| Objective:To investigate the effects of isoliquiritigenin,temozolomide,and the combination of two drugs on the proliferation and apoptosis of SHG44 human glioma stem cells and its possible mechanism.To provide a certain experimental basis for isoliquiritigenin as an adjuvant drug for glioma chemotherapy.Methods:(1)SHG44 human glioma cells were cultured in the petri dish using DMEM high glucose medium containing 10% fetal bovine serum.The MTS kit detected the control group(containing the same amount of DMSO as the experimental group),the isoliquiritigenin group(40?mol/L,80?mol/L,160?mol/L),the temozolomide group(50?mol/L,100?mol/L,200?mol/L).And the increase of the isoliquiritigenin+temozolomide group(40?mol/L+ 50?mol/L,80?mol/L+100?mol/L,160?mol/L+200?mol/L)after treatment with SHG44 human glioma cells,calculated based on absorbance values Value-added rate and inhibition rate,and then the drug combination effect evaluation was performed by Jin Zhengjun method;Western-blot analysis of the expression of apoptosis-related proteins Caspase-3,BAX,and anti-apoptotic protein Bcl-2 in cells after 48 hours of intervention,and further To explore the mechanism of synergistic effect of two drugs on SHG44 human glioma cell apoptosis.(2)Use DMEM / F12(1:1)serum-free medium(including B27,EGF,b-FGF)to extract SHG44 human glioma stem cells from adherent SHG44 human glioma cells.CD133 and Nestin were used to identify the characteristics of stem cells by immunofluorescence.(3)The MTS kit detects the increment of SHG44 human glioma stem cells treated by each treatment group,and observes the spheroid formation of glioma stem cells after drug treatment.Calculates the appreciation rate and inhibition rate based on the absorbance value,and then passes through both methods were used to evaluate the effect of drug combination;Western-blot analysis of the expression of apoptosis-related proteins Caspase-3,BAX,and anti-apoptotic protein Bcl-2 in stem cells after 72 hours of intervention,and then explore the mechanism of the combination of two drugs to promote SHG44 human glioma stem cell apoptosis.Results:(1)After the isoliquiritigenin group,temozolomide group,and the isoliquiritigenin + temozolomide group treated SHG44 human glioma cells for 12-72 hours,compared with the control group(containing the same amount of DMSO as the experimental group),the increase of the cells was inhibited as the concentration increased,and The difference was statistically significant between 24 and 72 hours after treatment(P <0.05),the difference was significant between 24 and 72 hours after temozolomide treatment(P<0.05),and the difference between 12 and 72 hours after treatment with both drugs was statistically significant(P <0.05).The q value was calculated by Jin Zhengjun method.After 24 hours of administration of different concentrations of isoliquiritigenin+temozolomide(80?mol/L+ 100?mol/L,160?mol/L+200?mol/L),the q values were greater than 1.15.The synergistic effect is shown,in which the synergistic effect of isogliocidin+temozolomide(80?mol/L +100?mol/L)for 48 h is the best;Western-blot results suggest that 80?mol/L isoliquiritigenin,100?mol/L temozolomide,and isoliquiritigenin+temozolomide(80?mol/L+100?mol/L)acted on SHG44 human glioma cells for 48 hours,compared with the control group.The expressions of death-related proteins Caspase-3 and BAX increased,while the expression of anti-apoptotic protein Bcl-2 decreased,and the difference was statistically significant(P <0.05).(2)Compared with the control group(containing the same amount of DMSO as the experimental group),the isoliquiritigenin group treated SHG44 human glioma stem cells for 12 to 48 hours,and the value-added activity of the cells increased with increasing concentration.Combined with the results of previous experiments,the reason for the increased proliferation activity is isoliquiritigenin promoted stem cell differentiation;the value-added activity of the isoliquiritigenin group was inhibited after 72 hours of treatment,and the difference was statistically significant(P <0.05);the temozolomide group was treated with SHG44 human glioma stem cells for 12 to 72 hours.Cell proliferation activity was inhibited with increasing concentration,and there was a statistically significant difference in treatment at each concentration at 72 hours(P<0.05);while the isoliquiritigenin+ temozolomide group treated SHG44 human glioma stem cells for 12 to 72 hours,cells increased with concentration Proliferative activity was inhibited,and the difference in treatment at each concentration was statistically significant at 48 to 72 hours(P <0.05);the q value was calculated by the Jin Zheng-Jun method.In the isoliquiritigenin+temozolomide group(80?mol/L+100?mol/L,160?mol/L+200?mol/L)for 72 hours,the q values were all greater than 1.15,and the combination of drugs showed a synergistic effect,of which the synergistic effect was the best when the concentration was 160?mol/L+200?mol/L for 72 hours;Western-blot results suggest that 160?mol/L isoliquiritigenin,200?mol/L temozolomide,and isoliquiritigenin+temozolomide(160?mol/L+200?mol/L)act on SHG44 human glioma stem cells for 72 hours,and then wither compared with the control group.The expressions of death-related proteins Caspase-3 and BAX increased,while the expression of anti-apoptotic protein Bcl-2 decreased,and the difference was statistically significant(P <0.05).Conclusions:(1)Isoliquiritigenin combined with temozolomide can inhibit the growth of SHG44 human glioma cells and SHG44 human glioma stem cells.The combination of the two has a synergistic effect.(2)Isoliquiritigenin and temozolomide can promote the apoptosis of SHG44 human glioma cells and SHG44 human glioma stem cells by up-regulating the expression of apoptosis-related proteins Caspase-3 and BAX,and down-regulating the expression of anti-apoptotic protein Bcl-2. |
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