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The Effect of Intermedin (IMD) on Hypoxia/Reoxygenation-induced NRK-52E Apoptosis

Author: CuiXiaoYan
Tutor: ZhaoHePing；ZhouZuo
School: Shanxi Medical
Course: Internal Medicine
Keywords: Intermedin Rat proximal tubular epithelial cells Hypoxia and reoxygenation Transfection Apoptosis
CLC: R96
Type: Master's thesis
Year: 2010
Downloads: 43
Quote: 1
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Abstract

Objective: rat proximal tubular epithelial cells (NRK-52E) hypoxia reoxygenation (H / R) model simulates in vivo ischemia-reperfusion (I / R) injury, high expression by transfection IMD plasmid explore in interleukin (IMD) on hypoxia / reoxygenation-induced proximal tubular epithelial cells (NRK-52E) apoptosis mechanism. Methods: (1) the use of a three-gas incubator adjust the nitrogen pressure to the formation of hypoxic conditions, hypoxia 4h, reoxygenation 12h after detection NRK-52E viable cell count, cell viability and culture supernatants of lactate dehydrogenase (LDH) to determine The model prepared success of. (2) using Fugene HD transfection reagent, the pIRES2-EGFP/IMD expression plasmid pIRES2-EGFP empty plasmid transfected into NRK-52E cells, in inverted fluorescence microscopy and flow cytometry to detect the transfection efficiency, selected the most Priority transfection conditions; select high transfection efficiency of cells with containing G418 300μg/ml 10% of FBS DMEM/F12 complete medium to filter after 2 weeks to obtain a stable positive clone NRK-52E cells expressing the IMD. (3) were divided into control group, model group, empty plasmid group and IMD plasmid group of four groups, three groups of lines H / R experiments, specimens from cell supernatants and cell flow cytometry measurement of apoptosis rate, enzyme-linked immunosorbent assay measured serine - threonine kinase (Akt) activity, colorimetry Caspase-3 activity. Results: (1) after hypoxia 4h, 12h of reoxygenation, NRK-52E cells count the number of reduced cell viability decreased cell supernatant LDH levels increased significantly, modeling success. The gradual emergence of clones of cells (2) fluorescence microscopy and flow cytometry, transfection efficiency, the results show that a the plasmid 3μg: the FuGENE HD 12μl proportion of transfected the highest transfection efficiency in 48h; transfection positive from the first three days of G418 screening like proliferation, five days untransfected cells mass death, after gradually increasing positive cell clones, fused into a sheet of positive clone cells seen after 2 weeks. (3) The cell supernatant phosphorylated serine - threonine kinase (p-Akt) content, compared with the control group, H / R increased Akt phosphorylation levels of the cells in each group, which IMD plasmid group and the model group and compared to the empty plasmid group, Akt phosphorylation level was significantly higher compared to the model group and the empty plasmid group no significant difference. (4) cells Caspase-3 activity, compared with the control group, H / R groups of Caspase-3 activity increased, which IMD plasmid group were compared with the model group and the empty plasmid group, caspase-3 activity was significantly decreased was no significant difference in the model group with empty plasmid group. (5) The rate of apoptosis compared with the control group, H / R cells apoptotic cells was significantly increased IMD plasmid group were compared with the model group and the empty plasmid group, the apoptosis rate decreased significantly, the model group no significant difference in rate of apoptosis compared with empty plasmid group. Conclusion: NRK-52E the IMD to reduce H / R-induced apoptosis, the specific mechanisms and IMD enhanced Akt activity, reducing Caspase-3 activity related.

The Effect of Intermedin (IMD) on Hypoxia/Reoxygenation-induced NRK-52E Apoptosis

Abstract

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