| Summary: | 碩士 === 中國醫藥大學 === 醫學研究所碩士班 === 94 === Heat shock protein 70 (HSP70) is well induced during press. Previous reports indicated induction of inducible HSP70 response to stressful conditions, such as heat shock, epilepsy, ischemia, and trauma. HSP70 was found in hippocampus of the temporal lobe epilepsy (TLE), and the treatment with Kainic acid (KA) in vivo had shown, however, these mechanisms in the brain are still poorly elucidated. In this aim of research is to explore the mechanism of HSP70 in epilepsy.
This experiment is totally divided three kinds of research ways. First, human sample: collect 6 TLE patients’ and 3 non- epilepsy patients’.Second, animal model: Male Sprague–Dawley（SD） rats, KA was microinjected into the CA1 area of the hippocampus to induce seizure.Third, cell model: primary neuron culture of embryonic day 18 SD rat pups (E18), primary cortex astrocytes cultures were prepared from newborn rat brains (1–2-day-old SD rat), and neuron culture was transfected with HSP70-siRNA（small interfering RNA）.Treat them KA to induce HSP70.We use Hematoxylin & Eosin（HE） stain, immunohistochemical stain, double immunofluorescence assay and analysis of apoptosis to study cell morphology.
Our observations suggest that HE stain shows that neuronal degeneration and gliosis are found in the CA3 area of TLE patients and treated KA animal model. By using double-immunofluorescence of HSP70 with MAP2 show that HSP70 is found mainly within neuron.Proliferating Cell Nuclear Antigen (PCNA）and GFAP double immunostaining show that KA induced epilepsy is evoked and increase of PCNA, whereas glia cells proliferation are detected 5 days after epileptic seizures. According to TUNEL and FJB（FLUORO-JADE® B）, we found denatured neuron obvious after treating KA 5 days （TUNEL：control 2.625±1.06%、KA 2days 39.37±2.97%、KA 5days 62.25±12.4%；p＜0.001）（FJB：control 4.245±2.2%、KA 2 days 73.5±13.84%、KA 5 days 98.75±9.6%；p＜0.001）.By using double-immunofluorescence of FJB and HSP70, we found that HSP70 is not expression in degenerate neuron. In KA treated neuron culture transfected with HSP70-siRNA found that the neuron apoptosis are unrelated with existence of the HSP70. So we suggest that HSP70 is just a stress marker.
|
|---|
