| Part I :The Diverse Expression of Prostate Apoptosis Response-4 Gene inDifferent Brain Regions of Hypoxic-Ischemic Neonatal RatsObjective:To investigate the various expression of prostate apoptosis response-4 (Par-4) gene in different brain regions of hypoxic-ischemic neonatal rats.Methods:All neonatal rats were divided randomly into hypoxic-ischemic encephalopamy (HIE) group, pseudo-operation group, and normal control group. The animal models of HIE were made .At different time after hypoxic-ischemia , the expression of par-4 mRNA in frontal lobes cortex, hippocampus, cerebellum and hypothalami was measured by RT-PCR. Western blot was used to detect the expression of par-4 protein in hippocampus.Caspase-3 relative activity of hippocampus was determined by colorimetric assay.Results:1. In various brain regions measured, the par-4 mRNA levels of HIE group at 2h,6h,12h,24h after hypoxic-ischemia were significantly increased,and all peaked at 6h(P<0.05). But there were not changes in pseudo-operation group and normal control group(P>0.05).2. In HIE group at 6h after hypoxic-ischemia, the par-4 mRNA levels of hippocampus were the highest(P<0.05) , and these of hypothalami were the lowest (P<0.05).3. In HIE group at 12h,24h,36h,48h,72h after hypoxic-ischemia, the expression levels of par-4 protein in hippocampus were significantlyincreased, But there were not changes in pseudo-operation group and normal control group(P>0.05).4. In HIE group at 12h,24h,36h,48h,72h after hypoxic-ischemia, the caspase-3 activity was significantly increased compared with that of pseudo-operation group and normal control group (P<0.05), and peaked at 24h(P<0.05).5. In HIE group ,the par-4 protein levels were significantly correlated with caspase-3 activity after hypoxic-ischemia, (r=0.622,P<0.05).Conclusions:1. Par-4 gene may be involved in the hypoxic-ischemic damage of neonatal rats2. There may be some relation between the diverse levels of par-4 mRNA in various brain regions after hypoxic-ischemia and selective vulnerability in the neonatal brain.3. The pro-apoptosis mechanism of par-4 gene may be closely correlated with the caspase-3 activity.Part II:The inhibition effects of Par-4 Antisense Oligodeoxynucleotide on Apoptosis of PC 12 Cell Line Induced by Glutamate and Its Signal Transduction Mechanism.Objective:To investigate the inhibition effects of Par-4 Antisense Oligodeoxynucleotide on Apoptosis of PC 12 Cell Line Induced by Glutamate and its signal transduction mechanism.Methods:1. PC 12 cells were cultured in normal condition or in the presence of glutamate.2. Cationic lipid-mediated par-4 antisense Oligodeoxynucleotide (par-4-AS-ODN) was transfected into PC 12 cells before they were treated with glutamate.Sense Oligodeoxynucleotide (S-ODN) and mismatch Oligodeoxynucleotide (MS-ODN) were also transfected into cells as controls.3. Morphological observation and the detection of anti-apoptosis effects of par-4-AS-ODN on PC 12 cells were done with fluorescence light microscope by double staining the cells with acridine orange/ethidium bromide (AO/EB) or Hoechest 33258/propidium iodide (Hoe/PI), addition to with flow cytometry.4. Double-fluorescense immuncytochemisty staining method was used to determine subcellar localization of par-4 protein.5. Western blot was used to detect the protein levels of par-4 and phosphorylated Aktl at threonine -308 (Thr-308).6. Caspase-3 relative activity was determined by colorimetric assay .7. Electrophoretic mobility shift assay (EMSA) was applied for evaluating the change of STAT3 DNA binding activity.8. The mRNA levels of X-linked apoptosis protein (XIAP) and calcium-dependent protease Calpain 10 were measured by RT-PCR.9. Intracelluar calcium concentration was detected using the fluorescent dye Fura-2/AM under the Laser Scanning Confocal Microscope.Results:1.The apoptosis of PC 12 cells was successfully induced by glutamate.2.The par-4 prot... |
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