D-氨基酸氧化酶基因在K562细胞中的表达及其介导的细胞毒性作用的研究  

Expression of D-amino acid oxidase gene in K562 cells and the cytotoxicity of D-Alanine to the cells

作　　者：翟勇平[1] 王健民[1] 周虹[1] 张雨生[1] 

机构地区：[1]第二军医大学附属长海医院血液科,上海200433

出　　处：《中华血液学杂志》2001年第12期646-648,共3页Chinese Journal of Hematology

基　　金：国家自然科学基金资助项目 ( 39870 710 ) ;上海市卫生系统"百人计划"资助项目 ( 98BRO2 9)

摘　　要：目的　探讨逆转录病毒介导的红色酵母D 氨基酸氧化酶 (DAAO)基因在K5 6 2细胞中的表达及其功能。方法　将DAAOcDNA克隆至逆转录病毒载体pLSN中 ,构建了载体pLDAAOSN ,经ΦNXA细胞包装后 ,用NIH3T3细胞测定病毒滴度 ,以重组逆转录病毒感染K5 6 2白血病细胞 ,G418筛选出抗性克隆 ,命名为KDAAO 。PCR、原位杂交分析外源基因整合和表达 ,并以不同浓度的D 丙氨酸 (D Ala)处理KDAAO 细胞。结果　重组逆转录病毒载体中含有完整的DAAO基因。包装细胞产生了高滴度病毒 (5 .2× 10 6cfu ml)。DAAO基因已整合至KDAAO细胞基因组中 ,并在mRNA水平表达。D Ala能明显杀伤KDAAO 细胞。结论　DAAO D Ala自杀基因系统可以进一步用于肿瘤的基因治疗研究。Objective To explore the feasibility of expression of R.gracilis D amino acid oxidase(DAAO) gene in leukemia cell line K562 and the cytotoxicity of D Alanine to the cells. Methods DAAO cDNA was cloned into retroviral vector pLSN and pLDAAOSN was generated. The vector was then packaged with ΦNXA and the virus titer was measured with NIH3T3 cells. Leukemia cell line K562 was infected with the viral supernatant. The positive clones were obtained by G418 selection and named K DAAO . PCR and in situ hybridization were used to identify the integration and expression of DAAO gene in K DAAO . K DAAO was treated with different concentrations of D Alanine. Results pLDAAOSN was confirmed containing the full length of DAAO cDNA. Infectious titer generated by the packaging cells was 5.2×10 6 cfu/ml. PCR and in situ hybridization analysis showed integration of DAAO gene in KDAAO and expression of DAAO mRNA. Preliminary observation suggested that D Ala could effectively kill KDAAO. Conclusion DAAO/D Ala suicide gene system might be useful in cancer gene therapy.

关 键 词：逆转录病毒载体 DAAO基因 基因表达 细胞株 K562细胞 细胞毒性作用 肿瘤 基因治疗 

分 类 号：R73-3[医药卫生—肿瘤]