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目的探讨去甲斑蝥素(NCTD)对大肠癌患者HT-29细胞增殖及Livin、Caspase-3表达的影响。方法体外培养人大肠癌HT-29细胞株,采用MTT法测定NCTD对HT-29细胞的增殖抑制情况,以免疫细胞化学法和RT-PCR法检测NCTD对HT-29细胞中Livin、Caspase-3表达的影响。结果浓度较低10μg/ml时,NCTD对HT-29细胞的抑制不明显,随着NCTD浓度增加,HT-29细胞抑制作用逐渐增强,NCTD浓度为100μg/ml,抑制率达90.82%,IC50为65.28μg/ml。NCTD作用12 h后即出现抑制效应,随着时间延长抑制率逐渐增强,36 h抑制率达55.84%,呈时间效应关系,最佳作用时间为36 h。将NCTD作用HT-29细胞前设为对照组,Livin多呈阳性表达,Caspase-3多呈阴性。IC50NCTD作用36 h后,NCTD组Livin蛋白表达显著低于对照组,Caspase-3蛋白表达高于对照组(P<0.05)。IC50NCTD作用36 h后,NCTD组Livin mRNA表达显著低于对照组,Caspase-3 mRNA表达显著高于对照组(P<0.01)。结论 NCTD可抑制HT-29细胞生长,能够降低细胞凋亡抑制因子Livin表达,其机制还可能与细胞凋亡信号通路中的重要环节Caspase-3有关。
Objective To investigate the effects of norcantharidin (NCTD) on the proliferation of HT-29 cells and the expression of Livin and Caspase-3 in colorectal cancer patients. Methods Human colorectal cancer cell line HT-29 was cultured in vitro. MTT assay was used to determine the inhibitory effect of NCTD on HT-29 cells. Immunocytochemistry and RT-PCR were used to detect the effect of NCTD on the expression of Livin, Caspase-3 The impact of expression. The results showed that the inhibitory effect of NCTD on HT-29 cells was not obvious at the lower concentration of 10μg / ml. With the increase of NCTD concentration, the inhibition of HT-29 cells gradually increased. The NCTD concentration was 100μg / ml, the inhibition rate was 90.82% 65.28 μg / ml. The inhibitory effect of NCTD appeared after 12 h, with the time increasing inhibition rate gradually increased, 36 h inhibition rate of 55.84%, showing a time-dependent relationship, the optimal duration of 36 h. The NCTD before HT-29 cells as a control group, Livin mostly positive expression, Caspase-3 mostly negative. After IC50NCTD for 36 h, the expression of Livin protein in NCTD group was significantly lower than that in control group, and Caspase-3 protein expression was higher than that in control group (P <0.05). After IC50NCTD treatment for 36 h, the expression of Livin mRNA in NCTD group was significantly lower than that in control group, and Caspase-3 mRNA expression was significantly higher than that in control group (P <0.01). Conclusions NCTD can inhibit the growth of HT-29 cells and decrease the expression of Livin, which may be related to Caspase-3, which is an important part of apoptosis signaling pathway.