| Objectives: Heart failure(HF) is the results of many cardiovascular diseases. The functions of left ventricle slipping down progressively are partially because of the loss of cardiac myocytes. Apoptosis in cardiac myocytes may be the main reason for this. More recent studies have developed and validated animal models available to investigate the mechanisms of apoptosis in cardiac myocytes. Since the limitations of animal models,in vitro culturing cardiac myocytes has become an important method to study pathophysiologic functions in cardiac myocytes. It is difficult to large-scale culture cardiac myocytes bacause of them are terminally differentiated cells. But H9C2, derived from rat cardiomyoblasts,can substitute primary culture cardiac myocytes in some extend.Oxidative stress is increased in the failing heart, and this contribute to the pathogenesis of heart failure. Reactive oxygen species(ROS) have been proposed that they could damage intracellular biomacromolecule (DNA, protein, lipid) and induce apoptosis. Norepinephrine (NE) is main neurotransmitter of sympathetic nerve and play an important role in cardiac myocytes pathophysiologic process. In myocardial cell, NE can increase intracellular ROS producing by time-dependent and dose-dependent way. With low dose of NE, myocardial cell could be induced to be hypertrophic phenotype. But with high dose of NE, they became apoptotic phenotype.PrxⅢ(PeroxiredoxinⅢ), a mitochondrion-specific peroxidase, serves as a thioredoxin-dependent peroxidase with its partners, an organelle-specific thioredoxin (Trx2) and NADPH-linked thioredoxin reductase (TrxR2). Mitochondria are the major intracellular sites of oxygen consumption producing reactive oxygen species (ROS) as toxic by-products of oxidative phosphorylation, primarily via electron leakage from the respiratory chain. Accumulating data suggest that the generation of ROS plays important roles in apoptosis. PrxⅢacts as an key scavenger of reactive oxygen species (ROS) under oxidative stress, preserves the cell to avoid damage from ROS, and produces a marked effect in anti-apoptosis.In this study, we used a cell culture model system-H9C2 cell line treated with apoptotic doses of NE (200μmol/L) for 24h to object the morphology changes during apoptosis process. Then we detect the expression changes of PrxⅢduring process of apoptosis in H9C2 cell line to understand the mechanisms of apoptosis and to lay a foundation for the dependablity between PrxⅢand the apoptosis in H9C2 .Methods1 Cell culture.H9C2 cell line is cultured with complete medium (Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5g/L sodium bicarbonate and 4.5g/L glucose, 90%; fetal bovine serum, 10%) in temperature 37.0℃, the 95% air and 5% carbon dioxide (CO2). When 70-80% fusion, cells were digested with 0.25% (w/v) Trypsin- 0.53 mM EDTA 2 mins and subcultured.2 To set up the cardiac myocytes apoptosis model.Replace the complete medium with the medium without FBS 24 hours after subculture and continue to culture for 24 hours. After removing the medium, add the medium with 0.5%FBS in NE control group and the medium with 0.5%FBS, 200μmol/L NE in NE group. Then culture continuously for 24 hours.3 Observe the morphology changes of the cell with phase contrast microscope .4 Observe the ultrastructure changes of the cell with transmission electron microscope (TEM).5 Observe the morphology changes of the cell after stained by Hoechst 33258 with fluorescence microscope, count the number of apoptosis cells ,then compute the rate of cell apoptosis.6 Measure the value of OD490 with MTT.7 The expression of PrxⅢmRNA in the cell.Results1 Observe the morphology changes of the cell with phase contrast microscope :In NE control group and normal control group, the cell morphology did not change expressly. In NE group, there can be found some round, half adherence or float cell and their refraction strongly; Cell membrane shrinking; The blank between cells became wider; Cell volume deflated and chromatin concentrated.2 Observe the ultrastructural changes of the cell under transmission electron microscope (TEM):In NE control group and normal control group, there was no change of the cell: intract nuclear membrane and uniformity chromatin. In NE group, apoptotic changes were found in some cells; chromatin clumps aggregated near the nuclear membrane; disrupted nuclear membrane.3 Observe the morphology changes of the cell after stained by Hoechst 33258 with fluorescence microscope, count the number of apoptosis cells:After specific nuclear being stained with Hoechst 33258, there revealed blue bright pyknotic, shrunken, distorted nuclei in the apoptosis cell.4 Measure the value of OD490 with MTT.The the value of OD490 in NE group(0.111±0.054) was much lower than that in NE control group(0.690±0.070) and in normal control group (0.430±0.095)(P < 0.05). But there was no difference between the value of OD490 in NE control group and that in normal control group (P>0.05).5 The expression of PrxⅢmRNA in the cell:The expression of PrxⅢmRNA in NE group (0.3121±0.0697) was higher than that in NE control group (0.2049±0.0374) (P<0.05). The expression of PrxⅢmRNA in NE group was higher than that in normal control group (0.1937±0.0468) (P<0.05). But there was no difference between the expression of PrxⅢmRNA in NE control group and that in normal control group (P>0.05).Conclusions1 The cell apoptosis model can be set up successfully through treatment the H9C2 cell with apoptotic doses of NE (200 microM) for 24h.2 Following the increase of apoptosis rate, the expression of PrxⅢmRNA augmentated significantly.3 Mitochondrial Dysfunction may be the main reason of the apoptosis of the H9C2 cell inducing by NE.
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