Mechanisms of a delayed calcium uptake in mouse ischemic model

我们已经报道，钙调神经磷酸酶和嗜胞菌素D的信号转导通路是开发抗缺血药物的新靶点。为了进一步分析这两种酶的作用，我们测量了在低氧和低糖刺激下，在有或没有免疫抑制剂环孢素A或FK506的情况下，海马鞋中锥体神经元细胞内钙的变化。此外，我们还发现了钙调神经磷酸酶与缺氧反应因子(HIF)之间的新关系，该关系在血管生成、糖酵解代谢、细胞凋亡和红细胞增多症的诱导因子中起关键作用。这些低氧因素深入参与了低氧在缺血性脑损伤和退行性疾病(如帕金森病和阿尔茨海默病)中发挥重要作用的机制。在我们的实验中，钙调神经磷酸酶可以稳定…更多地表达并激活HIFs的转录。我们通过酵母双杂交来发现调控HIF的新因子，并发现了抑制HIF2转录活性和表达的新的阳性克隆。其中一个已鉴定的FISD基因是Int6/eIF3e，它与小鼠哺乳动物肿瘤病毒(MMTV)引起的乳腺癌有关。MMTV在多个整合位点整合到基因组中，破坏了目的基因。Int6是目的基因之一。重组Int6突变体的进一步分析表明，Int6通过与HIF2直接结合和降解而具有肿瘤抑制作用。我们证明了注射了特异性抑制HIF2的si RNA后，小鼠皮肤中有强大的血管生成。在此基础上，我们利用转Int6显性负性突变体或siRNA表达载体的小鼠进行分析，根据本研究计划的特定目的，我们试图建立小鼠缺血模型(小鼠前脑缺血模型)。有两个因素很关键，一个是保持体温。我们配备了Acryl边界显微镜，以保持37℃。另一个因素是结扎颈动脉后缺血小鼠的选择。用多普勒血流仪观察到约20%的手术小鼠有足够的血供。遗憾的是，我们不能评估PLC D1KO小鼠和NCX2KO小鼠，但我们计划在完成该项目后进行Int6 siRNA或显性阴性TG小鼠的评估。较少

We have reported that signal transduction pathway involving calcineurin and cydophilin D is a novel target for developing anti-ischemic drugs. For further analysis to clarify the role of both enzymes, we measured the intracellular calcium in pyramidal neuronal cells in hippocampal shoes after stimulation of hypoxia and low glucose with or without immunosuppressants cydosporin A or FK506. The results suggested that calcineurin activity might regulate the uptake of calcium through unknown calcium channel existed on plasma membranes.In addition, we found the new relation between calcineurin and hypoxia responsive factor (HIF), which was reported to be critical in inducing factors in angiogenesis, glycolytic metabolism, apoptosis, and erythrocytosis. This hypoxic factors deeply involved in mechanisms by which hypoxia played an important role in ischemic brain damage and degenerative diseases such as Parkinson disease and Alzheimer disease. In our experiments, calcineurin can stabilize the … More expression and activate the transcription of HIFs. We performed yeast two hybrid to discover novel factors regulating HIF and found novel positive clones which suppressed HIF2 transcription activity as well as expression. One of fished done identified is Int6/eIF3e, which involed in breast cancer caused by mouse mammalian tumor virus (MMTV). MMTV was integrated into genome at several integration sites and damaged the target genes. Int6 was one of the target gene. Further analysis using recombinant Int6 mutants showed that Int6 was a tumor suppressor by direct binding to HIF2 and the degradation. We demonstrated the potent angiogenesis in mouse skin after injection of si RNA which specifically suppressed HIF2. Under these results, we started analysis using mice transgened with a Int6 dominant negative mutant or siRNA expression vector.According to our specific aims in this research plan, we tried to establish a mouse ischemic model (forebrain mouse ischemic model). Two factors were critical One was the maintenance of body temperature. We equipped acryl bords surrounding microscope to keep 37℃. Another factor was selection of ischemic mouse after ligation of carotid arteries. Around 20 % of operated mice was observed to have enough blood supply through co-lateral blood flow by Doppler flow meter. It was regret that we could not evaluate PLC D1 KO mice nor NCX2 KO mice, but we plan to perform evaluation of Int6 siRNA or dominant negative Tg mice even after finishing this project. Less