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目的 为了特异封闭白血病细胞survivin的表达,抑制其功能,本实验构建了survivin反义核酸载体并导入白血病细胞系中。方法 应用RT-PCR获得survivin的cDNA片段,反向插入pcDNA3质粒载体中;经限制性酶切和测序鉴定所构建的反义核酸是否正确;采用电转染方法将重组体导人HL-60细胞中;RT-PCR技术检测转染细胞sunrivin表达的变化。结果 经限制性酶切和测序鉴定证明survivin反义核酸已成功构建;RT-PCR产物电泳结果显示,与转染前细胞、空质粒转染细胞相比,转染survivin反义核酸的细胞survivin mRNA水平明显降低。结论 本实验已成功建立了survivin反义核酸真核表达载体,而且在白血病细胞系中发挥了特异封闭作用,为进一步研究survivin反义核酸在白血病治疗中的作用提供了实验基础。
In order to specifically block the expression of survivin in leukemia cells and inhibit its function, we constructed the survivin antisense nucleic acid vector and introduced it into the leukemia cell line. Methods The cDNA fragment of survivin was obtained by reverse transcription polymerase chain reaction (RT-PCR) and inserted into the pcDNA3 plasmid vector. The antisense oligonucleotides were identified by restriction enzyme digestion and sequencing. The recombinant plasmid was transfected into HL-60 cells The changes of sunrivin expression in transfected cells were detected by RT-PCR. Results The antisense oligonucleotides of survivin were confirmed by restriction analysis and sequencing. The results of RT-PCR showed that the expression of survivin mRNA was significantly higher than that of transfected cells Significantly lower the level. Conclusion The experiment has successfully established the survivin antisense nucleic acid eukaryotic expression vector, and it has a specific blocking effect in leukemia cell lines, which provides the experimental basis for further study on the role of survivin antisense nucleic acid in the treatment of leukemia.