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

1.研究了外源性雌激素物质通过转录因子、雌激素受体（er）和Pit-1对催乳素（PRL）基因表达的影响。己烯雌酚（DES）处理小鼠垂体prl和pit -1阳性细胞增加，FSH/ lh阳性细胞减少。对照组小鼠的PRL细胞和FSH/LH细胞的细胞核中存在ER α，而des处理小鼠的ER α阳性PRL细胞减少，ER β阳性PRL细胞先诱导后增加。在ER α-敲除小鼠中，ER β定位于PRL细胞核，且ER β、PRL或FSH/LH阳性细胞的数量不变。野生型小鼠经DES处理后，PRL基因转录增加，PRL细胞核中Pit-1和ER β表达增加，ER α表达减少。提示des2调控转录因子的数量和质量。我们研究了人工调控对产生PRL的GH3培养细胞中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诱导入细胞核阻断1P结合位点时，是否抑制了PRL基因的转录。高压灭菌法可有效增强核Pit-1和核内质网的信号，这些信号分别通过西南组织化学和过氧化物酶标记抗体法在光镜和电镜水平上检测到。在GH3细胞中，Pit-1和ER α位于异染色质附近的常染色质区域，具有PRL。在GH3细胞中，Pit-1和ER似乎分布在基因表达的特定核区。考察了探针反诱导后的最佳浓度和调谐。

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.