大剂量Bcl-2反义硫代磷酸寡核苷酸对细胞系的毒性作用

The toxic effects of high dose Bcl-2 antisense phosphorothioate oligodeoxynucleotides incubation on cell line

目的 探讨大剂量Bcl 2反义硫代磷酸寡脱氧核苷酸 (AS PS ODN ,ASPO)对HL6 0细胞系的毒性作用 ,同时了解毒性与剂量的关系。方法 应用四氮唑蓝比色法、台盼蓝拒染试验 ,CFU HL6 0克隆培养法观察大剂量Bcl 2ASPO对HL6 0细胞的毒性作用 ,同时应用免疫细胞化学染色及流式细胞仪检测P2 6Bcl 2蛋白表达 ,吖啶橙染色及DNA片段电泳对细胞凋亡的观察。并以其正义PS ODN(SPO)为反义序列非特异性毒性对照。用SPSS软件包进行剂量 效应曲线直线化回归分析。结果 (1)当ASPO、SPO剂量为 2 0 μmol/L时 ,即出现对HL6 0细胞生长的非特异性毒性作用 ;在 16 0μmol/L时 ,其非特异性毒性出现明显增加。对HL6 0细胞生长的抑制率分别为 40 %、18%。 (2 )量效曲线回归分析表明当剂量为 2 6 2 μmol/L时 ,ASPO与SPO的毒性作用无差别。 (3) 16 0 μmol/LASPO、SPO对Bcl 2蛋白表达率分别为 2 8%、78% ,将ASPO、SPO作用后的HL6 0细胞进行克隆传代发现Bcl 2表达阳性率反跳分别达 99.6 %、97.8%。结论 Bcl 2ASPO虽为低毒药物 ,但仍存在剂量依赖性毒性作用。其对HL6 0细胞作用的适合剂量以不超过

Objective To investigate the toxic effects of high dose Bcl 2 antisense phosphorothioate oligodeoxynucleotides (AS PS ODN, ASPO) incubation on HL60 cells and to understand the relationship between the dose of ASPO and the toxicity. Methods Cellular viability and toxicity were detected by trypan blue exclusion, colony forming unit HL60 cells and MTT assay. The expression of Bcl 2 protein was determined by immunocytochemistry and flow cytometry analysis. The proportion of apoptosis cells was tested by acridine orange (AO) standing. The sense (PS ODN, SPO) was chosen for the toxic control of independent sequence. The curves of dose effect and dose toxicity were transfered into straight line and the reguession analysis was done by computer with the SPSS soften ware. Results When the dose of Bcl 2 PS ODN was higher than 20 μmol/L, the non specific effect of suppressing cell proliferation could occur. When the dose of Bcl 2 PS ODN was more than 160 μmol/L, the non specific effect (toxic effect) increased significantly. When the dose of Bcl 2 ASPO was higher than 80 μmol/L, the specific effect of inhibitting the expression of Bcl 2 proteion increased slowly. When the dose of PS ODN reached 262 μmol/L, the toxic effect had no significant differences between Bcl 2 ASPO and Bcl 2 SPO. In the presence of ASPO or SPO at 160 μmol/L, the expression of Bcl 2 protein in the two groups of HL60 was decreased to 28% and 78% respectively, after 72 hours of incubation. The expression of Bcl 2 protein in the groups of HL60 which were passaged after incubation with ASPO or SPO was reinereased to 99.6% and 97.8%, respectively. Conclusion Although ASPO is a kind of low toxic drug, the toxic effects of Bcl 2 ASPO seem to be dependent on the dose increasing to a certain extent. It is clear that it can be used over a wide range of doses, however. Our results may also provide some referring concentrations of Bcl 2 ASPO for the pharmacodynamic and toxicological study in vivo in future.