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Deng H.,Xinxiang Medical University | Cheng Y.,Xinxiang Medical University | Guo Z.,Key Laboratory of Henan Province for Medical Tissue Regeneration | Zhang F.,Xinxiang Medical University | And 6 more authors.
Experimental and Therapeutic Medicine | Year: 2014

Hypoxia is a primary mediator for cell survival, and has been reported to inhibit cardiomyocyte proliferation in fetal and neonatal hearts. CyclinA2 is a key regulator of cell proliferation. Whether CyclinA2 affects cardiomyocyte proliferation in hypoxic conditions remains unexamined. This study was designed to investigate the roles of CyclinA2 expression on hypoxia‑impaired cardiomyocyte proliferation. Cardiomyocytes were isolated from neonatal rats and randomly separated into six groups: Control, hypoxia, enhanced green fluorescent protein (EGFP)‑Adv, EGFP‑Ccna2, EGFP‑Adv + hypoxia and EGFP‑Ccna2 + hypoxia. The cells in the control group were cultured in a general cell incubator; the cells in the hypoxia group were placed in a hypoxic chamber for 12 h; the cells in the EGFP‑Adv and EGFP‑Ccna2 groups were separately transfected with EGFP‑adenovirus capsids or EGFP‑adenovirus capsids with CyclinA2 cDNA for 18 h, and then placed in a general incubator for an additional 12 h; the cells in the EGFP‑Adv + hypoxia and EGFP‑Ccna2 + hypoxia groups were separately transfected with EGFP‑adenovirus capsids or EGFP‑adenovirus capsids with CyclinA2 cDNA for 18 h, and then placed in a hypoxia chamber for an additional 12 h. CyclinA2 expression was measured using immunochemical staining and western blot analysis, and cardiomyocyte proliferation was measured using the cell counting kit 8. GFP fluorescence indicated a high transfection efficiency (>80%), and immunochemical staining showed that CyclinA2 was mainly distributed in the nucleus. CyclinA2 expression was downregulated following exposure to hypoxia for 12 h. Cardiomyocyte proliferation was also significantly decreased following exposure to hypoxia for 12 h. However, compared with the EGFP‑Adv group, CyclinA2 expression and cardiomyocyte proliferation was markedly increased in the EGFP‑Ccna2 group. Furthermore, compared with the EGFP‑Adv + hypoxia group, CyclinA2 expression and cell proliferation were markedly increased in the EGFP‑Ccna2 + hypoxia group. These findings indicate that CyclinA2 upregulation improves cardiomyocyte proliferation in hypoxic conditions. © 2014, Spandidos Publications. All rights reserved. Source


Yang C.Q.,Northwest University, China | Li X.Y.,Xinxiang Medical University | Li Q.,Key Laboratory of Henan Province for Medical Tissue Regeneration | Fu S.L.,Xinxiang Medical University | And 5 more authors.
Genetics and Molecular Research | Year: 2014

To investigate the variance of exogenous gene expression driven by different promoters by in vivo electroporation, 3 plasmid vectors carrying different promoters were selected, and their driving strength was compared in developing chicken embryos. The 3 promoters included: 1) the CAG promoter (containing the cytomegalovirus (CMV) immediate early enhancer and the chicken β-actin promoter), 2) the CMV promoter (the human CMV immediate early region enhancer), and 3) the SV40 promoter (Simian virus 40). The intensity of GFP expression driven by the 3 promoters was detected by fluorescence microscopy. The results clearly showed that the expression intensity of the reporter gene differed significantly among the 3 promoters. Chicken β-actin promoter induced the highest intensity of GFP expression, while SV40 promoter induced the lowest intensity. Our results indicate that plasmids with appropriate promoters should be carefully selected to obtain strong exogenous gene expression by in vivo electroporation. © FUNPEC-RP. Source

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