壁虎活性单体衍生物腺嘌呤对人肝癌细胞作用的实验研究

Empirical Research of Gecko Active Monomer Derivative Adenine on Human Hepatic Carcinoma Cells

目的探讨壁虎活性单体衍生物腺嘌呤对人肝癌细胞的抑制作用及机制。方法 2014年9月—2015年4月,收集对数生长期的人肝癌Bel-7402细胞进行实验,将96孔板的第2~9列作为腺嘌呤组〔加入腺嘌呤的浓度分别为0.500 00、0.250 00、0.125 00、0.062 50、0.031 25、0.015 63、0.007 81、0.003 91 mg/ml（分别记为A组~H组）〕,第10列作为对照组（加入含10%胎牛血清的RPMI-1640培养液）,第11列作为空白组。采用噻唑蓝（MTT）法计算不同浓度腺嘌呤组不同时间（培养24、48、60 h）抑瘤率及腺嘌呤组半抑制浓度（IC50）。将对数生长期的人肝癌Bel-7402细胞分为腺嘌呤组（加入0.500 00 mg/ml的腺嘌呤）和对照组（加入含10%胎牛血清的RPMI-1640培养液）,采用透射电镜观察细胞超微结构。将对数生长期的人肝癌Bel-7402细胞分为腺嘌呤组（加入0.500 00 mg/ml的腺嘌呤）和对照组（加入含10%胎牛血清的RPMI-1640培养液）,采用流式细胞仪检测细胞周期。结果 24 h时,B组~H组抑瘤率均低于A组,C组~H组抑瘤率均低于B组,D组~H组抑瘤率均低于C组,E组~H组抑瘤率均低于D组,E组~G组抑瘤率均高于H组（P〈0.05）;48 h时,B组~H组抑瘤率均低于A组,C组~H组抑瘤率均低于B组,D组~H组抑瘤率均低于C组,D组、E组抑瘤率均高于H组（P〈0.05）;60 h时,B组~H组抑瘤率均低于A组,C组~H组抑瘤率均低于B组,D组~H组抑瘤率均低于C组,D组~F组抑瘤率均高于G组和H组（P〈0.05）。C组、E组~H组24 h时抑瘤率均低于48 h时抑瘤率,A组~F组24 h时抑瘤率均低于60 h时抑瘤率,G组24 h时抑瘤率高于60 h时抑瘤率,A组~D组48 h时抑瘤率均低于60 h时抑瘤率,G组、H组48 h时抑瘤率高于60 h时抑瘤率（P〈0.05）。不同时间IC50比较,差异有统计学意义（F=16.816,P〈0.001）;48 h、60 h时IC50低于24 h时,60 h时IC50低于48 h时（P〈0.05）。腺嘌呤作用48h后,肝癌细胞出现明显的线粒体改变。对照组G0/G1期、G2/M期人肝癌Bel-7402细胞数高于腺嘌呤组,S期人肝癌Bel-7402细胞数低于腺嘌呤组（P〈0.05）;腺嘌呤组S期人肝癌Bel-7402细胞数高于G0/G1期,G2/M期人肝癌Bel-7402细胞数低于S期（P〈0.05）。结论腺嘌呤抑制人肝癌细胞的增殖且呈一定的浓度、时间依赖性。腺嘌呤可引起人肝细胞线粒体改变,可将肿瘤细胞阻滞于S期并进一步诱导其凋亡进而发挥抗肿瘤作用。

Objective To study the inhibitory effect of gecko active monomer derivative adenine on human hepatic carcinoma cells. Methods Human liver cancer Bel-7402 cells in logarithmic phase and conducted experiments were collected in the study from September 2014 to April 2015. The 2nd to 9th columns of the 96 hole plate was assigned into the adenine group （adenine concentrations were 0. 500 00,0. 250 00,0. 125 00,0. 062 50,0. 031 25,0. 015 63,0. 007 81 and 0. 003 91 mg/ml,which were respectively recorded as group A to group H）. The 10th column was taken as the control group（RPMI-1640 culture medium containing 10% fetal bovine serum）. The 11th column was taken as the blank group. Thiazolyl blue method （MTT）was used to observe the tumor inhibition rate of each group（at 24 h,48 h and 60 h of the culture）and the 50%inhibiting concentration（ IC50 ）of the adenine group. The human liver cancer Bel-7402 cells in logarithmic phase were dividednbsp;into adenine group（0. 500 00 mg/ml adenine）and control group（ RPMI -1640 culture medium containing 10% fetal bovine serum）, and the cell ultrastructure was observed by transmission electron microscope. The collected human liver cancer Bel-7402 cells in logarithmic phase were divided into adenine group（0. 500 00 mg/ml adenine）and control group（RPMI-1640 culture medium containing 10% fetal bovine serum）,and tumor cell cycle was detected by flow cytometry. Results At 24 h,tumor inhibition rates of group B-group H were lower than those of group A;tumor inhibition rates of group C-group H were lower than group B;tumor inhibition rates of group D-group H were lower than group C;tumor inhibition rates of group E-group H were lower than group D;tumor inhibition rates of group E-group G were higher than H group（ P〈0. 05 ） . At 48 h,tumor inhibition rates of group B -group H were lower than those of group A;tumor inhibition rates of group C-group H were lower than group B;tumor inhibition rates group D-group H were lower than group C;tumor inhibition rates of group D and group E were higher than group H（P〈0. 05）. At 60 h,tumor inhibition rates of group B-group H were lower than that of group A;tumor inhibition rates of group C-group H were lower than group B;tumor inhibition rate of group D-group H were lower than group C;tumor inhibition rates of group D-group F were higher than group G and group H（ P 〈0. 05 ） . Tumor inhibition rates of group C and group E-group H at 24 h were lower than those at 48 h. Tumor inhibition rates of group A-group F at 24 h were lower than those at 60 h. Tumor inhibition rate of group G at 24 h was higher than that at 60 h. Tumor inhibition rates of group A-group D at 48 h were lower than those at 60 h. Tumor inhibition rates of group G and goupr H at 48 h were higher than those at 60 h（P〈0. 05）. IC50 varied significantly at different time points（F=16. 816,P=0. 000）. The IC50 at 48 h and 60 h was lower than that at 24 h;the IC50 at 60 h was lower than that at 48 h（P〈0. 05）. After 48h,the changes of mitochondria in liver cancer cells were obvious. The numbers of cells in G0/G1 and G2/M phase in control group were higher than those in adenine group;the number of cells in S phase in control group was lower than that in adenine group（ P〈0. 05 ） . In adenine group,the number of cells in S phrase was higher than that in G0/G1 phrase,and the number of cells in G2/M phrase was lower than that in S phrase（ P〈0. 05 ） . Conclusion Adenine could inhibit the proliferation of human hepatic carcinoma cells in a time and dose dependent manner. Adenine lead to changes of mitochondria. Adenine could inhibit tumor cells in S phase and further induce tumor cell apoptosis,thus playing an anti-tumor effect.