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

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 应用转录病毒转染技术获得稳定表达DAAO的高致瘤性K562e单克隆细胞KDfGC，用ＰＣＲ、原位杂交技术对ＤＡＡＯ基因修饰的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Ｈ２Ｏ２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。１２.5／Ｌ Ｄ－Ａla作用２４h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Ｈ２Ｏ２产生水平与杀伤转基因细胞作用相一致。ＫDfGC与不同比例的Ｋ５６２e细胞混合时，被１５.0mmol/L D-Ala杀死的细胞比例不超过ＫＤfGC细胞的比较。结论 ＤＡＡＯ／Ｄ－Ａla系统可有效杀伤Ｋ６５２e白血病细胞，在该模型未观察到明显旁观者效应。

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

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.