臺灣博碩士論文加值系統

松果體與腦下垂體後葉均由間腦區的中央神經管發育而來，所以與非神經性來源之腺體組織結構不同。分泌褪黑激素的松果體內具有各種神經元特質的細胞，但即至目前為止，對此類細胞的屬性, 功能與發育一直未有完整的了解。相對的，腦下垂體後葉主要由下視丘中分泌催產激素之室旁核和分泌抗利尿激素之視上核所延伸的軸突與腦下垂體細胞所組成。 同樣的我們對於腦下垂體後葉的發育與腦下垂體細胞所扮演的角色亦仍有諸多不明瞭之處。神經元中間絲包含了α-internexin, peripherin以及神經三合體蛋白(NFL, NFM和NFH)。在發育過程中，α-internexin 與 peripherin會在周邊神經系統與中樞神經系統中大量表現。即至成體時，α-internexin多存在於中樞神經系統中; 而peripherin則存在於周邊神經系統內。在神經發育的時程中，較低分子量的神經元中間絲較早出現，先形成起始的架構，陸續較高分子量的神經元中間絲繼而產生聚合的作用。本研究的目的即以神經元中間絲為觀點，探討同樣由中央神經管為發生起源的松果體與腦下垂體後葉於發育時之表現。
利用免疫組織化學法與西方點漬法所得之結果，α-internexin 與 peripherin均於出生後21天時在松果體與腦下垂體後葉內之表現趨穩定。神經三合體蛋白，則在出生後90天的松果體與出生後30天的腦下垂體才開始有穩定的表現。不同年齡層的鼷鼠松果體內，均具有可以同時表達α-internexin 與 peripherin的細胞。 細胞體內產生神經元中間絲堆積之現象則發生於趨近老年鼷鼠的松果體中。以原位雜交與RT-PCR所得結果，證實鼷鼠松果體內確實具有自行合成α-internexin 與 peripherin的細胞。觀察發育變化的時程，松果體內測定出α-internexin- 與 peripherin- mRNA的細胞比例與蛋白質表現的結果相符，也於出生後7天時為最高表達期。綜合標定蛋白質與測定mRNA之結果，得知鼷鼠松果體中，不論在何種年齡層，表現α-internexin的細胞所佔之比率均高於Peripherin。運用原位雜交法觀察鼷鼠的腦下垂體後葉，意外的發現pituicytes具有α-internexin 與 peripherin的mRNA。再以RT-PCR的方法，也測得腦下垂體後葉具有α-internexin與NFM mRNA的表現。
由神經元中間絲在發育中松果體與腦下垂體內的表現，得知鼷鼠的腦下垂體後葉開始發育及其發育成熟的時間點均較松果體早，但鼷鼠松果體內之細胞多樣性與複雜性則較腦下垂體後葉高。至於分佈於周圍神經系統的peripherin，同時也存在於由中樞神經組織發育而來的松果體與腦下垂體後葉中，對此現象我們需有更多的觀察與後續之研究來加以探討。

