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研究生:游美珠
論文名稱:鼷鼠輪廓乳突味覺接受細胞之發育與成熟:PGP9.5和Gustducin免疫組織化學之研究
論文名稱(外文):Development and Maturation of Taste Receptor Cells in Mouse Vallate Papillae : Immunohistochemical Studies on PGP9.5 and Gustducin
指導教授:黃怡仁黃怡仁引用關係
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:醫學研究所
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:2002
畢業學年度:90
語文別:中文
中文關鍵詞:神經纖維輪廓乳突PGP 9.5免疫染色synaptophysin免疫染色味蕾
外文關鍵詞:PGP 9.5α-gustducinsynaptophysintaste budsimmunohistochemistry
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本實驗應用神經標示物─PGP 9.5 、突觸前膜蛋白質─Synaptophysin和G蛋白α次單元家族蛋白質─α-gustducin的間接免疫過氧化氫酵素染色以及雙重免疫螢光方法來探討鼷鼠輪廓乳突發育成熟過程中味覺接受細胞分化形成時機。
PGP 9.5免疫組織化學染色實驗結果顯示:在胚胎第13天(E13)可清楚看到PGP 9.5免疫染色的神經纖維分布於新形成的輪廓乳突上皮層下方的結締組織,有少量的神經纖維穿入胚胎第14天(E14)輪廓乳突頂部的上皮層(apical epithelium),之後神經纖維再穿入胚胎第17天(E17)之輪廓乳突壕溝內外兩側壁的上皮層;PGP 9.5免疫染色的梭形細胞,首先被發現分佈於出生後第7天(P7)之乳突壕溝內外兩側壁上皮層內,雖然數量不多,但是PGP 9.5免疫染色之味蕾細胞數量隨發育年齡而逐漸增加,出生後第21天(P21)之輪廓乳突內PGP 9.5免疫染色的味蕾細胞和神經纖維分佈形式與成鼠輪廓輪廓乳突內PGP 9.5免疫染色之結果極為相似。另外,PGP 9.5和synaptophysin雙重免疫螢光染色結果: 在發育中及成熟鼷鼠輪廓乳突內PGP 9.5免疫染色的細胞同時具有synaptophysin免疫染色。
α-gustducin免疫組織化學染色結果顯示:在發育成熟過程中,鼷鼠輪廓乳突內沒有α-gustducin免疫染色的神經纖維。而在出生後第3天(P3)之輪廓乳突壕溝內側壁上皮層內有很少量梭形α-gustducin免疫染色的味蕾細胞,免疫染色之細胞數量隨動物發育年齡增長而逐漸增加。α-gustducin和synaptophysin雙重免疫螢光染色結果:雖然出生後第3天至第6天(P3~P6)之鼷鼠輪廓乳突內,α-gustducin免疫螢光染色的味蕾細胞未同時具有Synaptophysin免疫螢光染色,但是出生後第7天(P7)之鼷鼠輪廓乳突內,α-gustducin免疫螢光染色的細胞同時具有synaptophysin免疫染色。
根據本實驗研究結果,可以得到四個結論: (1)鼷鼠輪廓乳突的發育過程中神經纖維較味蕾細胞更早形成分佈於乳突內。(2) PGP 9.5免疫染色的味蕾細胞為味覺接受細胞並且會和神經纖維形成突觸。(3)鼷鼠輪廓乳突味蕾內味覺傳導機制,主要是在出生後之發育階段才逐漸分化完成。 (4) 與甜味和苦味傳導有關的味蕾細胞的分化,主要是在出生後之發育階段才開始進行。

The maturation of mouse vallate taste buds was investigated by means of indirect immunoperoxidase and double immunostaining methods using antibodies against neuronal marker, protein gene product 9.5 (PGP9.5), synaptic protein, synaptophysin, and G-protein  subunit, -gustducin.
PGP9.5 immunohistochemical studies revealed that nerve fibers distributed in the connective tissue below the epithelium of the newly forming papilla at E13. More nerve fibers penetrated both medial and lateral trench epithelium at E17. Only a few PGP9.5 immuoreactive cells were distinctly observed in addition to the nerve fibers at P7. However, the number of the PGP9.5 immuoreactive cells increased from P7 to P21. At P21, PGP9.5 immunoreactivities of nerve fibers and taste cells in the mouse vallate papilla resembled those of the adult mouse. Double-labeled studies showed that PGP9.5 and synatophysin colocalize.
-gustducin immunoreactive nerve fibers are not observed, but only a few taste cells are clearly seen in the medial trench epithelium of P3 mouse vallate papilla. More -gustducin immunoreactive cells are observed at P7, the number of α-gustducin immunoreactive taste cells increases from P7 to P21. Double-labeled studies show that α-gustducin and synatophysin almost coexist. In contrast, no synatophysin immunoreative taste cells were seen in vallate papillae before P7.
We suggest that, in the developing mouse vallate papilla, 1) nerve fibers in the trench wall epithelium of vallate papillae appear prior to taste cells, 2) PGP9.5 immunoreactive taste cells are bona fide taste receptor cells that form synaptic contacts with nerve fibers, 3) functional maturation, i.e. taste transduction, of vallate taste buds completes at the postnatal stage, and 4) the differentiation of taste cells concerning bitter and sweet taste transduction mainly begins to proceed at the postnatal stage.

目錄
壹、中文摘要.................................................1
貳、英文摘要.................................................3
參、緒論.....................................................5
肆、研究材料與方法
實驗動物.................................................16
組織準備與收集...........................................17
組織學觀察─Epon Embedded Tissuses.......................18
免疫組織化學染色法.......................................19
伍、結果.....................................................22
陸、討論.....................................................32
柒、圖片與說明...............................................43
圖1......................................................44
圖2......................................................45
圖3......................................................46
圖4......................................................47
圖5......................................................48
圖6......................................................49
圖7......................................................50
圖8......................................................51
圖9......................................................52
圖10.....................................................53
圖11.....................................................54
圖12.....................................................55
圖13.....................................................56
圖14.....................................................57
捌、參考文獻.................................................58

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