黄芩素和LY294002对人肝癌细胞系SMMC-7721细胞增殖和凋亡的影响研究

摘要

目的：探讨黄芩素和LY294002对人肝癌细胞系SMMC―7721细胞增殖和凋亡的影响。方法2015年3月—2016年1月，取人肝癌细胞系SMMC―7721，制备细胞悬液。加入黄芩素（1、2、5、10、20、50、100、200、300μmol/L，分别命名为A1、A2、A3、A4、A5、A6、A7、A8、A9组）或LY294002（1、2、5、10、20、30μmol/L，分别命名为B1、B2、B3、B4、B5、B6组），设定空白对照组和二甲基亚砜（ DMSO）对照组，采用CCK8试剂盒检测各组细胞增殖水平；采用20μmol/L黄芩素（ C1组）单独处理，20μmol/L黄芩素联合10μmol/L LY294002（ C2组）处理人肝癌细胞系SMMC―7721，设定空白对照组和DMSO组，采用流式细胞术检测各组细胞周期；采用2、5、10、20μmol/L黄芩素（分别命名为D1、D2、D3、D4组）处理人肝癌细胞系SMMC―7721，设定空白对照组和DMSO组，采用显微摄影检测各组细胞数量；采用20μmol/L 黄芩素（ E1组）单独处理，20μmol/L 黄芩素联合10μmol/L LY294002（E2组）处理人肝癌细胞系SMMC―7721，设定空白对照组和DMSO组，采用流式细胞术检测各组早期凋亡和晚期凋亡情况；采用20μmol/L黄芩素（ F1组）、10μmol/L LY294002（ F2组）或20μmol/L黄芩素联合10μmol/L LY294002（F3组）处理人肝癌细胞系SMMC―7721，设定空白对照组和DMSO组，采用实时荧光定量PCR（Real―time PCR）法检测细胞外调节蛋白激酶（ERK）1/2、周期素D1（CyclinD1）、糖原合成酶激酶―3β（GSK―3β）、丝氨酸/苏氨酸蛋白激酶（ AKT）mRNA表达水平；采用20μmol/L黄芩素（ G1组）、10μmol/L LY294002（ G2组）或20μmol/L黄芩素联合10μmol/L LY294002（ G3组）处理人肝癌细胞系SMMC―7721，设定空白对照组和DMSO组，采用Western blotting法检测磷酸化ERK1/2（ P―ERK1/2）、CyclinD1、磷酸化GSK―3β（ P-GSK―3β）、磷酸化AKT （ P―AKT）表达水平。结果 A8组、A9组、B6组细胞增殖水平低于空白对照组、DMSO对照组（ p〈0.05）。C2组G0/G1期、G2/M期细胞比例高于空白对照组、DMSO组，S期细胞比例低于空白对照组、DMSO组（p〈0.05）。D4组细胞数量少于空白对照组、DMSO组（p〈0.05）。空白对照组、DMSO组、E1组、E2组早期凋亡和晚期凋亡比较，差异无统计学意义（ p〉0.05）。F3组ERK1/2、CyclinD1、GSK―3β、AKT mRNA表达水平均低于空白对照组、DMSO组（p〈0.05）。G3组P―ERK1/2、CyclinD1、P-GSK―3β、P―AKT表达水平低于空白对照组、DMSO组（p〈0.05）。结论黄芩素和LY294002可抑制人肝癌细胞系SMMC―7721细胞增殖，但不影响其凋亡。%Objective To study the effects of baicalein and LY294002 on proliferation and apoptosis of human hepatocellular carcinoma cell line SMMC―7721. Methods From March 2015 to January 2016,human hepatocellular carcinoma cell line SMMC―7721 was cultured in vitro to produce cell suspension. The cells were treated by baicalein(1,2,5,10,20, 50,100,200,300 μmol/L,and were respectively named A1,A2,A3,A4,A5,A6,A7,A8,and A9 group),or LY294002(1,2,5,10,20,30 μmol/L,and were respectively named B1,B2,B3,B4,B5,and B6 group). The blank control group and DMSO control group were set up,and the cell proliferation level of SMMC―7721 was measured by CCK8 kit;20 μmol/L baicalein alone(C1 group)or combined with 10 μmol/L LY294002(C2 group)was added to SMMC―7721, the blank control group and DMSO group were set up,and the cell cycle was detected by flow cytometry;2,5,10,20μmol/L baicalein(D1,D2,D3,D4 group were named respectively)was added to SMMC―772,the blank control group and DMSO group were set up,and the cell amount in each group was analyzed by microscopy;20 μmol/L baicalein alone( E1 group)or combined with 10 μmol/L LY294002(E2 group)was added to SMMC―7721,the blank control group and DMSO group were set up,and the early and late apoptosis in each group were measured by flow cytometry;20 μmol/L baicalein alone ( F1 group),10 μmol/L LY294002 alone( F2 group)or combination of these two( F3 group)was added to SMMC ―7721,the blank control group and DMSO group were set up,and the expression level of ERK1/2,CyclinD1,GSK―3β,AKT mRNA was detected by real ― time fluorescence quantification PCR( Real ― time PCR);20 μmol/L baicalein alone( G1 group),10μmol/L LY294002 alone(G2 group)or combination of these two(G3 group)was added to SMMC―7721,the blank control group and DMSO control group were set up,and the expression level of P―ERK1/2,CyclinD1,P-GSK―3β,and P―AKT was analyzed by Western blotting method. Results The cell proliferation level in A8 group,A9 group and B6 group was lower than that in blank control group and DMSO control group(p〈0. 05). The cell ratio at G0/G1 and G2/M period in C2 group was higher than that in blank control group and DMSO group,while the ratio at S period was lower than that in blank control group and DMSO group(p〈0. 05). The cell amount in D4 group was fewer than that in blank control group and DMSO group(p〈0. 05). There was no significant difference in early and late apoptosis among blank control group,DMSO group,E1 group and E2 group(p〉0. 05). The expression level of ERK1/2,CyclinD1,GSK―3β,AKT mRNA in F3 group was lower than that in blank control group and DMSO group(p〈0. 05). The expression level of P―ERK1/2,CyclinD1,P-GSK―3β and P―AKT in G3 group was lower than that in blank control group and DMSO group ( p〈0. 05 ) . Conclusion Baicalein and LY294002 can inhibit the cell proliferation of human hepatocellular carcinoma cell line SMMC ― 7721 , but they cannot influence its apoptosis.