Fas靶向RNA干扰质粒载体的构建及生物活性鉴定

目的　构建Fas靶向的RNA干扰质粒载体,为探讨抑制Fas表达在急性再生障碍性贫血治疗中的意义奠定基础。方法　PCR法获得U6启动子序列以及产生siRNA的相应DNA模板序列,将其克隆入改造后的pcDNA3.1,获得RNA干扰载体pU6 siFas。脂质体法将pU6 siFas导入P815 细胞,免疫组化法检测对P815 细胞Fas表达的抑制情况。结果　所构建的载体pU6 siFas能够干扰P815细胞中Fas的表达,并且具有新霉素抗性,能够用G 418 筛选稳定转化的细胞株。结论　成功构建了抑制小鼠Fas表达的RNA干扰质粒载体。

The construction and identification of Fas-targeting siRNA-expressing plasmid

Objective To construct Fas-targeting siRNA-expressing plasmid, and explore the significance of Fas inhibition in the treatment of acute aplastic anemia. Methods U6 promoter cassette and siFas sequence were obtained by PCR method, and cloned into modified pcDNA3.1 to produce plasmid pU6-siFas, which was transfected into P815 cells with limpofectin 2000, and then Fas expression was detected by immunohistochemistry. Results The plasmid pU6-siFas could efficiently reduce the expression of Fas and confer G-418 resistance in P815 cells. Conclusion The successful construction of the siRNA expressing plasmid will facilitate the application of RNA interference technique, and lay foundation for further studies of the therapeutic effect of Fas inhibition on acute aplastic anemia.