叠氮胸苷对胶质母细胞瘤细胞系TJ905细胞p33ING1b表达和细胞衰老及凋亡的影响

目的 观察叠氮胸苷对恶性胶质瘤细胞系TJ905细胞p33ING1b表达的影响及该影响与细胞凋亡和衰老的关系.方法 将体外培养的TJ905细胞系分为对照组,50、100及200 μmol/L叠氮胸苷组,在后三组的培养液中分别给予相应终浓度的叠氮胸苷并继续传代培养;各组取第1、3、6代细胞以半定量逆转录聚合酶链反应(RT-PCR)和衰老相关β-半乳糖苷酶染色检测p33ING1bmRNA表达水平及衰老细胞;取第3、6代细胞以TUNEL法和单细胞凝胶电泳法检测凋亡细胞.结果 从用药后第1代起叠氮胸苷即呈时间和剂量依赖性地诱导TJ905细胞p33ING1b mRNA表达和细胞衰老;200 μmol/L叠氮胸苷组第6代TJ905细胞的p33ING1b RT-PCR扩增条带相对灰度(1.44±0.23)显著高于同组第1代(0.95±0.13)、第3代(1.35±0.23)及同代50μmol/L组(0.85±0.24)和100 μmol/L组(1.23±0.34),其衰老相关β-半乳糖苷酶标记指数(45.62±6.74)亦显著高于同组第1代(7.82±2.40)、第3代(26.27±7.17)及同代50 μmol/L组(27.37±6.41)和100 μmol/L组(35.49±5.12),上述差异均有统计学意义(P＜0.01);而叠氮胸苷促凋亡作用出现在稍晚的第6代.结论 叠氮胸苷可上调TJ905细胞p33ING1b的表达水平,这可能是叠氮胸苷诱导TJ905细胞衰老和凋亡的重要分子机制.

Effects of azidothymidine on p33ING1b expression, apoptosis and senescence of TJ905 human glioblastoma cell line

Objectives To investigate the pharmacological effects of azidothymidine (AZT) on p33ING1b expression, senescence and apoptosis of TJ905 glioblastoma cells. Methods TJ905 cells were treated with AZT at a serial concentrations of 50, 100 and 200 μmol/L. Semi-quantitative RT-PCR and cytochemical staining of senescence related-galactosidase (sβ-Gal) were used to evaluate the expression of p33ING1b mRNA and to label the senescent cells at the 1st, 3rd and 6th generations, respectively. In situ cell death detection and single cell gel electrophoresis were used to detect the apoptosis at the 3rd and 6th generations. Results AZT induced the expression of p33ING1b mRNA and senescence of the tumor cells of the 1st generation in a dosage and time dependent manner. At the 6th generation, the relative amount of p33ING1b RT-PCR product (1.44 ±0. 23 ) and s3-Gal labeling index of 200 μ mol/L group (45. 62 ±6. 74)were significantly higher than those of the 1st( 0. 95 ± 0. 13 and 7. 82 ± 2. 40 ) and the 3rd generation cells (1.35 ± 0. 23, 26. 27 ± 7. 17 ) of the same group, and cells of the same generation in the 50 μmol/L(0.85 ±0.24, 27.37 ±6.41) and 100 μmol/L groups (1.23 ±0.34, 35.49 ±5.12, P＜0.01 ). Therewas a significant positive correlation between the p33ING1b mRNA expression and the labeling index of sβ-Gal. Pro-apoptotic effects of AZT became obvious at the 6th generation. Conclusion AZT upregulates the expression of p33ING1b, a possible mechanism in regulating senescence and apoptosis of the TJ905 cells.