丁酸钠对Hep-2细胞增殖、细胞周期及端粒酶活性的影响

目的研究丁酸钠(sodium butyrate,SB)对人喉癌Hep-2细胞增殖、细胞周期、端粒酶活性及其3个亚单位表达水平的影响。方法采用噻唑蓝(methylthiazoletrazolium,MTT)比色法和倒置显微镜观察不同浓度SB对Hep-2细胞的抑制作用,流式细胞术分析细胞周期变化,免疫组化SP法检测细胞Ki-67表达情况,端粒重复序列扩增(telomeric repeat amplification protocal,TRAP)-银染法检测端粒酶活性变化,逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)分析端粒酶各组分的mRNA表达情况。结果SB对Hep-2细胞的抑制作用具有时间和剂量依赖性。5mmol/LSB作用24小时,G0/G1期细胞比例由作用前的51.52%增至68.70%,S期细胞由28.34%减至10.56%,细胞增殖指数(proliferation indexes,PI)由48.48%降至31.30%。2.5mmol/LSB作用24小时,Ki-67阳性细胞MOD值由作用前的0.243±0.010降至0.185±0.009。2.5mmol/LSB作用24、48、72小时,端粒酶活性分别下降23.60%、60.91%、88.45%,端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)表达水平分别下降21.85%、56.50%、77.23%,而端粒酶RNA(human telomerase RNA,hTR)模板和端粒酶相关蛋白(human telomerase associated protein,hTP1)表达无明显变化。结论SB能通过下调hTERT表达而有效地抑制Hep-2细胞端粒酶活性,抑制细胞增殖,并参与细胞周期调控,具有较好的应用前景。

Effect of realgar on telomerase activity and hTERT-mRNA expression in NB4 cells

Objective: To evaluate whether realgar could down-regulate human telomerase reverse transcriptase (hTERT) gene expression and telomerase activity in acute promyelocytic leukemia cell line-NB4 cells. Methods: The expression of hTERT-mRNA was analyzed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). Flow cytometry using PI staining was applied to analyze the cell cycle and apoptosis. Results: Treatment of NB4 cells with 155, 300, 600 μg/L realgar reduced telomerase activity significantly accompanying with decrease of hTERT-mRNA and increasing cell apoptosis. G2/M phase arrest appeared when treated with realgar in 300, 600 μg/L. Conclusion: It is suggested that telomerase activity of NB4 cells can be specifically inhibited by realgar through the down-regulation of hTERT gene expression. G2/M phase arrest and apoptosis by realgar in NB4 cells might be related to the reduction of telomerase activity and hTERT-mRNA expression.

RADIATION- INDUCED PROGRESSIVE DECREASING IN THE EXPRESSION OF REVERSE TRANSCRIPTASE GENE OF hEST2 AND TELOMERASE ACTIVITY

Objectives. In order to identify the relationship between telomerase and the biological effect of radiation injury,and investigate the role of human telomerase catalytic subunit gene (hEST2) reverse transcriptase(RT) segment in the expression of telomerase activity. Methods. Tumor HeLa cells, KB cells and A431 cells were employed to measure the change in telomerase activity after 60Co- ray irradiation at RNA level and protein level. Quantitative PCR and Northern blotting were used to determine the expression of hEST2 RT segment that encodes seven motifs of the human telomeres, a PCR- based telomeric repeat amplification protocol (TRAP)was used to assay telomerase activity after exposure to radiation. Results. Both of telomerase activity and the expression hEST2 RT segment were decreased with increasing dosage of radiation. In addition, testing the expression of motifs domain is similar to the measurement of telomerase activity. Conclusion. The detection of the hEST2 RT segment by Northern blotting and quantitative PCR are new methods for testing telomerase activity. Furthermore, radiation can cause a dose- dependent decrease in telomerase activity. The effect of radiation on telomerase is one possible reason for the death of cancer cells after irradiation.