| Objective: To study the effect of Zinc on the viability of HepG2 cells cultured in virto and research the effect of Zinc on HepG2 cells growth and mechanism for providing the experiment ground for treating liver cancer.Method: HepG2 cells, which were cultured in virto and experimented with cells in increased logarithmic phase, were cultured for 24 hours in two groups: the control and the high concentrations of 50, 100, 200, 250μmol/L respectively. The cell vigor was determined by MTT assay, and the morphology changes observed by a phase-contrast microscope and by AO/EB assay, for estimating the effects of Zinc of different concentrations on morphology changes of HepG2 cell; The cell cycle and apoptotic changes were studied by the flow cytometry, the level of Bax and Bcl-2 measured by the immunocytochemistry, the level of Bax, Bcl-2 and P53 measured by the western blot, the activity of SOD and the content of MDA in supernatant fluid detected by the xanthine oxidase and the Thiobituric acid reaction, for investigating the zinc's functional mechanism in HepG2 cells. The results were analyzed by the software SPSS13.0.Results:1. Zinc of high concentration restrained the viability of HepG2 cells. MTT results showed that the higher concentration of Zinc is, the lower the survival rate of HepG2 cells is. The survival rate of HepG2 cells was 69% in solution with Zinc concentration of 250μmol/L apparently lower than that in solution of control group(p<0.01), which was set as the experiment concentration.2. The changes of HepG2 cells were observed by a phase-contras microscope and AO/EB assay. Compared with the control cells, decreased number and changes of morphological characteristics including prolonged and shrank shapes of cells, refraction and split decrease happened in the HepG2 cells exposed to Zinc (250μmol/L) for 24 hours. More apoptotic cells were found.3. Zinc(250μmol/L) could cause cell apoptosis at the rate of 9.8% measured by the flow cytometry. No apparent change of cell cycle was observed.4. The western blot and Immunocytochemistry showed that Zinc (250μmol/L) increased the level of Bax and decreased the level of Bcl-2 and restrained the level of wt-P53 after 24 hours(p<0.05).5. Zinc (250μmol/L) enhanced the activity of SOD and decreased the content of MDA after 24 hours. The results bear statistics significance compared with that of control group(p<0.05).Conclusions:1. Zinc (250μmol/L) could influence the viability of HepG2 cells after 24hs.2. Zinc (250μmol/L) could inhibit the proliferation of HepG2 cells by causing apoptosis after 24 hours.3. Zn2+ could increase the level of Bax and decrease the level of Bcl-2 and increase the ratios of Bax/Bcl-2 to induce the HepG2 cells apoptosis.4. Zn2+ could increase the level of wt-P53 and the activity of SOD, decrease the content of MDA, and enhance the ability of resisting oxidize. Zn2+ could not induce the the HepG2 cells apoptosis by wt-P53. |
