https://scholars.lib.ntu.edu.tw/handle/123456789/163783
標題: | 行政院國家科學委員會專題研究計畫 期中精簡報告:細胞凋亡調控基因(sFRP2)在犬乳腺腫瘤之細胞凋亡調控角色及相關調節因子的研究(1/3) | 作者: | 林中天 | 關鍵字: | 分泌性細胞凋亡基因;分泌性frizzled蛋白基因;基因表現;基因轉染;乳腺腫瘤;secreted apoptosis related protein secreted frizzled related protein;apoptosis;gene expression;gene transfection;mammary neoplasia | 公開日期: | 2003 | 出版社: | 臺北市:國立臺灣大學獸醫學系暨研究所 | 摘要: | 乳腺腫瘤對動物與人類皆是重要且常見的腫瘤,其產生的病因是複雜為多因子牽涉的結果。Secreted frizzled related proteins (sFRPs)是最近被發現與Wnt-Frizzled訊息傳遞傳導路徑的調節和細胞凋亡(apoptosis)的調節中扮演著雙重角色的蛋白質。我們實驗室最近發現sFRP2基因在人類與犬隻乳腺腫瘤中有大量的表現及活化,但是在正常犬乳腺組織中則沒有表現。我們為了有系統地進一步研究犬隻乳腺腫瘤中的sFRP2在功能上的角色與腫瘤分子生物學上的機制,擬定了下列的幾項研究策略。 這個計畫包括了六個主要的部分,需要3年的研究時間:第一年首先主要的工作在於建立並分析多種新犬隻乳腺腫瘤組織的初代培養(primary culture)細胞株並且純化、分析乳腺上皮細胞。這一年我們已成功地建立並分析數個本地病例之犬乳腺腫瘤細胞株。這些細胞利用下列技術分析其特性,包括增殖速度(by MTT assay)、 反轉錄鏈聚合脢反應(RT-PCR)、原位雜交法(in situ hybridization)與免疫化學染色(immunohistochemistry)及西方墨點法偵測sFRP2的表現。結果發現sFRP2基因之mRNA及蛋白質在犬乳腺腫瘤細胞株有大量之表現,然而在犬正常乳腺細胞及其他非MGT細胞株則無表現。在第二階段,犬sFRP2被轉殖入含有GFP基因與CMV啟動子的哺乳類細胞表現載體,藉由lipofection方式將GFP-sFRP2穩定地轉染入(transfect)犬隻乳腺腫瘤初代培養與商品化乳腺腫瘤的細胞株,以進行更進一步的sFRP2功能分析。 本階段之研究結果,預期將提供重要之學術資訊,以了解 sFRP 基因族在犬乳腺腫瘤細胞之表現情形。此外,此計劃也為未來下一階段進一步研究 sFRP 基因族不同成員之各種功能,及了解犬乳腺腫瘤複雜之病因,提供進一步研究分析之基礎。 Mammary neoplasms are important and common tumors in both animals and humans and the etiology is complex. The secreted frizzled related proteins (sFRPs) are newly identified proteins and implicated to have dual roles of modulation of Wnt-Frizzled signal transduction pathway and regulation of apoptosis. We have recently found that sFRP2 was expressed abundantly in human and canine mammary gland tumors (MGT) tissues but was undetectable in normal canine mammary gland. To systematically investigate the functional roles and molecular mechanisms of sFRP2 in canine MGT, several strategies are to be carried out as described below. The project is comprised of six major parts for a period of 3 years: In the 1st year, new primary cell cultures from native canine MGT tissues has been established and purified for mammary epithelial cells. We have successfully established and analyzed more native primary MGT cell lines from surgically excised MGT specimens. The cells are characterized for their cell origins, proliferation rate (by MTT assay), expression of sFRP2 by RT-PCR, in situ hybridization, and immunohistochemistry, and Western blotting. Expression experiments revealed the sFRP2 was abundantly expressed in canine MGT cell lines, but not expressed in normal canine MG cells nor other commercial non-MGT cell lines. Canine sFRP2 is cloned into a mammalian expression vector with GFP reporter gene and CMV promoter. The GFP-sFRP2 is stably transfected into primary canine MGT and commercial MGT cell lines by lipofection for further analysis feom the next stage of the project. The results of this stage of the project should offer important scientific basis and information to understand the roles of sFRP gene family in canine neoplastic cells. It also provides a basis for further analysis of functions of different members of the sFRP gene family and elucidation of the complex etiology of canine model of mammary tumors. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/28722 | 其他識別: | 912313B002404 | Rights: | 國立臺灣大學獸醫學系暨研究所 |
顯示於: | 獸醫學系 |
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912313B002404.doc | 1.16 MB | Microsoft Word | 檢視/開啟 |
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