丁酸钠对Hep-2细胞的生长抑制和诱导凋亡作用及其对端粒酶活性的影响

Effects of sodium butyrate on growth,apoptosis and telomerase activity in Hep-2 cells

目的研究丁酸钠对 Hep-2细胞的生长抑制和诱导凋亡作用及诱导凋亡过程中对端粒酶活性的影响。方法采用四甲基偶氮唑蓝(methyl thiazolyl terazolium,MTT)比色法观察不同浓度丁酸钠对 Hep-2细胞的生长抑制作用,透射电镜下观察其形态学变化,未端脱氧核苷酸转移酶介导的dUTP 缺口末端标记(terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling,TUNEL)法、琼脂糖凝胶电泳和流式细胞仪检测丁酸钠作用后的细胞凋亡情况及细胞周期变化,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,RT-PCR 分析端粒酶各组分的 mRNA 表达情况。结果丁酸钠对 Hep-2细胞的生长抑制作用呈时间和剂量依赖性。透射电镜下可见典型的凋亡细胞形态学改变。琼脂糖凝胶电泳可见特征性的凋亡细胞 DNA 梯状条带。TUNEL 法显示,2.5 mmol/L 丁酸钠作用72 h,细胞凋亡指数由作用前的2.27±1.18增加至33.50±2.75。流式细胞仪显示,2.5 mmol/L 丁酸钠作用72h,细胞凋亡率由作用前的2.86%增加至31.28%,G0/G1期细胞比例由50.38%增加至70.88%,S 期细胞比例由 27.40%减少至8.20%,细胞增殖指数由49.62%降低至29.12%。TRAP-银染法显示,丁酸钠作用后细胞端粒酶活性下降,且具有一定时间效应关系。RT-PCR 显示端粒酶逆转录酶表达下降而端粒酶 RNA 模板和端粒酶相关蛋白表达无明显改变。结论丁酸钠对 Hep-2细胞具有生长抑制和诱导凋亡作用,并可能通过下调端粒酶逆转录酶表达而抑制端粒酶活性。

Objective To study the effects of sodium butyrate （SB） on growth, apoptosis and telomerase activity in Hep-2 cells. Methods Growth inhibition effect of SB on Hep-2 cells was assessed by methyl thiazolyl terazolium（MTT） assay. Morphological alterations were observed by electronic microscope. Cell apoptosis was confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling （TUNEL） method, DNA fragmentation and flow cytometry （FCM）. Cell cycle was analyzed by FCM. Telomerase activity was examined by telomeric repeat amplification protocal （TRAP）-sliver staining. The expression status of telomerase subunits was analyzed by reverse transcription-polymerase chain reaction（ RT- PCR）. Results A time-and dose-dependent inhibition was detected in cells treated with SB. Typical morphological changes of apoptotic cells were observed under electronic microscop. The characteristic DNA fragmentation of apoptotic cells was detected by agarose gel electrophoresis. Apoptosis and the changes of cell cycle were confirmed by TUNEL method and FCM. The apoptosis indexes of the cells before treatment and at 72 h after SB （ 2. 5 mmol/L） treatment were 2. 27 ± 1.18 and 33.50 ±2. 75 respectively, the apoptosis rates were 2. 86% and 31.28% respectively, the proportion of the cells at G0/G1 stage were 50.38% and 70. 88% respectively, the proportion of the ceils at S stage were 27.40% and 8.20% respectively, and the proliferation indexes of the cells were 49.62% and 29. 12% respectively. Telomerase activity and expression level of human telomerase reverse transcriptase （hTERT）, the key subunit of telomerase, decreased after SB treatment. No significant changes were observed in the expression of human telomerase RNA （hTR） and human telomerase associated protein （hTP1）, the other two subunit of telomerase. Conclusion SB could inhibit growth of Hep-2 ceils and induce apoptosis in the ceils, and inhibit telomerase activity by decrease expression level of hTERT.