林中天2006-07-262018-07-092006-07-262018-07-092002http://ntur.lib.ntu.edu.tw//handle/246246/28698分泌性細胞凋亡蛋白或稱分泌性frizzled 蛋白(secreted frizzled related protein， sFRP)有調控Wnt-Frizzled 信號傳遞途徑及細 胞凋亡活動之雙重功能。Wnt-Frizzled 信號傳 遞途徑在維持正常發育扮演十分重要之角色。 此信號傳遞途徑之改變或異常，將導致腫瘤之 發生。 本計劃分為三個部份:第一部分進行自外 科切除之犬乳腺腫瘤組織之新細胞株建立，以 作為sFRP 基因轉染(gene transfection )之 用。這一年我們已成功地建立數個本地病例之 犬乳腺腫瘤細胞株。將一部份組織保存於DMEM 組織培養液(加入10% 胎牛血清及抗生素)，作 為初代細胞培養之用。第二部份分析sFRP 基 因群在犬乳腺腫瘤細胞之mRNA 及蛋白質表現方 面，利用northern blotting hybridization， RT-PCR，原位雜交法及免疫組織化學染色等分 析技術。結果發現sFRP2 基因之mRNA 及蛋白質 在犬乳腺腫瘤細胞有大量之表現，然而在犬正 常乳腺細胞則無表現。 在計劃之第三部份，分析乳腺細胞之細胞凋 亡現象，包括受sFRP 轉染之犬乳腺腫瘤細胞、 犬正常乳腺細胞及未受sFRP 轉染之細胞，以了 解在犬腫瘤細胞之細胞凋亡現象是否受到sFRP 基因調控之影響而產生改變。利用 lipofection 方法把sFRP 基因傳送(gene delivery)入乳腺細胞並給予紫外光誘發細胞 凋亡，結果發現經sFRP2 基因轉染之犬乳腺腫 瘤細胞具有顯著抗紫外光造成之細胞凋亡現 象，相較於未經sFRP2 轉染之犬乳腺腫瘤細胞 及犬正常乳腺細胞，sFRP2 基因在犬乳腺腫瘤細 胞具有顯著抗細胞凋亡之功能。 本計劃之研究結果，預期將提供重要及創 新之學術資訊，以了解sFRP 基因族在犬乳腺 腫瘤細胞之表現及調控細胞凋亡情形。此外， 此計劃也為未來進一步研究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。The secreted apoptosis related protein (also named secreted frizzled related protein, sFRP) family is implicated to have dual roles of modulation of Wnt-Frizzled signal transduction pathway and regulation of apoptosis. The Wnt-Frizzled signaling plays an important role in normal development and oncogenesis, particularly in mammary neoplasia. Therefore, members of the sFRP gene family are good candidates to investigate their roles in apoptosis regulation and mammary tumors of canine species. The project is comprised of three major parts: at the first stage, primary canine mammary gland tumor (MGT) cell lines were established from freshly excised MGT specimens. An aliquot of tumor tissues was rinsed by sterile PBS and kept in DMEM medium supplemented with 10% fetal calf serum for primary cultures. We have successfully established more native primary MGT cell lines from surgically excised MGT specimens. At the second stage, the primary MGT cell lines were analyzed for mRNA and protein expression of sFRPs. RNA was extracted from MGT cells. extraction. Expression analysis included mRNA in situ hybridization, immunohistochemistry, RT-PCR, and northern blotting hybridization. Expression revealed the sFRP2 was abundantly expressed in canine MGT cell lines, but not expressed in normal canine MG cells nor human breast cancer cell line MCF7. At the third stage, primary MGT cell lines have been prepared for gene transfection. sFRP genes were transfected into MGT cells using a mammalian expression vector by lipofection and UV-induced apoptosis (25 J/m2) was performed. The apoptosis activity was investigated in sFRP-transfected MGT and MG cells, untransfected MGT and MG cells, and human breast cancer cell line MCF7, respectively. Results showed the sFRP2 transfected MGT cells possessed marked anti-apoptotic activity, compared to cells from untransfected MGT cells, and normal MG cells. The results of the project should offer important and novel scientific information to understand the roles of sFRP gene family in apoptosis control of canine normal and 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.application/pdf109135 bytesapplication/pdfzh-TW國立臺灣大學獸醫學系暨研究所分泌性細胞凋亡基因分泌性frizzled蛋白基因細胞凋亡Wnt-Frizzled 信號傳遞途徑基因表現基因轉殖乳腺腫瘤secreted apoptosis related proteinsecreted frizzled related proteinapoptosisWnt-Frizzled signal transduction pathwaygene expressiongene transfectionmammary neoplasia[SDGs]SDG3reporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/28698/1/902313B002294.pdf