蛋白酶体抑制剂MG-132诱导K562细胞凋亡的实验研究

Induction of apoptosis by proteasome inhibitor MG-132 in human erythroleukemia cell line K562

摘要:

目的：观察蛋白酶体抑制剂MG-132对人红白血病细胞株K562的致凋亡作用及其对凋亡相关基因NF-κB与caspase-3表达的影响。方法：将蛋白酶体通路特异性阻断剂 MG-132 加入K562细胞，用AO/EB细胞形态和DNA琼脂糖凝胶电泳检测细胞凋亡，流式细胞仪检测凋亡率,逆转录-PCR检测NF-κB的转录水平，免疫组化法检测NF-κB与caspase-3的表达，比色法检测caspase-3活性。结果：流式细胞仪检测15 μmol/ L MG-132作用24 h后，凋亡率为(26.5±0.6)％，与对照组(1.2±0.1)％相比明显增多，差异有显著性(P<0．01)，MG-132诱导K562细胞凋亡具有量-效关系; RT-PCR检测发现NF-κB mRNA表达下调；免疫组化法检测发现MG-132可降低 NF-κB的表达，增加caspase-3蛋白的表达, 且表达量与MG-132的作用呈剂量相关。结论：MG-132能诱导K562细胞凋亡,其机制可能与MG-132抑制NF-κB信号转导通路,下调NF-κB表达继而上调caspase-3表达有关。［中国当代儿科杂志，2009，11（4）：255-258］

Abstract:

OBJECTIVE: To investigate whether proteasome inhibitor MG-132 induces apoptosis of human erythroleukemia cell line K562 and possible mechanisms. METHODS: K562 cells were incubated with RPMI 1640 and exposed to 0, 1, 5, 10, 15 μmol/L of MG-132 for 24 hrs, respectively. The apoptosis of cells were detected by fluorescence microscope, DNA fragments and flow cytometry. The NF-κB mRNA expression was quantified by reverse transcription-polymerase chain reaction (RT-PCR). Expression of NF-κB and caspase-3 was semiquantitatively analyzed with SABC techniques. Caspase-3 activities were measured with a colorimetric method. RESULTS: The growth of K562 cells was inhibited and the apoptosis of the cells increased after MG-132 treatment in a dose-dependent manner. After 24 hrs of 15 μmol/L MG-132 treatment, the percentage of apoptotic cells (26.5±0.6%) increased significantly when compared with the untreated controls (1.2±0.1%) (P<0.01). MG-132 treatment decreased the mRNA and protein expression of NF-κB, and increased the protein expression of caspase-3. CONCLUSIONS: MG-132 can induce apoptosis of human erythroleukemia cell line K562 through the down-regulation of NF-κB expression and up-regulation of caspase-3 expression.［Chin J Contemp Pediatr, 2009, 11 (4):255-258］