Effects of hypoxia induced by the cobalt chloride on the growth and apoptosis in hepatic cancer cell HepG2 / 国际外科学杂志

ABSTRACT

Objective To establish a human HepG2 cell growth model under the low oxygen environment induced by cobalt chloride in order to observe the impacts of human HepG2 cell proliferation,cellular cycles and apoptosis,namely cellular growth conditions,under the low oxygen environment induced by cobalt chloride with different concentrations and to study the HepG2 cell growth mechanism under the low oxygen environment induced by cobalt chloride.Methods The human HepG2 cells in the logarithmic phase were randomized grouping as control group and CoCl2 experimental group with different concentrations (50 μm/L,100 μm/L,150 μm/L and 200 μm/L).① HepG2 cell proliferation was tested by MTT assay to calculate cell's suppression rate and draw HepG2 cell growth inhibition curves.② The move ability of HepG2 cells was observed by scratch test.③ The cellular apoptosis and periodic changes were detected using the flow cytometer Annexin-V FITC/PI double-staining and PI single staining methods.④HepG2 cell's Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Bcl-2 protein expression were detected by Western Blot.Results ① The test results obtained via MTT assay showed that CoCl2 suppressed the human HepG2 cell proliferation within a certain amount of time and concentration and presented a time-dose dependent relation.② Scratch damage trial suggested that the cobalt chloride suppressed the HepG2 cell migration and wound repair capacity and presented a concentration dependent relation.③ Flow cytometer' s test results revealed that the apoptosis rates (％) in control group and experimental group with different concentrations (50 μm/L,100 μm/L,150 μm/L and 200 μm/L) were 3.42,7.74,13.07,20.56,28.53 and 44.45 (P ＜0.05),respectively.The apoptosis rate of the experimental group was significantly increased compared with the control group,as well as showing a concentration dependency.The results of cellular cycles revealed that the cobalt chloride significantly suppressed the HepG2 cell's periodic changes along with increases of concentration,as well as blocked the cell cycle staying in phase G1,thereby suppressing cell proliferation.④Western Blot testCompared with the control group,the Bcl-2 protein expression was significantly decreased in the experimental group after treatment of cobalt chloride with different concentrations.Conclusion Within a certain range,CoC12-indiuced low oxygen environment can suppress the human HepG2 cell proliferation and healing migration capacity,induce apoptosis and present a time-dose dependent relation.The mechanism is likely associated with decreases of Bcl-2 protein expression.