Effects Of Hypoxia On Biological Behavior Of CD133~+ Hepatoma Cells

Objective:The biological characteristics of CD133+ hepatoma cells were studied by in vitro and in vivo experiments.The effects of hypoxia on the biological behavior of CD133+ hepatoma cells were investigated by simulating hypoxia microenvironment in vitro.Methods:1、Using CD133 as a surface marker,CD133+ cells and CD133’ cells were isolated from human hepatoma Huh7 cell lines by immunomagnetic bead sorting technique;2、The expression of CD 133 in Huh7 control group(group N),group CD 133+ and group CD 133-were detected by flow cytometry.Three groups of cells were cultured respectively;3.The cell growth curves of Huh7 control group(group N),group C3133+ and group CD 133-were drawn by MTT method;4、The cell cloning ability of Huh7 control group(group N),group CD133+ and group CD 133-were detected by colony forming assay;5.Chemical hypoxia micro environment was simulated by cobalt chloride(CoCl2).The hypoxic tolerance of Huh7 control group(group N),group CD133+ and group CD 133’ were detected by MTT;6.MTT method was used to detect the cell adhesion of Huh7 control group(group N),CD133+ group and CD133-group under normoxic and anoxic conditions;7.The MTT method was used to detect the resistance of the Huh7 control group(group N),the CD 133+ group and the CD 133-group to the treatment of cisplatin under the condition of normoxia and hypoxia,respectively;8.The apoptosis,necrosis and cell cycle of Huh7 control group(group N),CD133+group and CD133-group were detected by flow cytometry under the condition of normoxia and hypoxia;9.The cells of the Huh7 control group(group N),the CD133+ group and the CD133-group were inoculated subcutaneously in nude mice,and the growth of the tumor was observed.Cisplatin treatment was used to observe the tumor changes;10.Data were collated by Excel2007 and analyzed by SPSS22.0 software.Measurement data is represented by x±s.Comparison of two groups of measurement data,the data of normal distribution were tested by T,the data that do not obey the normal distribution are tested by Wilcoxon signed rank sum test.The variance analysis is used to compare the multi group measurement data that follows normal distribution and is homogeneous.Multivariate statistical data that do not obey normal distribution or variance are tested using the Kruskal-Wallis H test.Comparison of repeated measurements are tested using repeated measures analysis of variance.Significant level alpha =0.05.Results:1、The expression of CD 133 in the Huh7 cells was 42.99%,the expression level of CD133 in CD133+ group was 91.21%,and the expression level of CD133 in CD133-group was 10.23%by flow cytometry.The difference was statistically significant before and after sorting(t=12.50;P=0.0002).The cells grew well in the course of follow-up,and the cell activity was not affected;2、The growth curve showed that the viability and proliferation of CD133+ hepatoma cells were significantly stronger than those of CD133-hepatoma cells(p=0.029);3、Cell cloning experiments showed that CD133+ hepatoma cells formed the largest number of cell clusters,and the ability of clone formation was stronger than that of CD133-hepatoma cells(p=0.000009);4、With the increase of cobalt chloride(CoCl2)concentration,the proliferation of cells was inhibited.The survival rate of CD133+ hepatoma cells was higher than that of CD133-hepatoma cells under the same concentration of cobalt chloride(CoCl2)(p=0.00346);5、With the concentration of cisplatin increased,cell proliferation was inhibited.Under normoxic conditions,the resistance of CD133+ hepatoma cells to cisplatin treatment is stronger than that of CD 133-hepatoma cells(p=0.00403).Under hypoxic conditions,the sensitivity of CD 133+ hepatoma cells to cisplatin treatment was better than that of CD 133-hepatoma cells(p=0.0025);6、Apoptosis and necrosis rate decreased under hypoxia.The apoptosis and necrosis rate of CD133+ hepatoma cells were lower than those of CD 133-hepatoma cells under normoxia and hypoxia;7、Under aerobic condition,54.92%of HCC cells were in stage G0/G1,and in hypoxia condition,CD133+ HCC cells showed the number of cells in phase G0/G1 decreased,and the number of cells in phase S and G2/M increased.Under aerobic condition,42.65%of CD 133-HCC cells were in G0/G1 phase,and in hypoxia condition,the number of cells in G0/G1 phase and G2/M phase of CD133-HCC cells increased,and the number of cells in phase S decreased;8、After cobalt chloride(C0Cl2)induced hypoxia for 24 hours,cell adhesion increased(p=5.16*10-11).There was little difference in cell adhesion between group CD133+ and group CD133-(p=0.89401);9、Each group of cells formed tumor subcutaneously in nude mice.The weight of nude mice in cisplatin treatment group was lower than that in control group,and the tumor volume decreased(p=0.005).The tumor inhibition rate of cisplatin in group CD133+nude mice was the highest.Conclusion:1、With CD 133 as a marker,a large number of CD133+ hepatoma cells with higher purity can be obtained by immunomagnetic bead sorting technique.After sorting,the cells grew well and the cell viability was less affected.They could be successfully passaged,cryopreserved and recovered,which could meet the needs of the following experimental cells;2、Compared with CD 133-hepatoma cells,CD133+ hepatoma cells have stronger cell activity,in vitro proliferation ability,clone formation ability,hypoxia tolerance,anti apoptosis ability,and resistance to cisplatin treatment under normoxic condition,consistent with some properties of tumor stem cells.But CD 133 is not suitable for isolated liver cancer cells individually;3、Hypoxia can promote the adhesion of CD 133+ hepatoma cells to basement membrane and extracellular matrix.Hypoxia can reduce the apoptosis and necrosis of CD133+ hepatoma cells.It is suggested that the improvement of hypoxia microenvironment is expected to be a new direction in the treatment of liver cancer.