臺灣博碩士論文加值系統

　　記憶可簡單的分為兩種形式：短期記憶和長期記憶，兩者最大的差別在於長期記憶的形成需要新的核糖核苷酸和蛋白質合成。本論文主要目的在探討大白鼠杏仁核中與情境之恐懼制約學習記憶形成相關的基因表現，以及具有差異表現的基因在大腦各個區域的表現位置。本論文採用抑制性躲避學習的行為模式，首先以聚合酶連鎖反應差異表現法尋找表現量有差異的基因。結果發現在學習後3小時和6小時記憶好的大白鼠，testican-1基因片段表現量比對照組多。雖然後續用real-time反轉錄聚合酶連鎖方法並沒有確認testican-1基因在學習後6小時有增加的現象，但經由北方點漬法發現，大鼠的testican-1基因只表現在大腦中，且以原位雜合法發現其在大腦中有特殊次區域的表現情形。於是我們進一步詳細觀察其在腦中的表現情形並比較同家族的testican-2和testican-3基因的表現情形。cDNA選殖的結果發現大鼠testican-1 cDNA全長4452個鹽基，可演譯出441個胺基酸，且此胺基酸序列分別與人類和小鼠的testican-1胺基酸序列有95%和97%的同源性。以原位雜合法觀察tesican基因在大腦中各個腦區的表現，發現testican-1和testican-2的mRNA表現在多種神經細胞的細胞質中，testican-1在杏仁核有明顯次核區表現量上的差異，特別是與此行為模式有關的底側核、側核及中央核的內側區內有明顯的表現。而testican-2和3在杏仁核表現量較testican-1少，且沒有明顯次核區表現上的差異。Testican-1及2 的mRNA在海馬回的CA3、CA4表現量很多但在CA1和CA2表現量少。在齒狀回的顆粒細胞層沒有testican-1基因表現，但testican-2和3則有。在大腦皮質層及腦島皮層，testican-1及testican 2 mRNA主要表現在第五層。在小腦區域，testican-1，2和3在Purkinje cells表現量最多。目前對testicans在神經系統中真正扮演的功能和其功能的作用機制尚不清楚，不過根據其蛋白質可能的結構和基因的表現位置，猜測在成熟大白鼠的神經系統中，testican基因可能具有突觸型態及結構可塑性的調節功能。

　　Memory can be divided into two distinct forms: short-term memory long-term memory. Unlike short-term memory, long-term memory requires de novo mRNA and protein synthesis. The specific aim of this study is to analyze gene expression associated with contextual fear conditioning memory formation in the rat amygdala and examine the regional and subregional expression of the differentially expressed gene in the brain by in situ hybridization. Inhibitory avoidance learning was used as the behavioral model. Total amygdaloidal RNA isolated from good memory and control rats were subjected to PCR differential display analyses. One cDNA fragment corresponding to the testican-1 gene showed higher expression at 3 and 6 hours after inhibitory avoidance learning. Full length cDNA of the rat testican-1 was cloned and this 4452-bp cDNA sequence encodes a putative protein sequence containing 424 amino acids, which shows 95% and 97% homology to the human and the mouse testican-1, respectively. Differential expression of testican-1 gene was not confirmed by real-time RT PCR analyses. However, Northern hybridization demonstrated that testican-1 was mainly expressed in the brain and in situ hybridization showed subregional expression of the testican-1 gene in the brain. The distinct expression pattern of testican-1 gene in the brain was then compared with two recently cloned genes, testican-2 and testican-3. The testican-1 and testican-2 were expressed in the cytoplasm of different kinds of neuronal cells. The testican-1 gene has distinct subregional expression in the amygdala, notably in the lateral and basolateral nuclei, and medial portion of the central nuclei, nuclei that are involved in this learning model. Nevertheless, testican-2 and testican-3 have lower expression than testican-1 and no distinct subregional expression in the amygdala. Expression of these three genes was also examined in other brain areas. The mRNA level of testican-1 and testican-2 genes were highly expressed in CA3 and CA4, but rare in CA1 and CA2. In the granular layer of dentate gyrus, the mRNA levels of testican-2 and testican-3 can be detected but not that of the testican-1. In the cortex and insular cortex, both testican-1 and testican-2 were mainly expressed in the layer V. In the cerebellum, testican-1, -2 and -3 genes were strongly expressed in the Purkinje cell layers. The hybridization signals of testican-3 gene in these regions were too weak to be detected. The functional insight about testican-1 was discussed through its putative protein structure and expression pattern of the mRNA in various brain tissues. It is suggested that testican genes may have the abilities to regulate the neuronal morphology and synaptic plasticity in the brain.

誌謝.............................................1
圖表目錄.........................................4
中文摘要.........................................6
ABSTRACT.........................................7
第一章　緒論......................................8
　一、學習與記憶的定義.............................8
　二、記憶之認知心理學範疇.........................8
　三、記憶的分類...................................8
　四、情緒記憶...............................................9
　　1、情緒記憶的重要性..............................9
　　2、情緒記憶的主軸..............................................10
　　3、恐懼制約和學習記憶之神經投射.................10
　五、記憶之生物學範疇............................12
　　1、細胞層次.....................................12
　　　A.神經細胞之可塑性（neuronal plasticity)12
　　　B.突觸結構的修飾 vs.學習記憶............13
　　2、分子層次.....................................13
　六、PROTEOGLYCAN VS. 突觸可塑性(SYNAPTIC PLASTICITY).......15
　七、實驗目的...................................16
第二章　實驗材料和研究方法......................17
　一、實驗動物...................................17
　二、抑制性躲避學習與記憶測試...................17
　四、聚合酶連鎖反應差異表現法（PCR DIFFERENTIAL DISPLAY）........20
　五、CDNA選殖（CDNA CLONING）及定序分析
　　　（SEQUENCE ANALYSIS）........................21
　七、噬菌體 CDNA庫篩選法（PHAGE CDNA LIBRARY SCREENING）...27
　八、SYBR GREEN I REAL-TIME RT PCR..............31
　九、北方點漬分析法 (NORTHERN BLOT ANALYSIS )..................................35
　十、放射線標定原位雜合法
　　　（RADIOACTIVE IN SITU HYBRIDIZATION）...............38
　十一、非放射線標定原位雜合法
　　　（DIG-LABELED RIBOPROBE IN SITU HYBRIDIZATION）.............44
第三章　結果....................................49
　一、差異表現法篩選與記憶形成相關的基因表現.....49
　二、選殖PG5-1片段和定序分析....................49
　三、大白鼠TESTICAN-1全長CDNA之選殖.............50
　四、SBRY GREEN I REAL-TIME PCR 定量杏仁核TESTICAN-1
　　　基因之表現量..................................52
　六、放射線和非放射線原位雜合分析TESTICAN-1基因在杏仁核區
　　　及其他腦區的表現.........................53
　七、北方染漬法分析大白鼠各週邊組織之TESTICAN-2
　　　和TESTICAN-3基因.................................55
　九、放射線原位雜合分析TESTICAN-3基因之訊息核糖核苷酸
　　　在大腦的表現.................................57
第四章　討論....................................58
參考文獻......................................65
圖表...........................................71
附錄一 研究方法補充..........................104
附錄二 溶液的配製和使用藥品產牌一覽表........110
自述..........................................115

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