川芎嗪联合阿糖胞苷对人白血病U937细胞的增殖、凋亡影响及相关机制的研究

Inhibitory effect of combination treatment with tetramethylpyrazine and Cytarabine on proliferation and Apoplosis in U937 cell and its mechanism

目的探讨川芎嗪(TMP)联合阿糖胞苷(Ara-c)对白血病细胞株U937增殖、凋亡的影响及相关机制的研究,为临床寻找有效的化疗增敏剂提供实验依据和理论基础。方法实验分为对照组、TMP组、Ara-c组及TMP联合Ara-c组。应用CCK-8法检测各组细胞的增值抑制率;流式细胞术(FCM)检测各组细胞凋亡率;蛋白质印迹方法检测各组细胞内凋亡相关蛋白Bcl-2、Bax的表达情况。结果 TMP(0.5mg·ml-1)组与Ara-c(0.02,0.04,0.08,0.16μg·ml-1)组联合作用后,U937细胞的增殖抑制率明显高于相同浓度下Ara-c组,差异有统计学意义(t=5.38,t=8.23,t=14.61,t=17.31,P均<0.05)。TMP(0.5mg·ml-1)联合Ara-c(0.08μg·ml-1)组的凋亡率(27.32±3.64)明显高于Ara-c组[(15.07±3.85)%]、TMP组[(3.17±2.20)%]和对照组[(1.8±1.18)%](t=12.25,P<0.05;t=24.15,P<0.05;t=25.52,P<0.05)。蛋白质印迹结果显示,Ara-c能在一定程度上下调Bcl-2蛋白的表达,上调Bax蛋白的表达;联合用药后Bcl-2、Bax蛋白表达水平改变更明显(t=6.32,P<0.05,t=2.60,P<0.05;t=39.06,P<0.05,t=41.67,P<0.05)。结论 TMP能够增强Ara-c抑制细胞增殖、诱导细胞凋亡的作用,从而提高U937细胞对Ara-c的化疗敏感性,其机制可能与下调抑凋亡蛋白Bcl-2表达,上调促凋亡蛋白Bax表达有关。

Objective To investigate the proliferation and cell apoptosis of combination treatment with tetramethylpyrazine（TMP）and Cytarabine（Ara-c）on human leukemia cell line U937 cell and provide experimental evidence and theoretical basis for finding effective chemotherapy sensitizer in clinical study.Methods The experiment was divided into TMP group,Ara-c group,TMP combined with Ara-c group,and the control group.The inhibitory rate of U937 cell proliferation was detected by CCK-8assay.The apoptotic rate was examined by Flow cytometry（FCM）.The expression of Bcl-2and Bax of U937 cells were measured with immunohistochemical approach.Results TMP（0.5 mg·ml-1）conbined with Cytarabine（0.02,0.04,0.08,0.16μg·ml-1）did be able to inhibited the cell growth,compared with the Ara-c group in corresponding concentrations,the difference was significant（t=5.38,t=8.23,t=14.61,t=17.31,P〈0.05）.The inhibition of combination treatment with Tetramethylpyrazine（0.5μg·ml-1）and Cytarabine（0.08μg·ml-1）was significantly higher than Cytarabine（0.08μg·ml-1）[（15.07±3.85）%],Tetramethylpyrazine（0.5mg·ml-1）[（3.17±2.20）%]and the control[（1.8±1.18）%],respectively（t=12.25,P〈0.05;t=24.15,P〈0.05;t=25.52,P〈0.05）.The result of Western blot show that the expression of Bcl-12 was up-regulated and the expression of Bax was down-regulated by Ara-c,to some extent.The expression levels of Bcl-2and Bax were changed more significantly by combination treatment with TMP and Ara-c than the Ara-c alone（t=6.32,P〈0.05,t=2.60,P〈0.05;t=39.06,P〈0.05,t=41.67,P〈0.05）.Conclusion Tetramethylpyrazine can apparently inhibit the growth of U937 cell and enhance apoptosis induced by cytarabine,promote the drug-sensitivity to cytarabine.And its molecular mechanisms might be related to down-regulation of the Bcl-2expression and up-regulation of the Bax expression.