| Currently, liver cancer is one of the malignant tumors that has the highest morbidity and mortality rate and is most difficult to be cured in China. According to statistics, approximately 130,000 people die from liver cancer every year in China, accounting for 43.7% of the death toll of liver cancer worldwide. With the establishment of Mas gene knockout mouse, a majority of researches have indicated that Mas is a kind of specific receptor of Ang-(1-7), and it constitutes a new branch of the renin angiotensin (RAS) with ACE2: ACE2-Ang-(1-7)-Mas shaft, which is to antagonize the biological activity of the key shaft ACE-Ang Ⅱ-AT1 in the traditional RAS, thus realizing biological effects of vascular dilation, inhibition of cell growth, anti-proliferation, tissue fibrosis resistance, anti-arrhythmia, and anti-insulin resistance.Functions of the ACE2-Ang-(1-7)-Mas shaft are intervened via the drug (Mas activator Ang-(1-7), Mas inhibitor A779) effect, so as to provide new possibilities for the treatment of primary hepatocellular carcinoma.Objectives:To investigate the effect of Ang-(1-7) in hepatocellular carcinoma (HCC) cells through Mas by studying biological activity changes of cells in terms of the proliferationthe, the expression difference, apoptosis and cycle by Mas.Methods:1. Drug intervention on HepG2 cellTo determine the effective concentration of the drug,10-5mol/L,10-6mol/L 10"7mol/L and 10-8mol/L Ang-(1-7) are used respectively to stimulate the cell for 24 hours in the dosage effect experiment. In the time effect experiment, 10"7mol/L Ang-(1-7) is applied to stimulate the cell for 24,48 and 72 hours respectively. The same steps are adopted for A779.To elucidate the role of Mas, the Mas inhibitor A779 is used for contrast. The experiment is divided into 4 groups (the control group, Ang(1-7) group, Ang(1-7)+A779 group, and A779 group). Each group is stimulated for 24 hours.2. Conducting real-time quantitative RT-PCR to detect the expression level of Mas after drug intervention.3. Using flow cytometry instrument to detect changes of apoptosis after drug intervention.4. Using flow cytometry instrument to detect changes of the cell cycle after drug intervention.Results:1. The effective working concentration of Ang-(1-7) and A779 is 10"7mol/L.2. After intervention on cells by Ang-(1-7) and A779, the cellular morphology does not change significantly.3. Ang-(1-7) can promote the expression of Mas. In the real-time quantitative RT-PCR, cells are stimulated for 24 hours with 10-7mol/L Ang-(1-7), and the expression level of Mas is higher than that in the control group.4. A779 can inhibit the expression of Mas. In the real-time quantitative RT-PCR, cells are stimulated for 24 hours with 10"7mol/L A779, and the expression level of Mas is lower than that in the control group.5. The apoptosis of Ang-(1-7) group is clearly up-regulated.Conclusions:1. By combining with Mas, Ang-(1-7) can boost the cell proliferation, and its effective working concentration is10-7mol/L for 24 hours. A779 can inhibit the cell proliferation despite of the insignificant effect. Its main feature is to antagonize functions of Ang-(1-7).2. There is no obvious change for the cellular morphology after intervention on cells by Ang-(1-7) and A779.3. Ang-(1-7) can promote the gene expression of cell Mas, while A779 can inhibit the expression of Mas. However, effects of the two are not obvious in contrast to the control group, so there is no significant difference.4. Up-regulated Mas can dramatically promote the apoptosis of human hepatocellular carcinoma cells. |
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