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Molecular Histo-cytochemical Analysis of Gene Expression Regulation by Artificial Control of Transcription Factors

通过人工控制转录因子进行基因表达调控的分子组织细胞化学分析

基本信息

批准号：
15590161
负责人：
IZUMI Shin-ichi
金额：
$ 2.24万
依托单位：
Nagasaki University
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

项目摘要

1.Effects of exogenously administered estrogenic substances on prolactin(PRL) gene expression through the transcription factors, estrogen receptors(ERs) and Pit-1,were examined. PRL-and Pit-1-positive cells were increased, whereas FSH/LH-positive cells were decreased in the pituitaries of mice treated with diethylstilbestrol(DES). ER α was localized in the nuclei of PRL and FSH/LH cells hi the control mice, whereas hi DES-treated mice ER α -positive PRL cells were decreased and ER β-positive PRL cells were induced and then increased instead. In ER α-knockout mice ER β was localized in PRL cell nuclei and population of ER β, PRL or FSH/LH positive cells was constant. Treatment of wild-type mice with DES seemed to increase PRL gene transcription with an increase of both Pit-1 and ER β as well as a decrease of ER α hi the PRL cell nuclei. It suggests that quantity and quality of the transcription factors are regulated by DES.2.We examined effects of artificial regulation on intranuclear distribution of the transcription factors in PRL producing GH3 culture cells. Pit-1 and ERs were localized to clarify whether PRL gene transcription was repressed when the double-stranded nucleotide sequences of PRL gene regulatory element (1P), which bind to Pit-1, were trans-inducted into the nuclei to block the 1P-binding sites. An autoclave retrieval was effective to enhance signals from the nuclear Pit-1 and ERs detected by Southwestern histochemistry and peroxidase-labeled antibody method, respectively, both at light- and electron microscopic levels. GH3 cells, in which Pit-1 and ER α were localized in the euchromatin region in the vicinity of the heterochromatin, possessed PRL. It appeared that Pit-1 and ER were distributed hi a definite nuclear region where the gene is expressed in GH3 cells. Optimal concentration and tune after the probe trans-induction were under examination.

1.检测外源性雌激素通过雌激素受体(ER)和Pit-1转录因子对催乳素(PRL)基因表达的影响。己烯雌酚(DES)组小鼠垂体PRL和Pit-1阳性细胞增多，FSH/LH阳性细胞减少。ERα定位于对照组小鼠催乳素和促黄体生成素/促黄体生成素细胞的胞核，而Hi-DES处理组小鼠ERα阳性催乳素细胞减少，ERβ阳性催乳素细胞诱导后增加。在ERα基因敲除小鼠中，ERβ定位于PRL细胞核，ERβ、PRL或FSH/LH阳性细胞数不变。经DES处理后，野生型小鼠PRL基因转录增加，核内Pit-1和ERβ表达增加，ERα表达减少。结果表明，转录因子的数量和质量均受DES2的调控。2.我们研究了人工调控对产生PRL的GH3细胞转录因子核内分布的影响。将与Pit-1结合的PRL基因调控元件(1P)的双链核苷酸序列反式导入细胞核，阻断1P结合部位，定位于Pit-1和ERs，以明确PRL基因转录是否受到抑制。在光镜和电子显微镜水平上，高压锅修复分别增强了西南组织化学法和过氧化物酶标记抗体法检测到的核Pit-1和ER的信号。Pit-1和ERα定位于异染色质附近常染色质区域的GH3细胞具有催乳素。似乎Pit-1和ER分布在GH3细胞表达该基因的特定核区。对探针反式诱导后的最佳浓度和时间进行了考察。