【摘要】 目的构建DNA损伤修复基因脱嘌呤/脱嘧啶核酸内切酶APE1siRNA表达载体pSilenceAPE1,观察其对骨肉瘤细胞HOS和9901APE1蛋白表达的抑制作用。方法设计合成APE1siRNAcDNA序列与线性化pSilence2.0-U6质粒连接后构建APE1siRNA表达载体pSilenceAPE1,经酶切鉴定和测序确认。应用免疫组化检测骨肉瘤细胞APE1蛋白的表达,同时应用免疫印迹检测其对APE1基因的敲低作用的量效和时效关系。结果通过双酶切鉴定和测序分析,成功构建了APE1siRNA表达载体。免疫组化和免疫印迹证实pSilenceAPE1对骨肉瘤细胞APE1基因具有特异性“敲低”作用,其抑制APE1基因表达效率为72%~95%,而且其抑制作用以3·0μgpSilenceAPE1转染第3天最为显著。结论成功构建了APE1siRNA表达载体pSilenceAPE1,并且该载体能有效地敲低骨肉瘤细胞APE1基因表达。更多还原

【Abstract】 Objective To construct DNA damage and repair gene apurinic/apyrimidinic endonuclease (APE1) siRNA expression vector pSilence APE1 and investigate its inhibitory effect on the expression of APE1 in osteosarcoma cell HOS and 9901 in vitro. Methods An expression plasmid of a short hairpin RNA target APE1, pSilence APE1 was constructed and transfected to 9901 and HOS cell by lipofectamine. The expression of APE1 protein in HOS and 9901 osteosarcoma cells posttransfected with pSilence APE1 was detected by immunohistochemistry. Meanwhile, the dose-effect and time-effect relationship of APE1 gene silence induced by pSilence APE1 were measured using Western blot analysis. Results After evaluation and sequencing, the APE1 siRNA expression vector pSilence APE1 was constructed successfully. The result of Western blotting and immunohistochemistry showed that APE1 protein in osteosarcoma cells could be knocked down specifically by pSilence APE1, and the inhibition rate of APE1 expression was 72%-95%. The best inhibition of expression of APE1 gene was 3.0 μg and at the 72 h using pSilence APE1. Conclusion APE1 siRNA expression vector pSilence APE1 is successfully constructed that can significantly knock down APE1 gene expression in osteosarcoma cells.更多还原