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

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K562细胞中的表达及其功能.方法将DAAO cDNA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K562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(5.2×106 cfu/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.

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

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 phiNXA 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 KDAAO. PCR and in situ hybridization were used to identify the integration and expression of DAAO gene in KDAAO. KDAAO 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 x 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.