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Molecular Histochemical Analysis on Nuclear Distribution of Transcription Regulatory Factors and the Transcriptional Activity of the Genes.

转录调控因子核分布和基因转录活性的分子组织化学分析。

基本信息

批准号：
09670018
负责人：
IZUMI Shin-ichi
金额：
$ 1.98万
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：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670018/
关键词：
Transcription Nucleus Prolactin Growth Hormone in situ hybridization Southwestern Placenta Pituitary Pit-1 遺伝子 DNA 染色質

项目摘要

1. The optimun conditions of a combination treatment with exogenous or endogenous DNase and followed with proteinase K (0.02 unit/ml) for leucocytes were successful in DNA in situ hybridization histochemistry using labeled-hunan X chromoosome DNA (Xp21, Xq13, Xq21) probes. This revealed that active and inactive X chromiatin DNA in cells were localized in nuclear euchromatin and heterochromtin, respectively.2. In rat anterior pituitary GH3 culture cells and rat anterior pituitary tissue sections Southwestern histochemistry (SWH), which localizes DNA binding proteins, was performed. The method demonstrated that Pit-1 transcription regulatory factor participating in activation of growth hormone (GH) gene was localized in nuclei of GH cells, whereas PREB transcription regulatory factor in suppression was not localized in the nuclei, and that Pit-1 also in activation of prolactin (PRL) gene as well as PREB were local ized in nuclei of PRL cal Is.3. Rats were intraperitoneally injected 10 Nm GH releasing hormone or 10 nM scmatostatin or control saline 4 hours before pituitary gland dissection. The anterior pituitary tissue sections from them were stained for Pit-1 and PREB by SWH No marked difference of intranuclear distribution and staining intensity could be recognized among them. When Pit-1 is neutralized in GH3 cells, whether transcription of GH gene is suppressed has not been confirmed yet.4. In rat placentas at around 14 day of gestation, some similar trophoblasts started localizing nuclear Pit-1 and cytcplasmic PRL.As gestational day goes on, number and staining intensity for both positive trophoblasts increased. However, at around 19 day of gestation, Pit-1 was also seen in the cytoplasm and staining intensity for PRL decreased in the trophoblasts by immuno-histochemistry. It is suggested that distributing nuclear Pit-1 in the cells of rat placentas may be associated in functional differentiation of trophoblasts that produce PRL-like substances.

1.用标记的湖南X染色体DNA(Xp21、Xq13、Xq21)探针进行DNA原位杂交组织化学研究，确定了外源或内源性DNA酶与蛋白酶K(0.02单位/毫升)联合处理白细胞的最佳条件。这表明细胞内活性和非活性X染色质DNA分别定位于核常染色质和异染色质。在大鼠垂体前叶GH3培养细胞和大鼠前叶组织切片上，进行了DNA结合蛋白的西南组织化学定位。该方法证明参与生长激素(GH)基因激活的Pit-1转录调控因子定位于GH细胞的细胞核内，而参与抑制PREB转录调控因子的转录调控因子Pit-1未定位于GH细胞的细胞核内，Pit-1也参与激活催乳素(PRL)基因，PREB也定位于PRL细胞的细胞核内。大鼠在切除垂体前4h分别腹腔注射10 NM生长激素释放激素、10 NM抑制素或生理盐水。用SWH法对脑垂体前叶组织切片进行Pit-1和PREB染色，发现两者在核内分布和染色强度上无明显差异。当Pit-1在GH3细胞中被中和时，GH基因的转录是否受到抑制尚未得到证实。在孕14天左右的胎盘中，一些类似的滋养层细胞开始定位于核Pit-1和细胞质PRL，随着妊娠时间的延长，这两种阳性滋养层细胞的数量和染色强度都增加了。但在妊娠19d左右，Pit-1也出现在细胞质中，免疫组织化学显示滋养细胞中PRL的染色强度减弱。提示核Pit-1在大鼠胎盘细胞内的分布可能与滋养层细胞产生PRL样物质的功能分化有关。