【摘要】 目的体外构建合成针对Nuc leostem in(核干细胞因子,NS)基因的短发夹状干扰RNA(shRNA),用于进一步研究以该基因为靶目标、RNA干扰(RNA interference,RNA i)为手段的白血病基因治疗可行性。方法从Nuc leostem in基因的三个变异体cDNA共同序列中筛选出2个3′端为AA的21个碱基片段作为shRNA合成的对应序列,9个碱基的5′-UCUCUUGAA-3′作为Loop环。以23个碱基的5-′GGATC-CTAATACGACTCACTATA-3′作为T7启动子(T7 Promoter)序列,构建shRNA的体外合成双链DNA模板,一条链5′端无AA,顺序为启动子、shRNA正义信息链、Loop环、shRNA反义信息链,共70个碱基;另一条是该链的互补链,5′端带有AA,共72个碱基。在T7 RNA聚合酶(T7 RNA polym erase)作用下合成shRNA,纯化后通过凝胶电泳比较灰度值确定shRNA浓度;shRNA作用于HL-60细胞,通过观察细胞形态变化和NS-mRNA抑制率验证干扰效果。结果体外合成的两条shRNA电泳无降解和弥散现象,浓度分别为5.24μmol.L-1和3.35μmol.L-1;干扰HL-60细胞48 h后,细胞密度和聚集程度下降,细胞之间大小相差悬殊,经shRNA-NS-2作用的HL-60细胞一部分由圆形变成了梭形并有明显的伪足;与对照组相比NS-mRNA抑制率分别达到37.82%和71.88%。结论体外成功构建和合成了两条针对Nu-c leostem in基因的shRNA,且抑制该基因表达的作用明显,shRNA-NS-2使HL-60细胞形态发生了改变。所合成的shR-NA-NS-2可作为进一步研究该基因和探索白血病的基因治疗的工具。更多还原

【Abstract】 Aim In order to further explore the feasibility of gene therapy with RNA interference（RNAi） for acute leukemia,we synthesized short hairpin RNA（shRNA）for nucleostemin（NS）in vitro.Methods The designed DNA template consists of the sequence complementary to target mRNA,which was screened out consensus motif of three variants of NS gene,using 5′-UCUCUUGAA-3′ as loop sequence,The sequence of 5′-GGATCCTAATACGACTCACTATA-3′ acts as T7 promoter.Two shRNA were produced by T7 RNA polymerase and named as shRNA-NS-1 and shRNA-NS-2,respectively.The purified shRNA was quantified by gel electrophoresis.The interference effect of shRNA-NS transfected into HL-60 cell was examined by resultant cell morphology and inhibition rate of NS-mRNA expression.Results The concentrations of two shRNA-NS without degradation and diffusion were 5.24 μmol·L^-1and 3.35 μmol·L^-1,respectively.There were significant decline in density and aggregation of cells and significant differences in size between cells after interfering HL-60 cell for 48 h.Furthermore,some of HL-60 cells treated by shRNA-NS-2 were changed from round to fusiform shape even with pseudopod.Compared with control group,the inhibition rates of shRNA-NS-1 and shRNA-NS-2 to NS-mRNA expression were 37.82% and 71.88%,respectively.Conclusion The two shRNA for NS gene were successfully constructed,which suppress NS gene expression significantly.shRNA-NS-2 also can chang HL-60 cell′s morphology and might be a useful tool to explore NS gene function and possible therapy for acute leukemia.更多还原