| Glutamate is a major neurotransmitter of excitatory synaptis in CNS. And it plays important role in maintain regular signals in neurons. Commonly, glutamate release,uptake,reabsorption keep in balance, but when it release excessively or meet uptake obstacle, glutamate would accumulate largely in the brain, then glutamate receptors will be over stimulated and lead to a series of disease response, finally lead to cell death. It is investigated that the concentration of glutamate is extremely high in cerebral ischemia injury and some neurodegenarative diseases such as AD,Vascular Dementia and so on. Thus, to study and understand the mechanisms of glutamate injury is helpful to know some diseases and for cure. It has been reported that there is a relation among cell apoptosis,calcium homeostasis disorder,oxidative injury and glutamate excitotoxicity. However the molecular mechanism of glutamate toxicity has not been clearly investigated. As a well neural model, neuroblastma SH-SY 5Y cell has been widely used. This dissertation aims to study glutamate excitotoxicity mechanisms on SH-SY 5Y cells.1. The research of mechanisms of glutamate-induced human neuroblastma cell apoptosisSH-SY 5Y cell viability was measured by MTT assay. Other damaged profile was detected by lactate dehydrogenase (LDH) release and by 4', 6-diamidino-2-phenylindole (DAPI) staining. The cytosolic calcium concentration was tested by calcium influx assay. The glutamate-induced oxidative stress was analyzed by cytosolic glutathione (GS) assay, superoxide dismutase (SOD) assay and extracellular malondialdehyde (MDA) assay. Glutamate treatment caused damage in SH-SY 5Y cells, including the decrease of cell viability, the increase of LDH release and the alterations of morphological structures. Furthermore, the concentration of cytoplasmic calcium in SH-SY 5Y cells was not changed within 20min following glutamate treatment, while cytosolic calcium concentration significantly increased within 24h after glutamate treatment, which could not be inhibited by MK801, an antagonist of NMDA receptors, or by LY341495, an antagonist of metabotropic glutamate receptors. And both the two antagonists have no protective effects. On the other hand, oxidative damage was observed in SH-SY 5Y cells treated with glutamate, including decreases in GS content and SOD activity, and elevation of MDA level.2. The effects of glutamate on CCE induced by Tg in SH-SY 5Y cells.High concentrations (lOmM) of glutamate can inhibit calcium release from ER caused by Tg (Thapsigargin), in addition inhibit calcium inflow from intercellular space (inhibit CCE) in SH-SY 5Y cells. While, LY341495, antagonist of mGluRs could not ameliorate the above effects.3. Tanshinoneâ…¡A (Tanâ…¡A) protects SH-SY 5Y cell from apoptosis induced by glutamate.MTT assay was used to find whether Tanâ…¡A affects the viability of SH-SY 5Y cells at the presence of glutamate and the concentrations of effective Tan II A. The damaged profile was detected by lactate dehydrogenase (LDH) release and morphological structures by 4', 6-diamidino-2-phenylindole (DAPI) staining, certain concentrations of Tan IIA can work. And can alleviate injuries indexes including cytosolic GS,SOD and intercellular MDA. But Tanâ…¡A failed to alleviate calcium increase in 24 hours.4. The protective effects of mutated recombinant human glucagon-like peptide-1 (mGLP-1) on glutamate-induced SH-SY 5Y cell apoptosis. As mGLP-1 and nGLP-1 both have protective effects on glutamate-induced SH-SY 5Y cell apoptosis. MTT,LDH,DAPI,cytosolic glutathione,SOD and MDA assays all proved the above conclusion. And mGLP-1 and nGLP-1 also have no effects on calcium increase caused by glutamate. There is almost no difference between the protective effects of mGLP-1 and nGLP-1.
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