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目的研究组蛋白去乙酰化酶抑制剂曲古菌素A(trichostatin A, TSA)抑制HL-60细胞端粒酶活性及亚单位hTERT的表达并诱导凋亡的机制。方法采用MTT法和倒置相差显微镜观察不同浓度曲古菌素A对HL-60细胞的抑制作用,流式细胞仪检测600 nmol·L-1TSA作用后的细胞凋亡情况,TRAP-ELISA法检测端粒酶活性变化,RT-PCR分析端粒酶三个亚单位的mRNA表达情况。结果 TSA对HL-60细胞的抑制作用具有时间和剂量依赖性,AnnexinV/PI双染色法检测凋亡显示,600 nmol·L-1TSA作用48 h后,细胞凋亡率为42·6%。600 nmol·L-1TSA作用12、24和48 h后,端粒酶活性分别下降到1·95 ±0·25、1·73 ±0·12和1·52 ±0·09。RT-PCR显示,端粒酶逆转录酶hTERT表达下降,而端粒酶RNA模板(hTR)和端粒酶相关蛋白(hTP1)表达无明显改变。结论 TSA抑制HL-60细胞中端粒酶活性,并诱导凋亡,其机制可能与曲古菌素A下调hTERT转录水平有关。
Objective To study the mechanism of histone deacetylase inhibitor trichostatin A (TSA) inhibiting telomerase activity and subunit hTERT expression in HL-60 cells and inducing apoptosis. Methods MTT assay and inverted phase contrast microscope were used to observe the inhibitory effect of different concentrations of trichostatin A on HL-60 cells. The apoptosis of HL-60 cells treated with 600 nmol·L-1 TSA was detected by flow cytometry. Granzyme activity changes, RT-PCR analysis of telomerase three subunit mRNA expression. Results TSA inhibited the proliferation of HL-60 cells in a time-and dose-dependent manner. Annexin V / PI double staining showed that apoptosis rate was 42.6% after treated with 600 nmol·L-1 TSA for 48 h. After treated with 600 nmol·L-1 TSA for 12,24 and 48 h, the telomerase activity decreased to1.95 ± 0.25, 1.73 ± 0.12 and1.52 ± 0.09, respectively. RT-PCR showed that telomerase reverse transcriptase hTERT expression decreased, while telomerase RNA template (hTR) and telomerase-related protein (hTP1) expression did not change significantly. Conclusion TSA inhibits the telomerase activity and induces apoptosis in HL-60 cells, which may be related to the down-regulation of hTERT transcription by trichostatin A.