DC 欄位 | 值 | 語言 |
dc.contributor.author | 林中天 | zh_TW |
dc.creator | 林中天 | - |
dc.date | 2005 | zh_TW |
dc.date.accessioned | 2006-07-26T05:38:15Z | - |
dc.date.accessioned | 2018-07-09T06:18:49Z | - |
dc.date.available | 2006-07-26T05:38:15Z | - |
dc.date.available | 2018-07-09T06:18:49Z | - |
dc.date.issued | 2005 | - |
dc.identifier | 932313B002022 | zh_TW |
dc.identifier.uri | http://ntur.lib.ntu.edu.tw//handle/246246/28735 | - |
dc.description.abstract | 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)是近年的報告發現與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(前NSC project, 已發表於期
刊Breast Cancer Research and Treatment)。
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。在第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 situhybridization)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,第一年的成果已發表刊登於期
刊In vitro cellular and developmental
biology-Animal(2003)。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,
在本年度之計畫中進行更進一步的sFRP2 調控
細胞凋亡的功能分析。
在第二年度之研究方面,我們已非常辛苦
地分析確認sFRP2 基因確具有抗細胞凋亡之功
能,且分析複雜的調控細胞凋亡之相關訊息傳
遞途徑為fibronectin-integrin signal transduction
pathway,此重要發現為此新基因族之首篇調控
細胞凋亡功能訊息傳遞途徑之新發現,研究成
果也已發表。
在第三年度之研究方面,研究sFRP2 之抗
細胞凋亡與其他訊息因子或轉錄因子之關聯
性。實驗結果發現,表現sFRP2 基因之細胞受
到紫外光誘發細胞凋亡後,FAK 之tyrosine 磷
酸化程度會增加,且有NF-κB 之活性增加及
JNK 之活性抑制的情況。
本研究之結果,預期提供重要之學術資
訊,以了解sFRP 基因族在犬乳腺腫瘤細胞之
細胞凋亡調控之訊息傳遞情形。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,及了解乳腺腫瘤複雜之病因,提供
進一步研究分析之基礎。 | zh_TW |
dc.description.abstract | 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 (previous
NSC project, published in the Breast Cancer
Research and Treatment). 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 from the next stage
of the project.
In the 2nd year, apoptosis regulation
mediated by SFRP2 was investigated by
overexpression of SFRP2 in MGT and MCF7
cells. DNA fragmentation and caspase 3 activity
analyses showed that the susceptibility of the cells
to UV-induced apoptosis decreased in the context
of SFRP2 overexpression. To analyze the
pathways through which SFRP2 transduces
anti-apoptosis signals, co-immunoprecipitation
and cell adhesion assays were carried out. SFRP2
was found secreted from cells and associated with
the fibronectin-integrin protein complex and
could promote cell adhesion. Moreover, by using
heparin to block the SFRP2-fibronectin
interaction or anti-integrin 51 antibody to
interrupt the fibronectin-integrin connection, the
anti-apoptosis activity of SFRP2 was decreased.
Taken together, these results suggest that SFRP2
exert its anti-apoptotic function in mammary
cancer cells through association with the
fibronectin-integrin signal transduction pathway,
not the Wnt signaling as previous thought. The
important data has been published. In the 3rd year,
analysis of the relation of sFRP2 transduced
anti-apoptosis with other signaling and
transcription factors, multiple-color
immunofluorescence staining, immunoprecipitation,
and immunoblotting were carried out.
SFRP2 was found co-localized in the extracellular
matrix of MGTs and the tyrosine phosphorylation
of FAK was enhanced. Moreover, JNK was
suppressed and NF-kB was activated in the cells
expressing SFRP2 after UV-induced apoptosis
analyzed by immunoblotting and electrophoretic
mobility shift assay (EMSA). Taken together,
these results suggest that SFRP2 exerts its
anti-apoptotic function in mammary cancer cells
with NF-κB activation or JNK suppression.
The results of this project should offer
important scientific basis and information to
understand the roles of sFRP-mediated signaling
in canine mammary tumor 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 and signaling pathways
of mammary tumors. | en |
dc.format | application/pdf | zh_TW |
dc.format.extent | 1814563 bytes | - |
dc.format.mimetype | application/pdf | - |
dc.language | zh-TW | zh_TW |
dc.language.iso | zh_TW | - |
dc.publisher | 臺北市:國立臺灣大學獸醫學系暨研究所 | zh_TW |
dc.rights | 國立臺灣大學獸醫學系暨研究所 | zh_TW |
dc.subject | 分泌性細胞凋亡基因 | zh_TW |
dc.subject | 分泌性
frizzled 蛋白基因 | zh_TW |
dc.subject | 細胞凋亡 | zh_TW |
dc.subject | 訊息傳遞 | zh_TW |
dc.subject | 乳
腺腫瘤 | zh_TW |
dc.subject | secreted apoptosis related protein
secreted frizzled related protein | en |
dc.subject | apoptosis | en |
dc.subject | signal
transduction | en |
dc.subject | gene transfection | en |
dc.subject | mammary
neoplasia | en |
dc.subject.classification | [SDGs]SDG3 | - |
dc.title | 行政院國家科學委員會專題研究計畫成果報告: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(3/3) | zh_TW |
dc.type | report | en |
dc.identifier.uri.fulltext | http://ntur.lib.ntu.edu.tw/bitstream/246246/28735/1/932313B002022.pdf | - |
dc.coverage | 計畫年度:93;起迄日期:2004-08-01/2005-07-31 | zh_TW |
item.languageiso639-1 | zh_TW | - |
item.cerifentitytype | Publications | - |
item.fulltext | with fulltext | - |
item.openairecristype | http://purl.org/coar/resource_type/c_93fc | - |
item.openairetype | report | - |
item.grantfulltext | open | - |
crisitem.author.dept | Veterinary Clinical Sciences | - |
crisitem.author.orcid | 0000-0002-3872-152X | - |
crisitem.author.parentorg | College of Bioresources and Agriculture | - |
顯示於: | 獸醫學系
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