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

在哺乳动物睾丸中，生殖细胞和体细胞绞痛在较低温度（32-34℃）下进行活跃的细胞增殖和分化。为了阐明睾丸细胞如何适应较低的温度，并对各种应激源具有更强的抵抗力，我们分析了冷休克反应的分子机制，特别是与冷休克蛋白alp（冷诱导rna结合蛋白）有关。通过酵母1-杂交筛选，我们未能确定与MCRE结合并在较低温度下激活转录的转录因子，但通过质谱分析，我们成功地鉴定了几种与MORE相关的蛋白。它们中的大多数似乎改变了难以捉摸的转录因子的活性。当在培养的卷中过表达时，鉴定出的几个“MCRE结合蛋白”增强了Cap以及由MCRE和CMV启动子驱动的报告基因的表达，其中一个基因在培养细胞中短暂过表达，并通过DNA微阵列鉴定出与Cirp相似的诱导基因。研究发现，当MCRE在1厘米拷贝数处整合到染色体中时，它作为冷反应增强子是无效的。为了在32℃下更好地诱导，我们优化了序列和拷贝数。我们制作了dip敲除小鼠，并证明Cirp参与了伤口愈合过程。通过酵母2-杂交筛选，我们发现了与Cirp相互作用的蛋白，包括一种激酶，并可能赋予细胞对凋亡应激的抗性。

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.