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

1)PC12细胞在去血清培养条件下发生细胞凋亡。在这种情况下，caspase-3样酶的活性升高，caspase-3的特异性抑制剂乙酰(AC)-DEVD-CHO可以维持存活率。在含有AC-DEVD-CHO的PC12细胞培养中，caspase-3样酶不被激活，组织蛋白酶B的特异性抑制剂CA074诱导细胞凋亡。CA074的这种能力也被组织蛋白酶B反义寡核苷酸所取代。TUNEL和活化的caspase-3双重染色显示，CA074处理的死亡细胞TUNEL染色为阳性，caspase-3为阴性。细胞超微结构较大，核染色质凝集，提示细胞死亡。因此，加入溶酶体天冬氨酸蛋白酶抑制剂胃抑素A或组织蛋白酶D反义基因可抑制CA074或组织蛋白酶B反义基因的细胞死亡效应。结果表明，…存在更多的新的凋亡途径，它由溶酶体组织蛋白调控，其中组织蛋白酶D作为死亡因子，但这种死亡诱导活性通常被组织蛋白酶B抑制。2)转人bcl2基因的PC12细胞在血清剥夺条件下培养可以存活。在这种情况下，转基因细胞延长了轴突样突起。转染细胞的培养液中含有S因子，可在去血清后挽救野生型PC12细胞。因此，我们对该因子进行了部分纯化，并用天然SDSPAGE对其进行了分离，确定了该因子的N-端氨基酸序列，发现该蛋白为新的蛋白质。按照常规的cDNA克隆方法，获得了该因子蛋白的全长2332bp的克隆，其开放阅读框架为768bp，编码256个氨基酸的蛋白质。预测的氨基酸序列有23个氨基酸的信号序列和233个氨基酸的活性形式序列。经SDSPAGE和Western blotting分析表明，该蛋白的相对分子质量约为35kD。将克隆的c DNA导入PC12细胞，结果表明，PC12细胞在去血清后仍能存活。因此，我们将该因素命名为PCTF35。较少

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