大剂量Bcl-2反义硫代磷酸寡核苷酸对细胞系的毒性作用

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ＨＬ６０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－ＨＬ６０克隆培养法观察大剂量Ｂｃｌ－２ ＡＳＰＯ对ＨＬ６０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Ｐ２６ Ｂｃｌ－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ＰＳＯＤＮ（ＳＰＯ

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

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 micromol/L, the non-specific effect of suppressing cell proliferation could occur. When the dose of Bcl-2 PS-ODN was more than 160 micromol/L, the non-specific effect (toxic effect) increased significantly. When the dose of Bcl-2 ASPO was higher than 80 micromol/L, the specific effect of inhibitting the expression of Bcl-2 proteion increased slowly. When the dose of PS-ODN reached 262 micromol/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 micromol/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.