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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳義雄 | |
| dc.contributor.author | Hsuan Chen | en |
| dc.contributor.author | 陳 玄 | zh_TW |
| dc.date.accessioned | 2021-06-14T17:21:38Z | - |
| dc.date.available | 2011-08-05 | |
| dc.date.copyright | 2008-08-05 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-07-24 | |
| dc.identifier.citation | 賴明龍（1993）國立台灣大學生化科學研究所博士論文
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41173 | - |
| dc.description.abstract | 胰蛋白酶抑制因子P12由57個胺基酸所組成，是一個分子量6126 Da的鹼性不含醣基的蛋白，具有3對雙硫鍵，屬於Kazal-type胰蛋白酶抑制因子。在生殖系統及附屬性腺中，P12只有在儲精囊、前列腺和凝固腺有表現，並且其表現受到雄性激素的調控，成熟的雄性小白鼠才開始有mRNA和蛋白表現。在射精之後，P12會專一性結合於精子頂體而影響精子的活性。P12已被證實具有抑制精子吸收鈣離子的功能，以及精子的獲能作用、頂體反應、活動力和高度活化作用也會受到P12抑制。本論文目標研究是在精卵結合之前，結合於精子頂體的P12如何被移除的生理意義。
以酶譜法及蛋白酶活性的測定來分析子宮液中與P12有交互作用的蛋白酶，發現子宮液中有一個可以與P12結合的蛋白酶，分子量約33 kDa，不同於胰蛋白酶並且此蛋白酶的活性可以受到P12的抑制。此蛋白酶活性也會受到PMSF（phenylmethylsulphonyl fluoride）所抑制，而得知是一個絲胺酸蛋白酶。由質譜儀的定序，推測該蛋白酶可能是airway trypsin-like protease。經由間接免疫染色分析法的結果，證實子宮液中的絲胺酸蛋白酶可以移除結合於精子頂體的P12。 | zh_TW |
| dc.description.abstract | Trypsin inhibitor P12 consists of 57 amino acid residues in which six cysteines are crosslinked into three disulfide bonds. It is a protein of Mr 6126 Da with no glycoconjugate and belongs to Kazal-type trypsin inhibitor family. Among the reproductive tracts of male and female mice, P12 is only present in seminal vesicle, prostate and coagulating gland of adult male. P12 can specifically bind to sperm acrosome region to affect sperm activaty after ejaculation and it’s gene expression in these accessory sexual glands is androgen-dependent. It has verified that P12 can suppress a great extent of Ca2+-uptake by spermatozoa and has the ability to suppress capacitation, acrosome reaction, motility and hyperactivation of mouse spermatozoa. This work aims to study how to remove P12 on sperm acrosome before sperm-egg encounter.
Results of Zymography and protease activity assay demonstrated a 33 kDa P12-inhibited protease in the mouse uterine fluid. It was a serine-type protease, because it could be inhibited by PMSF (phenylmethylsulphonyl fluoride). It was likely to be airway trypsin-like protease, as suggested by the partial peptide sequences determined by MS/MS analysis. Result of indirect immunofluorescence statining assay demonstrated the removal of P12 on sperm acrosome by the serine protease in uterine fluid. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-14T17:21:38Z (GMT). No. of bitstreams: 1 ntu-97-R95b46020-1.pdf: 3064048 bytes, checksum: 530bf900f8d6d1b1bee846d4679e3719 (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 目 錄
縮寫表……………………………………………………………………………… v 摘要………………………………………………………………………………… vi Abstract…………………………………………………………………………… vii 第一章 緒論 1.1 小白鼠雄性生殖系統………………………………………………………… 1 1.2 精子的發生與結構…………………………………………………………… 2 1.3 精子的獲能作用……………………………………………………………… 3 1.4 儲精囊分泌蛋白的生理研究………………………………………………… 4 1.5 胰蛋白酶抑制因子P12在生殖生理上的研究……………………………… 5 1.6 小白鼠雌性生殖系統………………………………………………………… 6 1.7 子宮液相關研究……………………………………………………………… 7 1.8 精子與子宮液的相關生理研究……………………………………………… 8 1.9 本論文的研究方向………………………………………………………… 8 第二章 實驗材料與方法 實驗材料………………………………………………………………………… 9 2.1 重組胰蛋白酶抑制因子RP12的純化………………………………………… 9 2.1.1. 