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Identification of estrogen-responsive cells involved in cell proliferation in response to estrogen

鉴定参与雌激素反应的细胞增殖的雌激素反应细胞

基本信息

批准号：
15590206
负责人：
ARITA Jun
金额：
$ 2.24万
依托单位：
University of Yamanashi
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590206/
关键词：
estrogen cell proliferation prolactin anterior pituitary gland estrogen receptor

项目摘要

Research project 11)Using rat anterior pituitary cells in culture, we established triple immunostaining for Ki67, estrogen receptor (ER), and prolactin (PRL).2)When estrogen stimulated proliferation of lactotrophs, the proportion of ER-imnumoreadive cells in proliferating lactctrophs was decreased. The proportion of the ER-immunoreactive cells was not changed when estrogen inhibited IGF-1-induced proliferation.Research project 21)We produced recombinant adenovirus vectors that carry PRL promoter-driven dominant negative ER mutants.2)Using an adenovirus vector that carries PRL promoter-driven luciferase reporter gene, infection conditions in pituitary cultures were addressed. A maximal infection efficiency was obtained by infection at 5 MOI during the early culture period in the presence of insulin. Even at this infection efficiency, the proportion of reporter gene-expressing cells in lactotrophs was no more than 30%.Research project 31)We produced several lines of transgenic rats carrying PRL promoter-driven GFP gene.2)Among the transgenic rat lines, we chose MI-11-90 line on the basis of the fluorescence intensity, the proportion of GFP-expressing cells in lactotraphs, and the lactotroph-specificity for GFP expression.3)Using FACS, we succeeded in selectively collect ting GFP-expressing pituitary cells, 90% of which were lactoarophs.4)The stimulatory action of estrogen on lactotroph proliferation was enhanced in this lactotroph-enriched population while its inhibitory action of proliferation in the presence of IGF-1 was completely eliminated.

研究项目11）利用培养的大鼠垂体前叶细胞，建立了Ki67、雌激素受体（ER）和催乳素（PRL）的三重免疫染色。2）当雌激素刺激泌乳素细胞增殖时，增殖的泌乳素细胞中ER免疫细胞的比例下降。当雌激素抑制IGF-1诱导的增殖时，ER免疫反应细胞的比例没有改变。研究项目21）我们生产了携带PRL启动子驱动的显性失活ER突变体的重组腺病毒载体。2）使用携带PRL启动子驱动的荧光素酶报告基因的腺病毒载体，解决了垂体培养物中的感染条件。在存在胰岛素的情况下，在早期培养期间以 5 MOI 进行感染可获得最大感染效率。即使在这种感染效率下，泌乳素细胞中表达报告基因的细胞比例也不超过30%。研究项目31)我们制备了几个携带PRL启动子驱动的GFP基因的转基因大鼠品系。2)在转基因大鼠品系中，我们根据荧光强度、表达GFP的细胞比例选择了MI-11-90品系。 3）利用FACS，我们成功地选择性收集了表达GFP的垂体细胞，其中90%是乳腺素。4）在富含泌乳素的群体中，雌激素对泌乳素增殖的刺激作用增强，而在IGF-1存在的情况下，雌激素对增殖的抑制作用增强。彻底消除。