The Effects Of Hypoxia Induced By The Cobalt Chloride On The Growth And Apoptosis In Hepatic Cancer Cell HepG2

ObjectiveTo establish the growth model of human hepatocellular carcinoma HepG2 cells of cobalt chloride, observe the hypoxia environment induced by cobalt chloride on human hepatocellular carcinoma HepG2 cells proliferation, cell cycle and apoptosis, then discusses the metabolism mechanism of human hepatocellular carcinoma HepG2 cells of the hypoxia induced by different concentrations of cobalt chloride on the growth,and then further to explore the tolerance changes of the tumor cells to the low oxygen environment which is helpful to find the theoretical foundation for the clinically treatment of tumor chemotherapy.MethodLogarithmic phase of HepG2 cell line were random divided into groups control group and experimental group which had five different concentrations(50μm/L、100μm/L、200μm/L、 300μm/L、500μm/L)。 MTT method assay was used to detect cell survival rate or cell proliferation capacity,Scratch test were used to observe the influence of cobalt chloride with different does on migration ability of HepG2 cell line. By flow cytometry of V FITC/PI double staining method and PI single staining method to detect cell apoptosis and cycles respectively, the expressions of Glyceraldehyde 3-phosphoric acid dehydrogenase and the Bcl-2 protein were analyzed by Western blot.Result1. By the results of MTT method assay the Cobalt chloride inhibit the proliferation of human hepatocellular carcinoma HepG2 cells, and the inhibition was on time-dose dependent relationship in a certain reacting time and concentrtions, namely the cell increment rate was gradually reduced with the increase of concentrations and time of the Cobalt chloride.2. The effects of the HepG2 cells was injured after Scratch test with the increased concentration of cobalt chloride the experimentl group cell migration ability was restrained obviously compared with control group, cobalt chloride restrained the migration ability and damage repair ability of HepG2 cell in the concentration of dependency relationship. (P< 0.05).3. By the Flow cytometry instrument test results:the cell apoptosis rate(%) of the control group, the experimental groups of different concentration (50μm/L、100μm/L、200μm/L、 300μm/L、500μm/L) were:3.42,7.74,13.07,20.56,28.53,20.56 respectively(P<0.05), compared with the control group the apoptosis rate of experimental groups increased significantly and in the concentration dependence.With the increased of cobalt chloride concentration, the percentage (%) of the cell cycle G1 phase of the HepG2 cells increased from 30.51 to 68.10, higher than the 28.38(%) of the control group and the proportion of S phase was reduced from 42.51 to 17.58, which was less than 42.91(%) of the control group. Thus the cobalt chloride inhibiting the cell proliferation by significantly block the HepG2 cell cycle at G1 phase.4. Western Blot method:compared with the control group, the Bel-2 protein expression decreased significantly on experimental group after treated with different concentrations of cobalt chloride.ConclusionWithin a certain range,the effects of hypoxia induced by cobalt chloride can inhibit the proliferation, the capacity of heal transfer and the apoptosis of human hepatocellular carcinoma HepG2 cells in time-dose dependent relationship, the mechanism may be related to the expression of Bel-2.