DAAO/D-Ala系统对高致瘤性K562e细胞体外杀伤效应及其机制的研究

Study on in vitro killing activity of DAAO/D-Ala system to K562e cells

目的 研究D 氨基酸氧化酶 (DAAO) /D 丙氨酸 (D Ala)自杀基因系统在基因治疗中的应用。方法 应用逆转录病毒转染技术获得稳定表达DAAO的高致瘤性K5 6 2e单克隆细胞KDfGC,用PCR、原位杂交技术对DAAO基因修饰的KDfGC细胞进行鉴定 ;通过细胞形态、活细胞计数观察细胞生物学特性 ;应用MTT法检测D Ala对KDfGC、不同比例DAAO表达阳性与阴性混合细胞的杀伤作用 ;用酚红氧化法测定培养上清H2 O2 水平。结果 PCR和原位杂交分析证明DAAO基因已整合至细胞基因组中 ,并在mRNA水平表达。KDfGC与未转基因的原肿瘤细胞相比 ,生长速度差异无显著性。 12 .5mmol/LD Ala作用 2 4h即可杀死近 90 %的KDfGC细胞 ,而且D Ala达到一定有效浓度杀伤效率可成倍提高。上清的H2 O2 产生水平与杀伤转基因细胞作用相一致。KDfGC与不同比例的K5 6 2e细胞混合时 ,被 15 .0mmol/LD Ala杀死的细胞比例不超过KDfGC细胞的比例。结论 DAAO/D Ala系统可有效杀伤K6 5 2e白血病细胞 。

Objective To investigate the in vitro killing efficiency of D amino acid oxidase(DAAO)/D alanine(D Ala) system on K562e cells. Methods K DfGC cell line stably expressing DAAO was obtained by retrovirus transfection technique. The integration and expression of DAAO gene were identified by PCR and in situ hybridization. The killing activities of D Ala to DAAO + cells alone or the mixtures of DAAO + and DAAO - cells in different ratios were observed. H 2O 2 production by K DfGC cell was measured by phenol red oxidation assay. Results PCR and in situ hybridization analysis confirmed the integration of DAAO gene in positive clone and its mRNA expression. There was no significant difference in cell proliferation between the two kinds of K562 cells. Ninety percent of K DfGC cells was killed by 12.5 mmol/L D Ala after 24 hour treatment and the H 2O 2 levels were in accord with the killing activities of D Ala. When K DfGC was mixed with K562e at different ratio, no significant bystander effect could be found after treating with 15.0 mmol/L D Ala for 24 hours. Conclusion The leukemia cell line K562e was sensitive to DAAO/D Ala system and there was no significant bystander effects observed within this cells.