| Objcective:In order to study the effection of NRF-1 gene on the function metabolism of mitochondria in rat cardiomyocytes(H9c2), the physical deficiency oxygen model of 1%O2+5%CO2 was established to investigate whether the NRF-1 gene can improve the growth state of rat myocardial cells and the level of energy metabolism level under hypoxic condition,which aims to provide a new way for the gene therapy of heart failure. Method:1. NRF-1 overexpression cell line(p CDH-NRF),interfering cell line(sh-NRF) and empty virus cell line(p CDH-vector) were constructed by using the third-generation lentivirus vector with a conditional packaging system. The expression level of NRF-1 was identified by Western blot and fluorescence quantitative PCR.2. The three cell groups were cultured under 5%CO2+95% air and 1%O2+5%CO2 conditions respectively,then the change of cell viability was measured by CCK-8 method.3. The three cell groups were cultured under 5%CO2+95% air and 1%O2+5%CO2 conditions for 48 hs respectively,then the changes of mitochondrial membrane potential were measured by flow cytometry.4. The oxygen consumption under normal and hypoxic conditions.of the cells in each group was detected by using cell mitochondria energy metabolism apparatus. Results:1. The NRF-1 gene overexpression lentivirus vectorc was successfully constructed and the results of fluorescence quantitative PCR showed that the expression level of NRF-1 gene in over expression cell line((4.08±0.3)) was higher than the empty virus groups(1.00±0.15)and the level in NRF-1 interference cell(0.35±0.14)s was lower than the empty virus group cell line;the results of Western blot showed that the expression level of NRF-1 protein(5.07±0.63)was higher than empty virus group(3.15±0.59)and interference group(1.66±0.22).2. After the cells was cultured in normal conditions for 24 h, the optical density(OD) of NRF-1 overexpression cells, NRF-1 interference cells and empty vector cells was 1.4365±0.0223?1.5125±0.0543?1.4087±0.0263 respectively;however after the cell was cultured in the condition of 1%O2+5%CO2 for 24 h,the optical density(OD) of three groups were 1.4605±0.0413?1.4833±0.1092?1.4365±0.0926 respectively,difference is not obvious.3. After the cells was cultured in normal conditions for 48 h, the optical density(OD) of NRF-1 overexpression cells, NRF-1 interference cells and empty vector cells was 1.8649±0.0132?1.8176±.03964?1.7872±0.0062, 1.4087 + 0.0263 respectively;however after the cell was cultured in the condition of 1%O2+5%CO2 for 48 h,the optical density(OD) of three groups were 1.5706±0.0251?1.0781±0.0717?1.4038±0.0375 respectively,difference is not obvious,the difference was significant, with statistical significance(p<0.05).4. The mitochondrial membrane potential depolarization level of NRF-1 overexpression cells,NRF-1 interference cells and empty vector cells in normal condition was 3.1%?5.1%?1.2% respectively;but after the cell of NRF-1 overexpression group,NRF-1 interference group and empty vector group was cultured in the 1%O2+5%CO2 hypoxia condition,The mitochondrial membrane potential depolarization level was 52.7%?28.1%?48.1%,which suggesting that the overexpression of NRF-1 gene can decrease the damage of mitochondrial membrane potential by hypoxia.5.After cell was treated by cobalt chloride and hypoxia, compared with the empty virus group,the oxygen consumption of NRF-1 overexpression groups was higher,and the oxygen consumption of NRF-1 interference group was lower. Conclusion:The overexpression of NRF-1 gene can reduce the damage of hypoxia to cells. The possible mechanism is that NRF-1 gene can decrease the reduction of mitochondrial membrane potential of the cell,increase the oxygen consumption,by that can protect the cell from the damage of hypoxia,which indicate that NRF-1 gene can be used as a candidate moleculefor the treatment of heart failure. |
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