FasL基因siRNA真核表达载体的构建及意义

Construction and signification of FasL siRNA eukaryotic expression vector

目的构建针对细胞凋亡基因FasL的小干扰RNA(siRNA)表达载体,探讨载体介导的RNA干扰技术用于肺间质纤维化基因治疗的可行性。方法依据siRNA靶序列的设计原则,以FasL mRNA为靶基因,合成两对编码短发卡结构的两条DNA序列,经退火合成DNA双链,再克隆至siRNA表达载体pSilencer2.0中,转化后进行PCR鉴定和DNA序列测定。将构建的载体用脂质体介导转染入人肺腺癌A549细胞株,采用实时荧光定量PCR法检测细胞FasL mRNA水平变化。结果成功构建发卡样FasL siRNA真核表达载体;其转染人肺腺癌A549细胞后,细胞FasL mRNA的表达水平显著下降。结论FasLsiRNA真核表达载体能显著抑制人肺腺癌A549细胞FasLmRNA的表达;本研究为肺间质纤维化的基因治疗奠定了基础。

Objective To clone the expression vectors of small interfering RNA（siRNA） against FasL gene and to evaluate its valuce in the gene therapy of PIF. Methods According to the criteria of designing siRNA target sequence, two FasL siRNA template DNA sequences were designed and synthesized. The annealed siRNA template was inserted into siRNA expression vector pSilencer-2. O, which was transfected into A549 cells with liposome mediation after PCR identification and DNA sequencing,and the effect of p2. OSi-FasL on FasL mRNA expression of human lung carcinoma A549 cells was detected by quantitative fluorescence PCR. Results The eukaryotic expression vector of short hairpin siRNA against FasL was constructed successfully and FasL mRNA expression was inhibited. Conelusion FasL siRNA eukaryotie expression vector pSi2.0-FasL can inhibit the expression of FasL mRNA in human hmg carcinoma A549 cells. This study provides basis for the gene therapy of PIF.