DNAzyme抑制H-ras基因表达的研究  

Inhibitory Effect of DNAzyme on Expression of H-ras Gene

作　　者：吕峰[1] 胡凝珠[1] 施海晶 瞿素[1] 刘国栋[1] 胡云章[1] 

机构地区：[1]中国医学科学院中国协和医科大学医学生物学研究所,昆明650118

出　　处：《中国生物制品学杂志》2006年第1期12-16,共5页Chinese Journal of Biologicals

摘　　要：目的研究DNAzyme特异切割H-ras mRNA的效率。方法针对H-ras mRNA序列,设计并合成了7个10-23DNAzyme,分别进行DNAzyme的细胞外酶切反应;通过荧光显微镜观察脂质体介导DNAzyme的转染效果,利用实时定量PCR、MTT法在细胞水平检测DNAzyme对H-ras基因表达的抑制。结果合成的7个10-23DNAzyme均可以细胞外近生理条件下对H-ras mRNA进行有效切割。利用LipofectamineTM可将荧光标记的DNAzyme No·1转染Hep-2细胞。转染后DNAzyme在细胞核及细胞质内均存在,且在细胞质中多位于核周围。实时定量PCR检测,DNAzyme No.1在转染后24h及48h能显著降低Hep-2细胞中H-ras RNA的含量,从而抑制ras基因的表达。利用MTT检测到DNAzyme No.1在转染Hep-2细胞24h及48h后对细胞增殖及分化均产生明显的抑制作用。结论DNAzyme在细胞内外均能够有效抑制H-ras基因的表达。Objective To study the inhibitory effect of DNAzyme on expression of H-ras gene. Methods Seven DNAzyme gene fragments containing motif 10-23 （ 10-23 DNAzyme, No. 1-7）, specific to H-ras mRNA sequence, were designed and synthesized, then used for extracellular cleavage test separately. The transfection efficiency of DNAzyme in the mediation of lipofectamineTM was observed by fluorescent microscopy. The inhibitory effect of DNAzyme on expression of H-ras gene was determined by real-time quantitative PCR and MTT methods at cellular level. Results All the seven 10-23 DNAzyme fragments effectively cleaved H-ras mRNA, Hep-2 cells were transfeeted with fluorescently-labeled DNAzyme No. I in the mediation of lipofeetamineTM. After transfeetion, DNAzyme was observed in both nucleus and cytoplasm. However,the DNAzyme content in perinuclear space was relatively low. Real-time quantitative PCR proved that the H-ras RATA content in Hep-2 cells 24 and 48 hours after transfection with DNAzyme No, 1 decreased significantly. The determination result by MTT method showed that the proliferation and differentiation of Hep-2 cells 24 and 48 hours after transfection with DNAzyme No. 1 was inhibited significantly. Conclusion DNAzyme showed both intracellular and extracellular inhibitory effects on the expression of H-ras gene.

关 键 词：脱氧核酶 RAS基因 抑制 

分 类 号：R512.62[医药卫生—内科学] R737.9[医药卫生—临床医学]