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

研究项目11）以培养的大鼠垂体前叶细胞为材料，建立Ki 67、雌激素受体（ER）和催乳素（PRL）的三重免疫染色。2）雌激素刺激催乳细胞增殖时，ER免疫阳性细胞在增殖的催乳细胞中所占比例减少。雌激素抑制IGF-1诱导的增殖时，ER免疫反应阳性细胞的比例没有改变。研究项目21）我们制备了携带PRL启动子驱动的显性负性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。在胰岛素存在下，在早期培养期间以5 MOI感染获得最大感染效率。即使在这种感染效率下，报告基因表达细胞在催乳细胞中的比例也不超过30%。研究项目31）我们生产了几种携带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，催乳细胞中GFP表达细胞的比例，和GFP表达的泌乳素特异性。3）使用流式细胞仪，我们成功地选择性收集了表达GFP的垂体细胞，4）雌激素对催乳素细胞增殖的刺激作用在这种催乳素富集的群体中增强，而其在IGF-1存在下的增殖抑制作用在此群体中增强。1被彻底淘汰。

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.