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目的研究阿霉素诱导白血病细胞凋亡的剂量及时间关系,探索其相关的分子机制。方法分别以0.1、0.2、0.5、1.0 mg/L的阿霉素处理人类Jurkat白血病细胞61、22、44、8 h。其中一份样本在加入0.2 mg/L阿霉素前用zVAD-fmk(苄氧羰-缬氨酰-丙氨酰-天冬氨酰-氟甲基酮)预处理。应用AnV/PI双染细胞,在流式细胞仪上分析AnV/PI双阳性的凋亡细胞。采用Western Blot技术检测FasL和FADD(Fas相关死亡域)的表达。结果6 h时所有剂量的阿霉素诱导的凋亡细胞无显著差异(P>0.05),在12 h,只有1.0 mg/L诱导细胞明显凋亡。当细胞与0.2和0.5 mg/L的阿霉素共同培养24 h或36 h,观察到调亡细胞显著增加(P<0.001)。在zVAD-fmk存在的情况下,当细胞与阿霉素一同培养时,由阿霉素诱导的细胞凋亡完全受到抑制(P<0.001)。随着阿霉素作用时间增加,FasL和FADD表达水平相应增加。结论在阿霉素诱导的白血病细胞凋亡中,阿霉素以剂量和时间依赖方式诱导细胞凋亡;上调FasL可能启动FasL信号通路的激话,而caspase是最终的执行者。
Objective To study the dose and time relationship of doxorubicin-induced leukemia cell apoptosis and to explore its molecular mechanism. Methods Human Jurkat leukemia cells were treated with doxorubicin at 0.1, 0.2, 0.5 and 1.0 mg / L for 61,22,44 and 8 h, respectively. One of the samples was pre-treated with zVAD-fmk (benzyloxycarbonyl-valyl-alanyl-aspartyl-fluoromethyl ketone) before addition of 0.2 mg / L doxorubicin. AnV / PI double staining cells were used to analyze AnV / PI double positive apoptotic cells on a flow cytometer. Western Blot was used to detect the expression of FasL and FADD (Fas-related death domain). Results All doses of doxorubicin-induced apoptotic cells had no significant difference at 6 h (P> 0.05). At 12 h, only 1.0 mg / L induced apoptosis. When cells were incubated with doxorubicin at 0.2 and 0.5 mg / L for 24 h or 36 h, a significant increase in apoptotic cells was observed (P <0.001). In the presence of zVAD-fmk, doxorubicin-induced apoptosis was completely inhibited (P <0.001) when cells were cultured with doxorubicin. With the increase of adriamycin, the expression of FasL and FADD increased accordingly. CONCLUSION: Adriamycin can induce apoptosis in doxorubicin-induced leukemia cells in a time-and dose-dependent manner. Upregulation of FasL may activate the reflex of FasL signaling pathway, and caspase is the final executor.