STRESS GENES IN BRAIN ISCHEMIA

应激基因在中枢神经系统(CNS)细胞中诱导 对缺血、癫痫、创伤和其他应激的反应。Hsp70家族 包含一大组相关蛋白质，包括HSP70(热 诱导型)、hsc70(结构性)和GRP78(葡萄糖调节，内质 网状)。我们选择研究这个家庭，因为他们是 最具诱导性和丰富的应激蛋白。热休克蛋白70的蛋白和mRNA 作图使人们对缺血和缺氧的病理生理学有了深刻的了解。 其他类型的脑损伤。应激蛋白的另一个令人兴奋的方面 研究发现，这种蛋白质的诱导，以及热休克蛋白70在 尤其是与抵抗随后的伤害有关。因此， 对hsp70家族的研究可能有助于理解基础 中枢神经系统损伤的机制，并可能对治疗有指导意义。 中风和其他形式的中枢神经系统损伤。 在初步实验中，寻找与hsp70相关的序列 通过简并引物法对大脑进行聚合酶链式反应，从而鉴定出新的成员 Hsp70家族。发现了两个这样的HSP70相关的cdna片段，命名为 Hsr1和hsr2。Hsr1编码高度相似的多肽序列 线粒体HSP70家族成员(尚未克隆到 脊椎动物)和主要细菌hsp70的dNAK。它是符合宪法的 表达，但也可能对细胞损伤有反应。NO的Hsr2代码 HSP7O相关多肽，没有主要的开放阅读框架。可能是Hsr2 代表与HSP70无关的基因的3‘或5’侧翼区域。 然而，与hsr2高度杂交的有1个次要的和2个主要的mrna物种。 在脑损伤期间，严格性被发现上调。其他内容 Hsp7O、hsc7O和GRP78mRNA对FOCUS反应的初步数据 脑缺血提示脑内信息积累的差异性失灵 纹状体缺血，GRP78信息继续上升，而hsp70和 Hsc7O并非如此。这一现象并不发生在大脑皮层中。 这项建议中概述的研究是针对 HSP70家族和hsr2在正常和缺血组中的表达 大脑。Hsr1和hsr2的特征首先是完成 它们的全长cDNA测序及其5‘端启动子的检测 利用文库筛选和聚合酶链式反应技术。特异性抗体 将产生hsr1、hsr2、hsc70和GRP78，组织、细胞和 我们将研究hsr1和hsr2的亚细胞分布。的表达 Hsr2和所有可用的hsp70相关基因将在 正常脑、局灶性和全局性脑缺血的在体模型 高热、低血糖、缺氧性和兴奋性毒性损伤 培养中的小脑神经元和星形胶质细胞。有关以下方面的区域信息 MRNA和蛋白质表达的相对数量和形式将是 利用Northern和Western分析得出。细胞和亚细胞 分布将通过原位杂交和 免疫细胞化学。伤情分级和时间进程 我们将对其表达进行研究。

Stress genes are induced in cells of the central nervous system (CNS) in response to ischemia, seizure, trauma and other stresses. The hsp7O family comprises a large group of related proteins including hsp70 (heat inducible), hsc70 (constitutive) and grp78 (glucose regulated, endoplasmic reticular). We have chosen to study this family, as they are among the most highly inducible and abundant stress proteins. Hsp70 protein and mRNA mapping has yielded insights into the pathophysiology of ischemia and other types of brain injury. Another exciting aspect of stress protein research is the finding that induction of such proteins, and hsp70 in particular, is associated with a resistance to subsequent injury. Thus, the study of the hsp7O family may contribute to the understanding of basic mechanisms of CNS injury, and may have implications for the therapy of stroke and other forms of CNS damage. In preliminary experiments, hsp7O-related sequences were sought in the brain by degenerate-primer PCR, in order to identify new members of the hsp7O family. Two such hsp70-related cDNA fragments were found, designated hsr1 and hsr2. Hsr1 codes for a peptide sequence which is highly similar to mitochondrial hsp70 family members (which have not been cloned in vertebrates) and dnaK the major bacterial hsp7O. It is constitutively expressed but may also be responsive to cell injury. Hsr2 codes for no hsp7O-related peptides and has no major open reading frame. Hsr2 likely represents the 3' or 5' flanking region of a gene unrelated to hsp70. Nevertheless, 1 minor and 2 major mRNA species hybridizing to hsr2 at high stringency were found to be upregulated during brain injury. Additional preliminary data on the responses of hsp7O, hsc7O and grp78 mRNA to focal ischemia suggested a differential failure of message accumulation in striatal ischemia, with grp78 message continuing to rise while hsp7O and hsc7O did not. This phenomenon did not occur in the cortex. The studies outlined in this proposal are directed towards the characterization of the hsp70 family and hsr2 in the normal and ischemic brain. Hsr1 and hsr2 will first be characterized by completing the sequencing of their full-length cDNAs and examination of their 5' promoter regions using library screening and PCR techniques. Specific antibodies to hsr1, hsr2, hsc70 and grp78 will be produced, and the tissue, cellular and subcellular distributions of hsr1 and hsr2 will be examined. Expression of hsr2 and all of the available hsp7O-related genes will be studied in normal brain,in vivo models of focal and global ischemia and in hyperthermic, hypoglycemic, anoxic and excitotoxic injury in cortical and cerebellar neurons and astrocytes in culture. Regional information on relative quantities and forms of expression of mRNA and protein will be obtained using Northern and Western analysis. Cellular and subcellular distributions will be examined by in situ hybridization and immunocytochemistry. Graded degrees of injury and time courses of expression will be studied.