钩吻总碱对人结肠癌细胞增殖、凋亡的影响及其机制

目的:探讨钩吻总生物碱提取物(钩吻总碱)促进人结肠癌HT-29细胞凋亡的机制。方法:钩吻总碱体外干预HT-29细胞24 h后,用噻唑蓝(MTT)比色法评估其对细胞增殖的影响,4,6-二脒基-2-苯基吲哚(DAPI)染色法及流式细胞术(Annexin V-FITC/PI)观察HT-29细胞的凋亡情况,逆转录聚合酶链反应(RT-PCR)观察HT-29细胞B型淋巴细胞瘤-2(Bcl-2)相关X蛋白(Bax),Bcl-2 mRNA表达情况,利用紫外分光光度法检测含半胱氨酸的天冬氨酸水解酶-3(Caspase-3)表达量的变化。结果:钩吻总碱可抑制HT-29细胞增殖,200 mg·L~(-1)钩吻总碱干预24 h后,对HT-29细胞增殖的平均抑制率达54.17%。DAPI染色及流式细胞术(Annexin V-FITC/PI)观察到钩吻总碱可促进HT-29细胞凋亡。RT-PCR观察到钩吻总碱能降低HT-29细胞Bcl-2 mRNA表达(P0.05),对Bax mRNA表达无明显影响,提高了Bax/Bcl-2 mRNA表达(P0.05)。利用紫外分光光度法检测到随着钩吻总碱浓度的升高,HT-29细胞的Caspase-3表达呈上升趋势(P0.05)。结论:钩吻总碱能有效地抑制HT-29细胞的增殖,促进HT-29细胞的凋亡,其机制可能与通过Bax/Bcl-2途径诱导细胞凋亡相关。

Effect and Mechanism of Total Alkaloids of Gelsmium elegans on Human Colonic Carcinoma Cells Proliferation and Apoptosis

Objective: To explore the mechanism of the total alkaloids of Gelsmium elegans (TAG) on the apoptosis of the human colonic carcinoma HT-29 cells. Method: The HT-29 cells were treated with TAG for 24 h and the cell proliferation was assessed by 3-(4,5-dimethylthiazol-zy1)-2,5-diphenyltetrazolium bromide (MTT) assay; 4,6-diamidino-2-phenylindole (DAPI) staining experiment and the flow cytometry (Annexin V-FITC/PI) experiment were used to observe the apoptosis of HT-29 cells. Reverse transcriptase polymerase chain reaction (RT-PCR) test was used to analyze the mRNA expression of B cell lymphoma-2 (Bcl-2) associated X protein (Bax) and Bcl-2 of HT-29 cells after 24 hours'' treatment of the TAG. Caspase-3 expression of HT-29 cells was detected by UV Spectrophotometry. Result: In the study, it was demonstrated that the TAG had the inhibition against the proliferation of HT-29 cells. The average inhibitory effect (54.17%) was observed after the HT-29 cells were treated with 200 mg·L^-1 of TAG for 24 h. The apoptotic effect of the TAG on HT-29 cells was also confirmed by the DAPI staining experiment and the flow cytometry (Annexin V-FITC/PI) experiment. RT-PCR assay showed that expression of Bcl-2 mRNA was lower in the TAG-treated group than that in control group(P<0.05), but the expression of Bax mRNA was not changed obviously. The mRNA expression level of Bax/Bcl-2 in TAG-treated group was higher than that in control group (P<0.05). Caspases-3 expression was increased with the increase of TAG concentration in UV spectrophotometry (P<0.05). Conclusion: The TAG inhibited the proliferation of HT-29 cells and induced the apoptosis of HT-29 cells, and the mechanism may be associated with activating the Bax/Bcl-2 pathway.