寡突前驅細胞的標記,所以推測TB2細胞為一種神經前驅細胞,可能具有產生神經元、星狀神經膠和寡突神經膠的能力。然而,由於本論文並未證實TB2細胞會表現神經幹細胞的專一性標記,所以仍需要更多的研究才能證實TB2細胞是是否為多能性的神經幹細胞。
The most difference between fish and mammalian central nervous system (CNS) is the fish produce new neurons throughout their lifespan. Therefore, it is suggested fish CNS is a well material to study CNS development. A cell line designated TB2 has been established from tilapia brain and has been subcultured more than 100 times. In this study, TB2 cells were characterized by using immunocytochemistry, Western blot and RT-PCR methods to verify if they have neuronal and glial characters.

The immunocytochemistry results showed TB2 cells express GABA, astrocyte markers glial fibrillary acidic protein (GFAP), vimentin, S100, oligodendrocyte marker galactocere- broside (GalC) and oligodendrocyte precursor marker (A2B5). Most of the cells were identified express GFAP (63%), vimentin (71%) and S100 (61%). GABA (12%), A2B5 (12%) and GalC (14%) were observed in some of the cells. Western blot analysis also verified TB2 cells can express tyrosine hydroxylase (TH), GFAP, vimentin, glutamine synthetase (GS), S100 and myelin basic protein (MBP) and the molecular weights are similar to those brain tissue. The RT-PCR results showed TB2 cells derived from tilapia brain consider that TB2 cells and tilapia brain have the same GFAP sequence. In addition, cytokeratin 8 that has been reported in nerve tissue was also demonstrated appear in TB2 cells by RT-PCR.

Since TB2 cells were identified coexpress the glia-specific and neuron-specific molecular markers, it is suggested TB2 cells might be the neural stem cells than can produce neurons, astrocytes and oligotentrocytes in the cultures. However, as no neural stem cell specific marker was evidence in TB2 cells, more studies are need to verify.
目錄
圖目錄
中文摘要
英文摘要
第一章 緒論
1.1 中樞神經細胞的種類及功能
1.1.1 神經元 (Neuron)
1.1.2 星狀神經膠細胞 (Astrocyte)
1.1.3 寡突神經膠細胞 (Oligodendrocyte)
1.1.4 微神經膠細胞 (Microglia)
1.1.5 室管膜細胞 (Ependymal cells)
1.2中樞神經細胞的發育和分化
1.2.1 GRP (glial restricted precursor)
1.2.2 OP (oligodendroglia precursor)
1.2.3 O2A (oligodendrocyte type 2 astrocyte progenitors)
1.2.4 Radial glia
1.2.5 WMPC (white matter progenitor cell)
1.2.6 APC (astrocyte restricted precursors)
1.2.7 MNOP (motoneuron-oligodendrocyte precursor)
1.3魚類和哺乳類CNS神經細胞的差異
1.4魚類的神經細胞體外培養
1.5 論文研究目的
第二章 實驗材料與方法
2.1 細胞繼代培養
2.1.1 實驗材料
2.1.2 實驗方法
2.2 細胞免疫螢光染色法
2.2.1 實驗材料
2.2.2 實驗方法
2.2.2.1 細胞固定
2.2.2.2 細胞染色
2.2.2.3 計算表現抗體細胞數目的百分比
2.3 西方點墨法
2.3.1 實驗材料
2.3.2 實驗方法
2.3.2.1 蛋白質濃度測定
2.3.2.2 膠體製備
2.3.2.3 電泳
2.3.2.4 膠體轉漬
2.3.2.5 免疫呈色反應
2.4 RT-PCR
2.4.1 實驗材料
2.4.2 實驗方法
2.4.2.1 萃取TB2 total RNA
2.4.2.2 萃取吳郭魚腦組織total RNA
2.4.2.3 Primers
2.4.2.4 cDNA合成
2.4.2.5 聚合酶連鎖反應
2.4.2.6 DNA膠體萃取
2.4.2.7 DNA定序,並作序列比對
第三章 實驗結果
3.1 TB2表現星狀神經膠細胞的標記GFAP、vimentin、S100
及GS
3.2 TB2表現寡突神經膠細胞的標記GalC、MBP及寡突神經
膠前驅細胞的標記A2B5
3.3 TB2表現神經元細胞的標記GABA和TH
3.4 TB2表現cytokeratin 8的mRNA

第四章 討論
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