融合蛋白質的表現與細菌蛋白粗抽液的取得…………………………… 9 2.1.2. 親合性管柱層析（affinity column chromatography）…………………… 10 2.1.3. 分子篩膠體層析（gel filtration chromatography）與RP12取得……… 11 2.2. 子宮液的純化………………………………………………………………… 13 2.2.1. 子宮液的誘發與取得……………………………………………………… 13 2.2.2. 分子篩膠體層析（gel filtration chromatography）……………………… 13 2.3 蛋白質定量法 --- BCA蛋白質定量法……………………………………… 14 2.4 十二酯硫酸鈉-聚丙烯醯胺膠體電泳（SDS-PAGE Gel Electrophoresis）…… 16 2.5 蛋白質染色 --- Coomassie Brilliant Blue G250染色……………………… 18 2.6 蛋白質染色 --- 硝酸銀染色（silver nitrate staining）…………………… 18 2.7 西方點墨法（Western Blotting）…………………………………………… 20 2.8 P12親合性膠體的製備…………………………………………………… 22 2.9 酶譜法（Zymography）……………………………………………………… 23 2.10 蛋白酶活性的測定………………………………………………………… 24 2.11 精子頂體完整性的觀察…………………………………………………… 25 2.12 間接螢光免疫染色法（indirect immunofluorescence statining）………… 27 第三章 實驗結果 3.1 P12的製備…………………………………………………………………… 29 3.2 子宮液中的蛋白酶活性受到12的抑制……………………………………… 29 3.2.1 以分子篩膠體層析法分離子宮液中的蛋白質…………………………… 29 3.2.2 藉由酶譜法（Zymography）對子宮液中蛋白酶的活性之分析………… 30 3.2.3 P12對子宮液中的蛋白酶活性的抑制作用分析…………………………… 30 3.3 子宮液中的蛋白酶移除結合於精子P12的情形…………………………… 31 3.3.1 細胞染色的分析…………………………………………………………… 31 第四章 討論與未來展望………………………………………………………… 54 參考文獻…………………………………………………………………………… 57 圖目錄 圖一：重組胰蛋白脢抑制因子RP12的純化結果................................................... 33 圖二：以分子篩膠體層析法分離子宮液中的蛋白質.............................................. 35 圖三：以酶譜法（Zymography）分析子宮液中蛋白酶的活性............................. 37 圖四：P12對子宮液抑制活性的分析....................................................................... 39 圖五：P12的濃度相依性對UF3蛋白酶活性的變化.............................................. 41 圖六：PMSF的濃度相依性對UF3蛋白酶活性的影響.......................................... 43 圖七：精子頂體的完整性.......................................................................................... 45 圖八：胰蛋白酶移除結合於精子頂體上的P12....................................................... 47 圖九：UF3移除結合於精子頂體上的P12............................................................... 49 圖10：UF3移除結合於精子的P12能力之統計..................................................... 51 附圖一：雄性小白鼠的生殖系統………………………………………………….. 69 附圖二：精子的發生與成熟……………………………………………………… 70 附圖三：小白鼠精子的結構……………………………………………………… 71 附圖四：雌性小白鼠的生殖系統………………………………………………… 72 附圖五：P12的三級蛋白質結構………………………………………………… 73 附錄一：SDS-PAGE配方………………………………………………………… 74 表目錄 表一：以胰蛋白酶以及不同子宮液部分處理已結合P12的精子，利用間接免疫螢光法觀察P12被移除的情形之統計…………………………………… 53 | |
| dc.language.iso | zh-TW | |
| dc.subject | 精子頂體 | zh_TW |
| dc.subject | 蛋白酶 | zh_TW |
| dc.subject | 子宮液 | zh_TW |
| dc.subject | 抑制因子 | zh_TW |
| dc.subject | 胰蛋白酶 | zh_TW |
| dc.subject | P12 | en |
| dc.subject | sperm acrosome | en |
| dc.subject | protease | en |
| dc.subject | uterine fluid | en |
| dc.title | 小白鼠子宮液之絲胺酸蛋白酶移除結合於精子表面P12的研究 | zh_TW |
| dc.title | Removal of P12 (a Kazal-type Trypsin Inhibitor) on Mouse Sperm by Serine Protease in the Uterine Fluid | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 朱善德,曾婉芳,林翰佳 | |
| dc.subject.keyword | 胰蛋白酶,抑制因子,精子頂體,蛋白酶,子宮液, | zh_TW |
| dc.subject.keyword | P12,sperm acrosome,protease,uterine fluid, | en |
| dc.relation.page | 74 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-07-26 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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