The mammalian epiphysis (pineal gland) and neurohypophysis (posterior lobe of pituitary gland) originated ependymal cell of the roof and the floor of the third ventricle from central nerve canal respectively. In certain species, the pineal gland contains neurons and/or neuron-like peptidergic cells, The nature and cytological features of these neuronal cells, in addition to the pinealocytes, interstitial cells need to be classified. Posterior pituitary consists of pituicytes, the modified astrocytes and the predominant cellular type in the neurohypophysis, Development and the role of pituicytes still has many aspects remain to be clarified so far; and axons, originating from neurons in supraoptic and paraventricular nuclei synthesizing vasopressin and oxytocin. The neuronal intermediate filaments include not only the neurofilament triplet proteins but also α-internexin and peripherin. Both α-internexin and peripherin are expressed in the neurons of CNS and PNS during development. In adult, peripherin is found abundantly in the neurons of peripheral nervous system, and α-internexin is present mainly in the neurons of central nervous system. We employ neuronal intermediate filaments as probes into developing central neural glands of mouse.
Results from the immunohistochemistry and Western blot indicated that, not only pineal but also posterior lobe of pituitary, both α-internexin and peripherin were start constants expression at P21 and the neuronal triplets proteins were steady display at P90 for pineal gland and at P30 for neurohypophysis. α-internexin and peripherin are colocalized in the perikarya of some cells in the different age of mouse pineal gland. In aged (P360) mouse pineal gland, the expression of neuronal intermediate filaments is augmented as compare with that of the other adult stages. Results from the in situ hybridization and RT-PCR, showed that the number of the mRNA labeling cells of α-internexin and/or peripherin at P7 were the most numerous among all the age stages in mouse pineal. Taken all data together, we concluded that α-internexin-mRNA positive cells are generally more numerous than peripherin-mRNA positive cells during development, and therefore, we suggested that α-internexin-mRNA positive cells were more widely spread than peripherin-mRNA positive cells in pineal gland. In the neurohypophysis, by the in situ hybridization, the mRNAs of α-internexin and peripherin were unexpectedly found in the pituicytes. α-Internexin and NFM mRNAs were also detected in neurohypophysis of developing mouse by mean of RT-PCR. More studies are needed to elucidate the neuronal nature of these specialized glial cells, pituicytes in the neurohypophysis.
In summary, neurohypophysis is earlier than epiphysis not only in start developing time but also in mature period. The phenomenon may cause by more variety and complexity of pineal gland than posterior lobe of pituitary. That needs more investigation about both central glands existing peripherin.

目 錄
目錄------------------------------------------------------------------------------II
英文縮寫與全文對照表-----------------------------------------------------III
中文摘要-----------------------------------------------------------------------1
英文摘要-----------------------------------------------------------------------3
壹、緒論-----------------------------------------------------------------------5
貳、材料與方法-------------------------------------------------------------16
參、結果
A. 發育中鼷鼠的松果體神經元中間絲之表現----------------------23
I. 一般光學顯微鏡觀察--------------------------------------------------------23
II. α-internexin與peripherin相關結果--------------------------------------24
1. 免疫螢光染色法------------------------------------------------------------24
2. 螢光雙重免疫染色---------------------------------------------------------25
3. 原位雜交(in situ hybridization)-------------------------------------------26
4. 蛋白質免疫螢光染色與mRNA表現之相關性-----------------------26
5. 統計分析---------------------------------------------------------------------27
III. 神經三合體蛋白相關結果------------------------------------------------27
1. 免疫組織化學染色法-----------------------------------------------27
2. 西方點漬法 (Western blot)---------------------------------------------- 29
B. 發育中鼷鼠的腦下垂體後葉神經元中間絲之表現--------------30
I. 一般光學顯微鏡觀察--------------------------------------------------------30
II. 免疫組織化學染色法-------------------------------------------------------31
1. α-internexin-------------------------------------------------------------------31
2. peripherin---------------------------------------------------------------------31
3. α-internexin與peripherin 雙重免疫螢光染色------------------------32
4. NFL----------------------------------------------------------------------------32
5. NFM---------------------------------------------------------------------------33
6. NFH----------------------------------------------------------------------------33
III. 西方點漬法 (Western blot) -----------------------------------------------34
IV. 原位雜交法 (in situ hybridization) --------------------------------------34
V. 反轉錄聚合酶鏈鎖反應，RT-PCR---------------------------------------35
肆、討論
A. 發育中鼷鼠的松果體神經元中間絲------------------------------- 36
B. 發育中鼷鼠腦下垂體後葉神經元中間絲--------------------------45
C. 發育中鼷鼠松果體與腦下垂體後葉之比較-----------------------49
伍、總結-----------------------------------------------------------------------52
陸、後續可再進行之實驗--------------------------------------------------53
柒、參考文獻-----------------------------------------------------------------54
捌、圖表說明-----------------------------------------------------------------62

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