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The Protective Effect of Nec-1 on Al-poisoned Nerve Cells

Author: XuLi
Tutor: NiuQiao
School: Shanxi Medical
Course: Occupational and Environmental Health
Keywords: Aluminum Nec-1 Necrosis Apoptosis Autophagy
CLC: R96
Type: Master's thesis
Year: 2010
Downloads: 51
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Abstract

[Objective] To observe and investigate the role of nerve cells Nee-1 on the stained aluminum (A1). [] 1. Primary cultured mouse neural cells: selection of newborn 1-3d Kunming mice, the brain, and separation of the cerebral cortex neuronal cultures. Manufactured Al damage the nerve cells Model: select a good growth of the nerve cells of the same batch, cells were cultured for 5 days, exposed to aluminum to give a final concentration of 2mmol / L 3. Join Nee-1 different dose 0 μmol / L, 30μmol / L, 60 μmol / L to intervene after 48 hours (1) Observation of the morphological changes of the nerve cells and nerve cell growth was detected by CCK-8 assay; (2) Flow cytometry (AnnexinV and PI double staining) to detect nerve cell necrosis and apoptosis rate; (3) fluorescence quantitative PCR detection of different doses of Nec-1 under the action of nerve cell necrosis, apoptosis and autophagy gene expression levels ; (4) Western blot (Western-blot) to detect neuronal necrosis, apoptosis and autophagy protein expression levels. [Results] 1.Nec-1 exposed to aluminum nerve cell morphology and cell viability effects: (1) light microscope observation of nerve cell morphological changes, the results show that the normal nerve cell growth strong, long and relatively uniform shaft process, the more frequently from the cell body to the peripheral tapering dendritic cells and synapses, the connection between the cell body and the cell body rich; exposed to aluminum (2mmol / L) to reduce the number of nerve cells, the nucleus split synaptic change little shorter or even disappear; With doses of Nec-1 to increase the number of nerve cells gradually increased, increased in volume, long axis mutation, and significantly increased the number of dendrites. (2) AO-EB staining results showed that the nuclear chromatin of normal cells with green and showed normal structure, adding Al3 (2mmol / L) nerve cells appear obvious phenomenon of apoptosis and necrosis, and with increasing doses of Nec-1 apoptosis and necrosis of nerve cells significantly decreased. (3) cell viability results show that compared with the dyed aluminum, Nec-1 (60μmol / L, 90μmol / L) Cell viability can be increased (P LT; 0.01). 2.Nec-1's role in nerve cell necrosis induced by aluminum: (1) flow results show A13 (2 mmol / L) role in nerve cells allows cell necrosis rate (P lt; 0.01), and as Nec-1 doses increase cell necrosis rate declined, and was statistically significant (P lt; 0.01). (2) fluorescent quantitative PCR (qRT-PCR) results show that the mRNA expression of the NFκB to A13 (2mmol / L) as a control, Nee-1 (60μmol / L, 90μmol / L) of nerve cells can be necrosis related gene RIP1 decreased (P lt; 0.01). (3) Western results show that cells NFκB protein group compared with A13 (2mM), NEC-1 (60 μmol / L, 90μmol / L) decreased, and there is a significant statistical significance (P LT; 0.01) ; Nec-1 (90μmol / L) also RIPl protein expression decreased (P lt; 0.01). 3.Nec-1 in neuronal apoptosis induced by aluminum: (1) JC-1 staining results show normal nerve cell mitochondrial membrane potential, resulting in red fluorescence; 2 mM dye aluminum group of neurons in the mitochondrial membrane potential low (early apoptotic change) to produce green fluorescent cells increased; Nec-1 (60μmol / L) and its elevated membrane potential, resulting in red fluorescence cell group increased significantly more exposed to aluminum. (2) flow of Al13 (2 mmol / L) role in the nerve cells in its apoptosis rate rise (P lt; 0.01), Nec-1 (60μmo1 / L, 90μm01 / L) increased cell apoptosis The rate of decline (P lt; 0.01). (3) qRT-PCR results showed that A13 (2mmo1 / L) for control, Nec-1 (60μmol / L, 90μmol / L) can neuronal apoptosis related gene caspase-3, caspase-8 and caspase-9 mRNA decreased (P lt; 0.01). (4) Western results showed that group compared with the A13 (2mmol / L), Nec-1 (60μmo1/L90μtmol/L) group the caspase-3, caspase-8 also showed a downward trend of caspase-9 protein expression and statistically significant (P lt; 0.01). 4.Nec-1 exposed to aluminum nerve cell autophagy: with A13 (2mmol / L) than, Nee-1 (60 μmol / L) LC3-Ⅱ mRNA expression decreased (P lt; 0.05); and Nee-1 (90μmol / L) LC3-Ⅱ protein expression was also found statistically decreased (P lt; 0.05). [Conclusion] Nec-1 can reduce the aluminum due to nerve cell death, especially necrosis and apoptosis, and thus play a role to protect nerve cells.

The Protective Effect of Nec-1 on Al-poisoned Nerve Cells

Abstract

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