【摘要】 甲胎蛋白(AFP)是原发性肝癌的标志物.本实验室前期研究表明,AFP的表达与肝癌细胞的增殖及细胞周期相关.为了建立高效稳定的AFPsiRNA细胞导入方法,本研究构建了AFP慢病毒siRNA干涉载体pRNAT-U6.2/AFPsiRNA.并将其转入HEK293细胞后,Western印迹表明,pRNAT-U6.2/AFPsiRNA的干扰效率为88.7%(P<0.05).pRNAT-U6.2/AFPsiRNA与4个包装质粒共同转染293T细胞包装成慢病毒后感染HepG2细胞,感染3 d时GFP的表达量达95%,感染35 d的子代细胞中GFP的表达量仍稳定在85%.GFP表达量的观察显示,慢病毒载体可以高效并稳定表达外源基因.Western印迹结果也显示,HepG2细胞被病毒感染3 d和25 d后,AFP表达水平均有降低,抑制率分别为74.8%和63.4%(P<0.05).克隆形成实验表明,AFP沉默后,细胞克隆形成个数降低63%(P<0.01),表明HepG2细胞增殖能力受到抑制.更多还原

【Abstract】 Alpha-fetoprotein(AFP) is a biomarker of primary liver cancer,which has been indicated to relate the proliferation and cell cycle of hepatoma cells.To establish an efficient and stable method to deliver AFPsiRNA,small interfering RNA expression vector pRNAT-U6.2/AFPsiRNA was constructed.Western blotting showed that the knock down efficiency on AFP was 88.7%(P<0.05)in transfected HEK293 cells.The four viral packaging vectors with pRNAT-U6.2/AFPsiRNA were used to transfect 293T cells to produce lentivirus particles.After infecting HepG2 cells with the obtained viruses for 3 days,more than 95% positive cells were found,and the transgene expression remained stable in 85% of the cells for 35 days.Western blotting showed that AFP expression was down regulated in HepG2 cells to 74.8% and 63.4% from 3 to 25 days post lenti-AFPsiRNA infections as compared to the controls.The colony formation assays showed that the number of colonies from lenti-AFPsiRNA-infected HepG2 cells was reduced to 63%(P<0.01),suggesting that the cell proliferation was inhibited.更多还原