CAJ | 학술논문

目的构建针对肿瘤凋亡基因FasL的小干扰RNA(siRNA)表达质粒,研究RNA干扰技术对人肺腺癌A549细胞FasL mRNA表达的抑制作用,探索Fas/FasI途径在肺癌转移中的作用机制.方法依据siRNA靶序列的设计原则,以FasL mRNA为靶基因,合成2对编码短发夹结构的两条DNA序列,经退火合成DNA双链,再克隆至siRNA表达质粒pSilencer2.0中,转化后进行PCR鉴定和DNA序列测定.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检测细胞FasL mRNA水平的变化.结果成功构建发卡样FasL siRNA真核表达载体,转染人肺腺癌A549细胞后,FasL mRNA的表达水平显著下降.结论成功构建的FasL基因siRNA真核表达载体pSi2.0-FasL能显著抑制人肺腺癌A549细胞FasL mRNA的表达.
목적 구건침대종류조망기인FasL적소간우RNA(siRNA)표체질립,연구RNA간우기술대인폐선암A549세포FasL mRNA표체적억제작용,탐색Fas/FasI도경재폐암전이중적작용궤제.방법 의거siRNA파서렬적설계원칙,이FasL mRNA위파기인,합성2대편마단발협결구적량조DNA서렬,경퇴화합성DNA쌍련,재극륭지siRNA표체질립pSilencer2.0중,전화후진행PCR감정화DNA서렬측정.용지질체개도전염입인폐선암A549세포주,채용실시형광정량PCR검측세포FasL mRNA수평적변화.결과 성공구건발잡양FasL siRNA진핵표체재체,전염인폐선암A549세포후,FasL mRNA적표체수평현저하강.결론 성공구건적FasL기인siRNA진핵표체재체pSi2.0-FasL능현저억제인폐선암A549세포FasL mRNA적표체.
Objective To clone the expression vectors of small interfering RNA(siRNA) against FasL gene and to evaluate its inhibition on the FasL mRNA expression in human lung carcinoma A549 cells. 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-2.0, which was transfected into A549 cells with liposome mediation after PCR identification and DNA sequencing, and the effect of p2.0Si - FasL on FasL mRNA expression of human lung carcinoma A549 cells was detected by quantitative fluorescence PCR. Results The eukaryotic ex-pression vector of short hairpin siRNA against FasL was constructed successfully and FasL mRNA expres-sion was inhibited. Conclusions FasL siRNA eukaryotic expression vector pSi2.0 - FasL constructed successfully could inhibit the expression of FasL mRNA in human lung carcinoma A549 cells.