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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 ,在该模型未观察到明显旁观者效应
Objective To study the application of DAO / D-Ala suicide gene system in gene therapy. Methods The Kao-K 2 6e monoclonal antibody KDfGC stably expressing DAAO was obtained by retroviral transfection. The DAAO gene-modified KDfGC cells were identified by PCR and in situ hybridization. The cell morphology, viable cell count Cell biological characteristics. The killing effect of D Ala on mixed KDfGC and DAAO positive cells was detected by MTT assay. H2O2 level in culture supernatant was determined by phenol red oxidation assay. Results PCR and in situ hybridization analysis showed that the DAAO gene has been integrated into the cell genome and expressed at the mRNA level. There was no significant difference in growth rate between KDfGC and non-transgenic original tumor cells. Nearly 90% of KDfGC cells can be killed after 12.5 mmol / L Ala treatment for 24 hours, and the effective killing rate of D Ala can be doubled. The level of H2O2 production in the supernatant is consistent with the effect of killing the transgenic cells. When KDfGC is mixed with different ratios of K5 6 2e cells, the proportion of cells killed by 15.0 mmol / L Ala does not exceed the proportion of KDfGC cells. Conclusion The DAAO / D Ala system can kill K6 5 2e leukemia cells effectively and no obvious bystander effect is observed in this model