曲古抑菌素A诱导Hela细胞凋亡过程中端粒酶逆转录酶表达的变化

The Change in Expression of Human Telomerase Reverse Transcriptase during Induction of Apoptosis by Trichostatin A in Hela Human Cervical Cancer Cells

目的:观察Trichostatin A(TSA)对子宫颈癌细胞株Hela细胞的体外作用,探讨TSA对子宫颈癌细胞的作用机制,以及细胞凋亡与端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)之间的关系。方法:倒置相差显微镜观察细胞形态变化;磺酰罗丹明B(sulforhodamine B;SRB)法检测药物动力学特征;流式细胞仪检测药物作用前后Hela细胞凋亡情况;RT-PCR检测hTERT和p21Waf1基因变化;免疫荧光检测TSA作用前后hTERT表达变化。结果:在0.5～2.0μmol/LTSA作用下,Hela细胞生长明显受抑制。倒置相差显微镜检测Hela细胞经TSA处理后形态发生明显变化。2.0μmol/LTSA作用于Hela细胞,48h以前表现细胞周期的变化,48h后则出现明显的凋亡。hTERT经1.0μmol/LTSA诱导后表达下调,呈时效关系。免疫荧光检测hTERT蛋白经1μmol/LTSA处理后表达下降;结论:一定浓度的TSA可以诱导子宫颈癌细胞株Hela凋亡,其作用机制可能与下调hTERT表达有关。

Objective： To investigate the in vitro effect of Trichostatin A （TSA） on Hela human cervical cancer cells in order to reveal its probable mechanism and to elucidate the relationship between apoptosis and the expression of human telomerase reverse transcriptase （hTERT）. Methods： Inverted phase contrast microscopy （IPCM） was used to observe morphologic changes in the cells. Sulforhodamine B was employed to determine the pharmacokinetic characteristics. Flow cytometry（FCM） was conducted to measure apoptosis before and after drug treatment. The expression levels of hTERT and p21/Wafl mRNA, before and after Trichostatin A treatment, were detected using semi-quantitative reverse transcription po!ymerase chain reaction （RT-PCR）. The hTERT protein expression level before and after TSA treatment was detected by fluorescence microscopy. Results： After being treated with 0.1uM, 0.5uM, 1.0uM, and 2.0uM Trichostatin A, the proliferation of Hela cells was significantly inhibited. IPCM showed that there were obvious morphologic changes after TSA treatment. Changes in the cell cycle occurred 48 h after treatment, and with a dose of 2.0umol/L TSA apoptosis was apparent.hTERT expression was down-regulated after treatment with 1.0umol/L TSA, in a time-dependent man- ner. Immunofluorescence detection showed that there was decreased expression of the hTERT protein after the 1 umol/L TSA treatment. Conclusion： Trichostatin A significantly inhibited growth of Hela cells by inducing apoptosis and the mechanism may be related to a down-regulation in hTERT expression.