Molecular mchanisms of stress tolerance observed at mildly low temperature environment and the clinical relevance of their abnormality in the testis

微低温环境下睾丸应激耐受的分子机制及其异常的临床意义

基本信息

批准号：
18390434
负责人：
FUJITA Jun
金额：
$ 10.17万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18390434/
关键词：
hypothermia environment testis transcription factor stress 遺伝子細胞・組織特殊環境 cirp

项目摘要

In the mammalian testes, germ cells and somatic colic undergo active cell proliferation and differentiation at mildly low temperatures (32-34℃). To elucidate how testicular cells adapt to the lower temperature and become more resistant to various stressors, we have analyzed the molecular mechanism underlying the cold shock response, especially in relation to the cold shock protein alp (cold-inducible RNA-binding protein).1. We failed to identify the transcription factor that binds to the mild-cold response element (MCRE) and activates transcription at lower temperatures by the yeast 1-hybrid screening, but successfully identified several proteins by mass spectrometry that associate with MORE in vitro and in vivo. Most of them seemed to modify activity of the elusive transcription factor.2. When over-expressed in cultured rolls, several of the identified "MCRE-binding proteins" enhanced the expression of Cap as well as the reporter gene driven by MCRE and CMV promoter One of the genes was transiently over-expressed in cultured cells and the induced genes similarly to Cirp were identified by using a DNA microarray.3. MCRE was found to be ineffective as a cold-responsive enhancer when it is integrated into the chromosome at a cm all copy numbers. We optimized the sequence and copy numbers for better induction at 32℃.4. We produced the dip knockout mice and demonstrated that Cirp is involved in wound healing process.5. By using the yeast 2-hybrid screening, we identified proteins, including a kinase, that interact with Cirp and potentially endow resistance to apoptotic stresses.

在哺乳动物睾丸中，生殖细胞和体绞痛在轻度低温（32-34℃）下经历活跃的细胞增殖和分化，为了阐明睾丸细胞如何适应较低温度并变得对各种应激源具有更强的抵抗力，我们分析了分子。冷休克反应的机制，特别是与冷休克蛋白alp（冷诱导RNA结合蛋白）相关的机制。1。我们未能识别与轻度冷反应元件（MCRE）结合的转录因子。并通过酵母1-杂交筛选在较低温度下激活转录，但通过质谱法成功鉴定了几种与体外和体内MORE相关的蛋白质。2。在培养的细胞中表达，几个已鉴定的“MCRE结合蛋白”增强了Cap的表达以及由MCRE和CMV启动子驱动的报告基因。其中一个基因在培养的细胞中瞬时过表达，诱导的基因类似于利用DNA微阵列对Cirp进行了鉴定。3．当MCRE以cm的拷贝数整合到染色体中时，其作为冷反应增强子是无效的。我们优化了序列和拷贝数以在32℃下更好地诱导。 .4. 我们培育了dip 敲除小鼠并证明Cirp 参与伤口愈合过程。5. 通过使用酵母2 杂交筛选，我们鉴定了与Cirp 相互作用并可能赋予其抗性的蛋白质，包括激酶。凋亡应激。