摘要
目的 探讨二甲双胍对人结肠癌HT29细胞增殖、凋亡的影响及可能机制.方法 体外培养人结肠癌细胞株HT29,随机分为2组,二甲双胍干预组采用5,10,20及40 mmol/L的二甲双胍进行干预,对照组加入等量PBS液,干预24,48及72 h后用MTT法检测细胞的增殖情况.二甲双胍干预组HT29细胞在上述各浓度的二甲双胍干预24 h后,于光镜下观察形态学改变;40 mmol/L的二甲双胍干预48 h后,行AnnexinV-FITC和PI染色,并采用流式细胞术检测分析凋亡情况.二甲双胍干预组二甲双胍干预浓度为5及40 mmol/L,对照组加入等量 PBS 液,干预 48 h,Western-blot 法检测 PI3K, AKT,P-AKT,GSK-3β 及 P-GSK-3β 蛋白的表达.结果 二甲双胍可抑制结肠癌HT29细胞的增殖,并呈时间-剂量依赖性,各组间抑制率差别有统计学意义(P均<0.05).光镜下可见细胞数目减少,由多边形变为圆形,体积缩小,核固缩,细胞核溶解,且随着二甲双胍浓度的增高,细胞凋亡比例升高.流式实验结果 显示,干预48 h的二甲双胍干预组凋亡率大于对照组(P<0. 05).Western-blot检测结果 显示,二甲双胍可抑制HT29细胞的PI3K,P-AKT及P-GSK-3β蛋白表达量,且呈浓度依赖性,而对AKT及 GSK-3β蛋白表达量无明显影响.结论 二甲双胍可抑制人结肠癌HT29细胞的增殖能力并促进细胞凋亡,其机制可能与抑制PI3K/AKT/GSK-3β通路相关蛋白的表达有关.
Abstract
Objective To investigate the effect of metformin on the proliferation and apoptosis of human colon cancer HT29 cells and its possible mechanism. Methods Human colon cancer cell line HT29 was cultured in vitro and randomly divided into two groups. In the experimental group, the concentration of metformin was 5,10,20,and 40 mmol/L, and the corresponding dose of PBS was added to the control group. The cell proliferation was detected by MTT assay at 24, 48,and 72 h. HT29 cells in the experimental group were treated with 5,10,20,40 mmol/L metformin for 24 h, and the morphological changes were observed under light microscope. 5,10,20 and 40 mmol/L metformin for 48 h after HT29 cells were treated with Annexin V-FITC and PI after flow cytometry. The concentration of metformin in the experimental group was low concentration ( 5 mmol/L ) and high concentration(40 mmol/L). The control group was treated with PBS solution for 48 h. Western-blot was performed to analyze the expression of P-AKT (phosphorylated AKT),GSK-3β,and P-GSK-3β (phosphorylated GSK-3β) protein. Results Metformin could inhibit the proliferation of colorectal cancer HT29 cells in a time-dose dependent manner, and the difference was statistically significant (P<0.05). The number of cells detectable by the light microscope was reduced. The number of cells in polygonal shape was decreased while there were more cells appeared in circular, volume reduction, nuclear pyknosis, and nuclear dissolution, with the increase of metformin concentration. The experimental results showed that the apoptotic rate of the experimental group was higher than that of the control group,and the apoptotic rate increased with the increase of the drug concentration (P<0.05). The results of Western-blot showed that metformin could inhibit the expression of PI3K,P-AKT,and P-GSK-3β protein in HT29 cells, and had no significant effect on the expression of AKT and GSK-3β protein. Conclusion Metformin inhibits the proliferation of human colon cancer HT29 cells and promotes apoptosis. The mechanism may be related to the inhibition of PI3K/AKT/3β pathway-related protein expression.
基金项目
河北省政府资助临床医学优秀人才培养和基础课题研究项目(冀财社[2017]46号)
NETL
NSTL
维普
万方数据