永久缺血缺氧對PC12細(xì)胞自噬的影響
[Abstract]:Objective to establish a cell model of ischemia and hypoxia injury and to explore the mechanism of nerve cell injury induced by permanent ischemia and hypoxia and its effect on autophagy of PC12 cells. Methods the classical nerve cell model PC12 cells were used as the research object. PC12 cells were cultured in sugar-free DMEM medium and hypoxia tank (95%N_2 and 5%CO_2) to simulate the ischemic and anoxic environment of nerve cells in vivo. The cells were divided into two groups: control group (cultured in complete medium under normal conditions) and glucose-oxygen deprived (OGD) group (cultured in OGD medium under hypoxia): 0.5h (OGD 0.5h), 2h (OGD 2h), 6 h (OGD 6 h), 12 h (OGD 12 h) and 24 h (OGD 24 h). The cell survival rate was detected by MTT, the apoptosis rate was detected by flow cytometry, the release rate of lactic dehydrogenase (LDH) was detected by colorimetric assay, the expression of hypoxia inducible factor-1 偽 (HIF-1 偽), cyclooxygenase-2 (COX2), microtubule-associated protein light chain 3 (LC3) and Beclin-1 were detected by immunofluorescence and Western blotting, and the ultrastructure of autophagy was detected by transmission electron microscopy. To investigate the effects of different permanent ischemia and hypoxia time on cell injury and autophagy. Results compared with the control group, the cell survival rate decreased in a OGD time-dependent manner, and decreased significantly since OGD 6 h, the difference was statistically significant (P 0.05), the apoptosis rate and death rate continued to increase, which was positively correlated with OGD time, and increased significantly after 12 h OGD, the difference was statistically significant (P 0.05). The expression of HIF-1 偽 and COX2 protein increased gradually with the prolongation of OGD time, and increased significantly after 12 hours of OGD, the difference was statistically significant (P 0.05). Compared with the control group, the number of green LC3 protein labeled with fluorescence increased and the green fluorescence increased. Beclin-1 protein expression showed that the expression of Beclin-1 was positively correlated with OGD time, and began to increase significantly by OGD6h, the difference was statistically significant (P 0.05). Conclusion ischemia and hypoxia can induce oxidative stress and autophagy activation in PC12 cells.
【作者單位】: 河南大學(xué)神經(jīng)生物學(xué)研究所;河南大學(xué)醫(yī)學(xué)院生物化學(xué)與分子生物學(xué)教研室;河南大學(xué)護(hù)理學(xué)院;
【基金】:國家自然科學(xué)基金(31070952,U1204311)
【分類號】:R743.3
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