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目的探讨尿多酸肽(CDA-2)抑制多发性骨髓瘤细胞株RPMI8226增殖作用及其机制。方法采用四甲基偶氮唑(MTT)比色法检测CDA-2对RPMI8226抑制作用并筛选研究浓度;通过Hoechst33258、Annexin-V/PI、细胞周期分析、DNA琼脂糖凝胶电泳等方法检测CDA-2诱导RPMI8226凋亡作用;使用Western Blot法检测caspase-8、caspase-3及其激活物的表达改变;半定量RT-PCR法检测TNF、FADD、TRAF3 mRNA表达。结果 CDA-2能够抑制RPMI8226细胞株增殖,并呈浓度依赖性,IC50为1.64 mg·mL-1;经CDA-2作用后,Hoechst33258荧光染色提示细胞核浓集及出现凋亡小体,Annexin-V/PI分析示早期凋亡细胞比例呈时间依赖增加,细胞周期分析提示呈浓度依赖上调凋亡峰及下调G1期比例,DNA凝胶电泳可见断裂成180~200 bp及其倍数的片断梯形条带,故CDA-2可诱导RPMI8226细胞株凋亡;Western Blot检测发现随药物作用时间延长caspase-8、caspase-3表达明显下降,而active-caspase8、active-caspase3表达上升;半定量RT-PCR证实凋亡相关基因TNF、FADD、TRAF3 mRNA表达上调。结论 CDA-2可抑制RPMI8226增殖,且可通过死亡受体途径诱导细胞凋亡。
Objective To investigate the proliferative effect of CDA-2 on multiple myeloma RPMI8226 cells and its mechanism. Methods MTT assay was used to detect the inhibitory effect of CDA-2 on RPMI8226 cells. The concentrations of CDA-2 were determined by Hoechst33258, Annexin-V / PI, cell cycle analysis and DNA agarose gel electrophoresis -2 induced the apoptosis of RPMI8226 cells. The expressions of caspase-8, caspase-3 and their activators were detected by Western Blot. The expressions of TNF, FADD and TRAF3 mRNA were detected by semi-quantitative RT-PCR. Results CDA-2 could inhibit the proliferation of RPMI8226 cells in a concentration-dependent manner with IC50 of 1.64 mg · mL-1. After treated with CDA-2, the fluorescence intensity of Hoechst33258 indicated the concentration of nuclei and apoptotic bodies, Annexin-V / PI analysis showed that the proportion of early apoptotic cells increased in a time-dependent manner. Cell cycle analysis suggested that the apoptotic peak was down-regulated and the proportion of G1 phase was down-regulated. DNA ladder electrophoresis showed that the DNA was broken into 180 ~ 200 bp fragments , So CDA-2 could induce apoptosis of RPMI8226 cell line. Western Blot showed that the expression of caspase-8 and caspase-3 decreased significantly and the expression of active-caspase8 and active-caspase3 increased with the prolongation of drug action time; The apoptosis related genes TNF, FADD and TRAF3 mRNA were upregulated. Conclusion CDA-2 can inhibit the proliferation of RPMI8226 cells and induce apoptosis through death receptor pathway.