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Apoptosis of CA1 pyramidal neurons in the hippocampus after brief forebrain ischemia and its prevention

前脑短暂缺血后海马CA1锥体神经元凋亡及其预防

基本信息

批准号：
07458201
负责人：
UCHIYAMA Yasuo
金额：
$ 4.8万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07458201/
关键词：
Apoptosis PC12 Cells Bcl-2 gene Neurotrophic factor Cathepsin B Cathepsin D Lysosomes Dorsal roof ganglion bcl-2遺伝子 bcl-2 遺伝子導入 cDNAクローニング nedd-2 脳虚血オートファジ-リソゾーム CAl錐体細胞

项目摘要

1) PC12 cells undergo apoptosis when cultured under serum deprivation. In this situation, the activity of caspase-3-like proteinases was elevated, and the survival rate could be maintained by treatment with acetyl (AC)-DEVD-CHO,a specific inhibitor of caspase-3. In a culture of PC12 cells with AC-DEVD-CHO,where caspase-3-like proteinases are not activated, CA074, a specific inhibitor of cathepsin B induced apoptosis of the cells. This ability of CA074 was also replaced by cathepsin B antisense oligonucleotides. By double staining of TUNEL and activated caspase-3, the dying cells treated with CA074 were positive for TUNEL staining but negative for caspase-3. Ultrastructures of the cells were relatively large and had nuclei with chromatin condensation, suggesting that they died by apoptosis. Thus cell death effect by CA074 or the cathepsin B antisense were inhibited by the addition of pepstatin A,a lysosomal aspartic proteinase inhibitor, or cathepsin D antisense. The results suggest tha … More t a novel pathway of apoptosis exists, which is regulated by lysosomal cathepsins, and in which cathepsin D acts as a death factor, but that this death-inducing activity is usually suppressed by cathepsin B.2) PC12 cells transfected with the human bcl-2 gene can survive when cultured under serum deprivation. In this situation, the transfected cells extended neurite-like processes. Culture media of the transfected cells contained factor (s) which rescued wild-type PC12 cells following serum withdrawal. We therefore partially purified the factor and separated it by native SDSPAGE.From this, the N-terminal amino acid sequence of the factor was decided and the protein was found to be novel. Following the routine method of cDNA cloning, the cDNA clones of the factor protein were isolated, consisting of 2332 bp of total sequence having an ORF of 768 bp, encoding a protein of 256 amino acids. The predicted amino acid sequence has a signal sequence of 23 amino acid and an active form sequence of 233 amino acid. Antibodies against synthesized peptides corresponding to several parts of the protein indicated that the molecular weight of the protein is approximately 35 kD by SDSPAGE and Western blotting. Transfection study of the cloned cDNA into PC12 cells demonstrated that the cells can survive following serum withdrawal. We, therefore, named the factor as PCTF35. Less

1)PC 12细胞在无血清培养条件下发生凋亡。在这种情况下，caspase-3样蛋白酶的活性升高，并且通过用caspase-3的特异性抑制剂乙酰基（AC）-DEVD-CHO处理可以维持存活率。在含有AC-DEVD-CHO的PC 12细胞培养物中，其中caspase-3样蛋白酶未被激活，组织蛋白酶B的特异性抑制剂CA 074诱导细胞凋亡。CA 074的这种能力也被组织蛋白酶B反义寡核苷酸取代。TUNEL和活化caspase-3双重染色显示，CA 074处理的凋亡细胞TUNEL染色阳性，caspase-3染色阴性。超微结构显示细胞较大，核染色质浓缩，提示细胞凋亡。因此，CA 074或组织蛋白酶B反义的细胞死亡效应通过添加胃酶抑素A（一种溶酶体天冬氨酸蛋白酶抑制剂）或组织蛋白酶D反义而被抑制。结果表明， ...更多信息结论：（1）人bcl-2基因转染的PC 12细胞在无血清培养条件下可以存活，但其死亡诱导活性通常被组织蛋白酶B抑制。（2）人bcl-2基因转染的PC 12细胞在无血清培养条件下可以存活。在这种情况下，转染的细胞延长神经突样突起。转染细胞的培养基含有在血清撤出后拯救野生型PC 12细胞的因子。因此，我们对该因子进行了部分纯化，并通过SDS-PAGE进行了分离，确定了该因子的N-末端氨基酸序列，发现该蛋白为新蛋白。按照cDNA克隆的常规方法，分离因子蛋白的cDNA克隆，其由具有768 bp的ORF的2332 bp的总序列组成，编码256个氨基酸的蛋白。预测的氨基酸序列具有23个氨基酸的信号序列和233个氨基酸的活性形式序列。针对合成肽的抗体对应于蛋白质的几个部分，表明蛋白质的分子量约为35 kD的SDSPAGE和蛋白质印迹。将克隆的cDNA转染到PC 12细胞中，证明细胞在血清撤除后可以存活。因此，我们将该因子命名为PCTF 35。少