摘要
目的观察肿瘤坏死因子相关凋亡诱导配体(TRAIL)联合5-Fu治疗结肠癌的增殖抑制作用,并探讨其机制。方法将结肠癌细胞株CT26消化处理成3×104/mL的悬液,接种于96孔板中,分为4组。TRAIL组分别加入5、10、50、100、200 ng/mL的TRAIL;5-Fu组分别加入5、10、15、20、25μg/mL的5-Fu;联合用药组加入5、50、200ng/mL的TRAIL和5-Fu 10μg/mL;对照组加入5、50、200 ng/mL TRAIL。分别培养24、48、72 h。MTT法检测CT26细胞增殖的抑制率;流式细胞术检测细胞周期和细胞凋亡率;Western-blot法检测周期相关基因p21WAF1和p27的表达;免疫组化法检测凋亡相关基因Bax、Bcl-2的表达。结果 TRAIL、5-Fu单药应用能够抑制CT26细胞增殖,且CT26细胞增殖抑制率与5-Fu浓度呈负相关关系。联合组CT26细胞的增殖抑制率高于余3组。与对照组相比,联合组G0/G1期细胞比例显著增加,S期细胞比例明显下降(P均<0.05),且联合组内,随着TRAIL浓度升高,G0/G1期细胞比例增加(P<0.05)。相比于对照组,联合组随TRAIL作用浓度提高,CT26细胞凋亡率逐渐上升(P<0.05)。与对照组、5-Fu组比较,联合组p21WAF1、p27蛋白的表达量明显增加,且随着TRAIL作用浓度升高而增加(P均<0.05)。联合组与对照组、5-Fu组相比,Bax蛋白的表达量增加,Bcl-2蛋白表达量减少(P均<0.05)。结论TRAIL与5-Fu联合使用可抑制结肠癌CT26细胞凋亡,两药联用具有协同抗肿瘤作用,可能与上调p21WAF1、p27、Bax蛋白表达、下调Bcl-2蛋白表达有关。
Objective To observe the inhibitory effect of TNF-related apoptosis-inducing ligand( TRAIL) combined with 5-fluorouracil( 5-Fu) on the proliferation of human colon carcinoma cells and to investigate the mechanism. Methods The colon cancer cell line CT26 was digested into suspension( 3 × 104/ mL),inoculated into 96-well plates,and then they were divided into 4 groups: TRAIL group which was added 5,10,50,100,200 ng / mL TRAIL,5-Fu group which was added 5,10,15,20,25 μ g / mL 5-Fu,combined treatment group which was treated with 5,50,200 ng / mL TRAIL and 5-Fu 10 μ g / mL and the control group which was treated with 5,50,200 ng / mL TAIL. After culture for 24,48 and72 h,the inhibitory rate of CT26 cell proliferation was detected by MTT; the cell cycle and apoptosis rate were detected by flow cytometry; the expression of p21WAF1and p27 was detected by Western blotting; and the expression of apoptosis-related genes Bax and Bcl-2 was detected by immunohistochemistry. Results The single use of TRAIL and 5-Fu inhibited the proliferation,and the inhibition rate was negatively correlated with the 5-Fu concentration; the inhibition rate of combined treatment group was higher than that of the other 3 groups. Compared with the control group,the proportion of cells in G0/ G1phase was significantly increased,while the proportion was significantly decreased in S phase of the combined treatment group( all P < 0. 05),and with the increase of TRAIL concentration,the proportion of cells in G0/ G1phase was increased( P < 0. 05). Compared with the control group,the apoptosis rate of CT26 cells in the combined treatment group was increased with the increase of TRAIL concentration( P < 0. 05). Compared with the control and 5-Fu groups,the expression levels of p21WAF1and p27 protein in the combined treatment group were significantly increased,and increased with the concentration of TRAIL( all P < 0. 05). The expression level of Bax protein was higher in the combined treatment group than those of the control and 5-Fu groups,while the expession level of Bcl-2 was lower( all P < 0. 05). Conclusion The combined use of TRAIL and 5-Fu can inhibit the apoptosis of colon cancer CT26 cells,and with a synergistic antitumor effect,whose mechanism may be related to the up-regulation of p21WAF1,p27 and Bax protein expression,down-regulation of Bcl-2 protein expression.
出处
《山东医药》
CAS
2013年第30期16-20,共5页
Shandong Medical Journal
基金
徐州市科技局科研基金资助项目(Y3014)