阿霉素对Tca8113细胞端粒酶活性及端粒结合蛋白表达的影响

The effects of Adramycin on telomerase activity and expression of telomere repeat binding factor proteins in Tca8113 cells

目的观察阿霉素诱导Tca8113细胞凋亡对端粒酶活性及端粒结合蛋白(TRF)表达的影响;探讨端粒酶和TRF在细胞凋亡途径中的作用机制。方法通过Tca8113细胞MTT实验,确定后续实验中阿霉素作用浓度。将Tca8113培养细胞分成用药组和对照组,培养5d和7d后,利用流式细胞仪检测细胞凋亡;采用Giemsa染色进行凋亡形态学观察;通过端粒酶酶联免疫吸附实验对端粒酶活性进行定性和定量分析;利用免疫组化和免疫荧光标记法分别检测TRF表达和表达水平。结果5μg/ml阿霉素作为后续研究的作用浓度。在5μg/ml阿霉素作用5d和7d后,用药组细胞凋亡率分别为(36.4±1.7)%和(54.2±2.1)%,与对照组比较差异有统计学意义(P<0.05);Giemsa染色显示用药组出现明显凋亡细胞;TRAP检测结果表明,用药组端粒酶活性降低,与对照组比较,差异有统计学意义(P<0.05);端粒酶活性随阿霉素作用时间延长而降低(P<0.05);用药组与对照组细胞均有TRF表达,在细胞核内呈均匀蓝染状态,而在诱导凋亡细胞核和凋亡小体内呈点状弥散分布,但两组间TRF表达水平差异无统计学意义(P>0.05)。结论阿霉素诱导Tca8113细胞凋亡使其细胞内端粒酶活性受抑制,TRF表达特点发生改变,但未改变TRF表达水平。

Objective To observe the effects of apoptosis of Tca8113 cells induced by Adramycin on the telomerase activity and expression of telomere repeat binding factors （TRF） and probe the mechanism by which telomerase and TRF proteins function during transmitting apoptotic signals. Methods The effective concentration of Adramycin in the following experiments was determined by using methyhhiazolyl tetrazolium （MTT） assay of Tca8113 cells. Tca8113 cells cultured in the medium were divided into two experiment groups, namely treated group containing Adramycin and untreated one containing sodium saline. After cultured for 5 days and 7 days, apoptosis of Tca8113 cells was examined by flow cytometer method; apoptotic morphology after Giemsa staining was observed under microscope; a qualitative and quantitative analysis of telomerase activity was performed by TRAP （telomeric repeats amplification protocol）-enzyme-linked immunosorbent assay; expression and expression level of TRF proteins from Tca8113 cells were detected with immunohistochemical staining and immunofluorescence label assay respectively. Results The effective concentration of Adramycin in the subsequent experiments was determined as 5 μg/ml by MTT assay. After 5 days＇ and 7 days＇ treatment with Adramycin at 5 μg/ml, apoptotic rates of the treated groups were 36.42±1.70% and 54.24±2.07%, respectively, which were significantly different from those of the untreated groups （P 〈0.05）; apoptotic cells obviously appeared in the treated groups after Giemsa staining; the immunosorbent assay indicated that telomerase activity from the treated groups dropped obviously, which was statistically significant compared with that from the untreated groups （P 〈 0.05）; the activity level decreased with increasing duration of Adramycin at 5 μg/ml （P 〈 0.05 ）; expression of TRF proteins which evenly appeared in the nucleus in blue were found in the cells of the two experiment groups; the expression in the plasma and apoptotic debris of induced apoptotic cells was characteristic of blue speckles distributed diffusely; there were no statistically difference on the expression level between the treated and untreated group （P 〉 0.05）. Conclusions Inducing TcaSll3 cells apoptosis by Adramycin inhibited the telomerase activity, changed the expressive characteristics of TRF, but had no influence on the expression level of TRF proteins.