STRESS GENES IN BRAIN ISCHEMIA

应激基因在中枢神经系统（CNS）细胞中被诱导， 对局部缺血、癫痫、创伤和其他应激的反应。HSP7O家族 包括一大组相关蛋白质，包括热休克蛋白70（heat 诱导型）、hsc70（组成型）和grp78（葡萄糖调节型、内质 网状的）。我们选择研究这个家族，因为他们是 最高度诱导和丰富的应激蛋白。Hsp70蛋白和mRNA 绘图已经产生了对缺血的病理生理学的见解， 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，诱导这种蛋白质，和热休克蛋白70在 特别是与对随后损伤的抵抗力有关。因此，在本发明中， 对hsp7O家族的研究可能有助于理解 中枢神经系统损伤的机制，并可能对治疗 中风和其他形式的中枢神经系统损伤。 在初步的实验中，我们在大肠杆菌中寻找hsp7O相关的序列。 通过简并引物PCR，以确定新成员的大脑， HSP7O家族发现了两个这样的hsp70相关的cDNA片段，命名为 HSR 1和HSR 2。Hsr1编码一种肽序列， 线粒体hsp70家族成员（尚未克隆， 脊椎动物）和dnaK主要细菌hsp70。它本质上是 表达，但也可能对细胞损伤有反应。HSR2代码，用于 hsp7O相关肽，没有主要的开放阅读框架。HSR2可能 代表与hsp70无关的基因的3 ′或5 ′侧翼区。 然而，1个次要和2个主要mRNA种类在高水平下与hsr2杂交， 发现严格性在脑损伤期间上调。额外 hsp7O、hsc7O和grp78 mRNA对局灶性脑缺血反应的初步数据 局部缺血表明，在缺血性脑损伤中， 纹状体缺血，grp78信息继续上升，而hsp70和 HSC7O没有。这种现象并没有发生在大脑皮层。 本提案中概述的研究旨在 正常和缺血性脑梗死中hsp70家族和hsr2的特征 个脑袋Hsr1和hsr2将首先通过完成 对其全长cDNA进行测序并检查其5'启动子 区域使用文库筛选和PCR技术。特异性抗体 将产生hsr1、hsr2、hsc70和grp78，并且组织、细胞和 将检查HSR 1和HSR 2的亚细胞分布。表达 hsr2和所有可用的hsp7O相关基因将在 正常脑、局灶性和全脑缺血的体内模型以及 高温、低血糖、缺氧和兴奋性毒性损伤， 小脑神经元和星形胶质细胞。区域信息， mRNA和蛋白质表达的相对量和形式将是 使用北方和西方分析获得。细胞和亚细胞 将通过原位杂交检查分布， 免疫细胞化学损伤分级和时间进程 表达将被研究。

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.