硫代反义寡核苷酸及脂质体对培养细胞的毒性作用

Effects of phosphothioate antisense oligodeoxynucleotide or cationic liposome on survival of rat renal epithelial cell line

目的 评价硫代反义寡核苷酸 (antisenseoligodeoxynucleotide ,AS ODN)及脂质体对肾小管上皮细胞的毒性作用。方法 针对大鼠骨调素 (osteopontin ,OPN)的cDNA序列设计合成与OPNcDNA序列互补的AS ODN ,其所有碱基均经硫代修饰 ;用不同浓度的游离AS ODN或阳离子脂质体 (DOTAP)处理大鼠肾小管上皮细胞株 (NRK5 2E)不同时间 ,采用噻唑蓝 (MTT)比色法测定细胞的存活率。结果 硫代AS ODN在 0～ 4 0 μmol/L浓度范围内或DOTAP在 0～ 10 0 μg/ml浓度范围内处理的NRK5 2E细胞存活情况良好 ;浓度 4 0 μmol/L的硫代AS ODN或浓度 10 0 μg/ml的DOTAP对NRK5 2E细胞的毒性作用较小 ,72h内无时间依赖性 ;浓度 2 0 0 μg/ml的DOTAP对细胞的毒性具有明显的时间依赖性。结论 正常使用浓度的DOTAP或硫代修饰的AS ODN对大鼠肾小管上皮细胞株均无明显毒性作用 ,建议在转染实验中 ,AS ODN ,DOTAP浓度分别不超过 4 0 μmol/L ,10 0 μg/ml,处理细胞时间小于 72h。

Objective To observe cytotoxicities of phosphrothioate antisense oligodeoxynucleotide or cationic liposome to rat renal epithelial cell line.Method An AS ODN (antisense oligodeoxynucleotide) complementary to rat OPN (osteopontin) cDNA sequence was designed and synthesized.Its all nucleotides were modified with phosphothioate.The cultured NRK52E cells (rat renal epithelia cell line) were treated with various concentrations of free AS ODN or DOTAP (dioleoyl trimethylammonium propane, a cationic liposome) for different time interval and cell survival was detected by MTT (thiazolyl blue) colorimetric assay.Results At low concentration (less than 40 μmol/L of ODN or 100 μg/ml of DOTAP) the cells well suvive.ODN with 40 μmol/L or DOTAP with 100 μg/ml just exerted few cytotoxicities to the cells.But at higher concentration (80 μmol/L of ODN or 200 μg/ml of DOTAP),the survival of cells was markedly reduced.40μ mol/L of AS ODN or 100 μg/L of DOTAP showed cytotoxicities to NRK52E cells without time dependence,but 200 μg/ml of DOTAP exerted increasing cytotoxicities with time dependence.Conclusion Routine concentration of phosphothioated AS ODN or DOTAP have few cytotoxicities to rat renal epithelia cell.It was recommended that the concentrations of AS ODN and DOTAP should be less than 40 μmol/Land 100 μg/ml respectively and the time of treatment should be less than 72 hour in transfection experiments.