人類同源箱基因 OTEX在正常睪丸組織及前列腺癌細胞株中之分子特性研究

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Title:	人類同源箱基因 OTEX在正常睪丸組織及前列腺癌細胞株中之分子特性研究 Molecular Characterization of Human Homeobox Gene OTEX in Normal Testis Tissues and Prostate Cancer Cell Line
Authors:	顏志宏 Jr-Hung Yan
Contributors:	醫學研究所
Keywords:	同源箱基因精子熟成睪丸演化 homeobox gene spermatogenesis testis evolution
Date:	2003
Issue Date:	2009-09-11 15:37:09 (UTC+8)
Abstract:	目前不孕症造成之原因，約略有五成來自男性，而其中之七成是由於精子生成過程中發生缺陷。在臨床上出現無精蟲（azoospermia）或精蟲極度稀少（severe oligozoospermia）的症狀。目前絕大多數仍舊原因不明，少部分是因賀爾蒙失調，或是因Y染色體異常。我們的長遠目標是希望了解基因與睪丸發育及精子成熟的關係，以便解開更多男性不孕的謎題。目前，人類基因定序計畫提供大量基因序列訊息，我們之前即利用degenerate oligonucleotide PCR方法找到一個在睪丸表現的新同源箱基因（homeobox gene）TSX1，我們在查詢TSX1之基因組序列時，發現TSX1位在人類的X染色體上，而且進一步意外地發現其上游30 Kb處有另一個同源箱基因，我們已經定序一個含有750 bp的clone並定名為TPX1 (Testis-Pairlike-Homeobox-1)，但這個基因去年2002年分別已被德國Christoph等人發表命名為OTEX及美國Wayen等人發表命名為hPEPP1。因此我們之後即以第一次發表之OTEX稱之。我們已利用北方轉漬法（Northern blot）的分析發現OTEX在人類的睪丸有明顯的表現。進一步我們利用原位雜交（in situ hybridization）的技術瞭解此基因OTEX在人類細精小管中有表現，因此推測此基因在精子生成中可能扮演重要的角色。再進一步利用較為敏感RT-PCR則發現OTEX在副睪、攝護腺及卵巢中也有表現。我們目前也了解OTEX 在2株不同的前列腺癌細胞(LNCaP, PC-3)及卵巢癌細胞(ES-2, PA-1)的表現情形，初步結果顯示OTEX會受到睪固酮荷爾蒙之刺激而表現，我們將進一步以一些可能調節睪固酮之藥物了解OTEX在前列腺癌細胞之表現情形。另外我們嘗試純化融合蛋白(fusion protein)以製備抗體再利用純化的抗體偵測OTEX 蛋白質在不同組織的表現，並且可藉以了解OTEX結合之DNA序列及蛋白質，讓我們更進一步確認OTEX可能扮演的角色。此外，我們利用Zoo blot分析，得知在不同物種中OTEX只在人類存在，這個結果說明OTEX可能是在演化後期才出現之基因，也暗示其在人類生理功能上之重要。 Approximately 50% of human infertility is attributable to male defects, 70~90% of which arises from impaired spermatogenesis. We have isolated a human testis specific homeobox gene, TSX1, through degenerate oligonucleotide PCR screen. Another testis expressed pair-like homeobox gene named TPX-1 (testis-pair-like-homeobox-1) was identified 30 kb upstream of TSX1 on the human X chromosome. This gene has been published with name OTEX by Christoph in April 2002 and hPEPP1 by Wayen in July 2002. We used RT-PCR and Northern blot analyses to confirm that OTEX is expressed in human reproductive organs and brain. In the LNCaP and PC3 prostate cell lines, we found OTEX is expressed in these cell lines. Zoo blot analysis was performed and showed that OTEX is only present in human genome. The results explained that the mouse ortholog of OTEX hasn’t been isolated after severed screenings of different mouse testis cDNA libraries. The expression pattern identified by in situ hybridization implied some functional information of OTEX. In the study of protein level, we constructed expression plasmids to generate OTEX-fusion protein from the bacteria. OTEX-fusion protein would be used to raise antibody, identify the target-binding site and immunohistchemistry study. We expect the study would provide more information for understanding genetic factors associated with male infertility
Data Type:	thesis
Appears in Collections:	[Graduate Institute of Medical Sciences] Dissertation/Thesis

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