异甘草素对SHG44人脑胶质瘤干细胞增殖和分化的影响

Effect of isoliquiritigenin on proliferation and differentiation of SHG44 human brain glioma stem cells

目的探讨不同浓度异甘草素对SHG44人脑胶质瘤干细胞增殖和分化的影响及机制。方法实验分为二甲基亚砜(dimethyl sulfoxide,DMSO)对照组,异甘草素(10~160μmol/L)诱导组,氮-[氮-(3,5-二氟苯乙酰)-L-丙氨酰]-S-苯基甘氨酸丁酯(N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-ph,DAPT)(2.0μmol/L)阻断剂组,异甘草素+阻断剂组(10~160μmol/L+2.0μmol/L DAPT),采用CCK-8法、免疫荧光染色、Western blot及Real-time PCR分别检测细胞抑制率、相关分化蛋白及Notch1通路相关基因表达情况。结果异甘草素在12~48 h,随着浓度增加,细胞抑制率减弱(P<0.05),且分化细胞越多,干细胞减少;72 h后随着浓度增加,细胞抑制率增强(P<0.05),分化细胞及干细胞同时减少;隔日加药至第7 d时,经统计分析胶质瘤干细胞球数目减少、直径减小(与对照组比),且P<0.05。异甘草素作用72 h后:与对照组比较,随着异甘草素浓度的增加Nestin蛋白表达量逐渐下调(P<0.05);与对照组比较,10、40、160μmol/L组GFAP蛋白表达水平均上调(P<0.05),且40μmol/L组GFAP蛋白表达量较其他浓度组均较高,(P<0.05);与对照组比较,10、40、160μmol/L组β-TubulinⅢ蛋白表达水平均上调(P<0.05),且10μmol/L组β-TubulinⅢ蛋白表达量较其他浓度组均较高(P<0.05)。Notch1通路阻断剂作用后,与对照组比较,各异甘草素组和阻断剂组Notch1、RBP-JK及Hes1基因表达均显著下调(P<0.05);与异甘草素组比较,Notch1、RBP-JK及Hes1基因表达在异甘草素加DAPT组及阻断剂组显著下调(P<0.05);与阻断剂组比较,Notch1、RBP-JK及Hes1基因表达在异甘草素加DAPT组显著下调(P<0.05)。结论异甘草素能诱导SHG44人脑胶质瘤干细胞向星形胶质细胞和神经元细胞分化,且能抑制其增殖,可能与下调Notch1信号通路中的Notch1、RBP-JK及Hes1有关。

Objective To investigate the effect of isoliquiritigenin on the proliferation and differentiation of SHG44 human brain glioma stem cells and related mechanisms.Methods SHG44 cells were divided into dimethyl sulfoxide （DMSO） control group,isoliquiritigenin （10-160 μmol/L） induction group,N-[N-（3,5-difluorophenacetyl）-1-alanyl]-S-phenylglycine t-butyl ester （DAPT,2.0 μmol/L） antagonist group,and isoliquiritigenin （10-160 μmol/L） ＋ antagonist DAPT （2.0 μmol/L） group.CCK-8,immunofluorescent staining,Western blot,and real-time PCR were used to measure cell inhibition rate and expression of differentiation proteins and Notch1 pathway genes.Results Within 12-48 hours,the cell inhibition rate of isoliquiritigenin decreased with the increasing concentration （P 〈 0.05）,and the number of stem cells decreased with the increase in differentiated cells;after 72 hours,cell inhibition rate increased with the increasing concentration （P 〈 0.05）,and there were reductions in differentiated cells and stem cells.The drugs were added every other day,and on the seventh day,there were significant reductions in the number and diameter of glioma stem cells （compared with the control group,P 〈 0.05）.After 72 hours of isoliquiritigenin treatment,the expression of Nestin protein was gradually downregulated with the increasing concentration of isoliquiritigenin （compared with the control group,P 〈 0.05）;compared with the control group,the 10,40,and 160 μmol/L groups had a significant increase in the expression of GFAP protein （P 〈 0.05）,and the 40 μmol/L group had significantly higher expression of GFAP protein than the other groups （P 〈 0.05）;compared with the control group,the 10,40,and 160 μmol/L groups had a significant increase in the expression of β3-Tubulin Ⅲ protein （P 〈 0.05）,and the 10 t.μmol/L group had significantly higher expression of β-Tubulin Ⅲ protein than the other groups （P 〈 0.05）.After the treatment with Notch1 pathway antagonist,the isoliquiritigenin groups and antagonist group had significant reductions in the expression of Notch1,RBP-JK,and Hes1 genes compared with the control group （P 〈 0.05）;compared with the isoliquiritigenin group,the isoliquiritigenin ＋ DAPT group had significant reductions in the expression of Notch1,RBP-JK,and Hes1 genes （P 〈 0.05）;compared with the antagonist group,the isoliquiritigenin ＋ DAPT group had significant reductions in the expression of Notch1,RBP-JK,and Hes1 genes （P 〈 0.05）.Conclusions Isoliquiritigenin can induce the differentiation of SHG44 human glioma stem cells into astrocytes and neurons and inhibit the proliferation of glioma stem cells,possibly by downregulating the expression of Notch1,RBP-JK,and Hes1 in the Notch1 signaling pathway.