| [BACKGROUND]In the mammalian central nervous system, zinc could function as a key component of numerous enzymes and as a neuroregulator. On the other hand, zinc also may result neuronal death caused by its over accumulation. The past studies have shown that intracellular homeostasis for zinc is achieved through the coordinate regulation of specific transporters engaged in zinc influx, efflux, and intracellular compartmentalization. The past studies found that three main zinc transporting gene family, the ZIP (Zrt-, Irt-like Protein), the MT (Metallothioneins) and CDF (Cation Diffusion Facilitator) families, were involved in the zinc homeostasis upon various zinc condition. The expression of ZIP mRNA will increased on low zinc status to enhance zinc influx; The expression of ZnTs and MT mRNA will increased to mediate zinc efflux by zinc transporter ZnT1(zinc transporter 1) or taken up into organelles, or sequestered by metallothioneins(MTs).The past studies shows that the overaccumulation of zinc in the synaptic cleft is closely related to episodes of ischaemia, epilepsy, brain trauma, Alzheimer Diseases, amyotrophic lateral sclerosis(ALS),et al. For example, in Alzheimer's disease (AD), mature amyloid plaques, but not preamyloid deposits, are found to contain high levels of zinc, suggesting the role of zinc in the process of plaque maturation. Under conditions of zinc toxicity, when extracellular zinc levels are high, zinc may enter into neurons via N -methyl- D -aspartate (NMDA) receptors, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) kainate receptors, voltage-dependent calcium channels, or transporter-mediated exchange with intracellular sodium. Although the zinc content usually fluctuates drastically around synaptic clefts or some neurodegenerative diseases, the mechanism of zinc homeostasis in never system is unclear till now. The discovery of zinc-transporting relating gene in neurons, such as zinc transporter, Divalent metal transporter, suggests that neurons could regulate zinchomeostasis by these genes. However, there are no systematic studies on the roles of these genes in zinc homeostasis in neurons.[OBJECTIVE]To determine the expressions of those confirmed zinc-transporting relating genes in new-born rats primary culture hippocampal neurons, their concentration-response and time course, their corresponding zinc concentrations in neurons, as well as the cell viability on zinc exposure. To analyze their relationships so as to find those main genes involved in zinc homeostasis.[METHODS]:1. The protocol to establish new-born rats primary culture hippocampal neurons .The hippocampi were removed from the newborn SD rat skull with care to remove meninges and blood capillary under aseptic condition. Then all the hippocampis were minced into approximately 1.0 1.0 mrn pieces using iris scissor, collecting the neurons as normal methods, then purified the neurons with B-27 media. Six days later, adjust the zinc level with N,N,N',N'-tetrakis (2-pyridylmethyl)-ethylenediamine (TPEN), a intracellular Zn(2+) membrane-permeant chelator or ZnSO4 to control the zinc concentration in B-27 media.2. The zinc toxicity to hippocampal neurons in primary culture:After 6 days of culture, the zinc toxicity was performed by changed to media containing various concentrations of Zn2+( 0 u M, 50 u M, 100 u M, 125 U M, 150 u M, 175 u M, 200 u M ). All treatment were added so that they were 1.0% of the total volume. After 48 hr, the cell numbers were counted in several representative culture fields, and the ratio of cells survived was calculated, judged by morphology and trypan blue staining.3. The zinc contents in neurons on zinc exposure.To determine the zinc contents by atom absorption spectrophotography: 1. The total neuron zinc contents on 10 uM TPEN and 0, 50, 100, 125, 150umol/L ZnSO4 exposure in 30, 180 and 360 min. 2. The zinc contents of cytoplasm and nucleus on 10 u M TPEN and 0, 125, 150 u mol/L ZnSO4 exposure in 180 min.4. The expression of ZnTK MTs, rZIP and DMT1 on zinc expos...
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