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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。
Objective To construct short hairpin RNA (shRNA) against Nuc leostem in (NS) gene in vitro for further study of the gene targeting RNA interference (RNAi) Feasibility of leukemia gene therapy. Methods Two 21-mer fragments of AA at the 3’-end were screened from the cDNA sequence of three variant cDNAs of Nuc leostem in as the corresponding sequences for the synthesis of shRNA. The 9-base 5’-UCUCUUGAA-3 ’ Loop ring. The in vitro synthesized double-stranded DNA template of shRNA was constructed with 23 bases of 5’-GGATC-CTAATACGACTCACTATA-3 ’as the T7 promoter sequence. One of the 5’- Information loop, Loop loop, shRNA antisense information chain, a total of 70 bases; the other is the complementary strand of the chain, 5 ’end with AA, a total of 72 bases. The shRNA was synthesized under the action of T7 RNA polym erase. After purification, the concentration of shRNA was determined by comparing the gray value by gel electrophoresis. The effect of shRNA on HL-60 cells was observed by observing the changes of cell morphology and NS-mRNA inhibition rate Interference effect. RESULTS: The two shRNAs synthesized in vitro showed no degradation and diffusion at the concentrations of 5.24μmol.L-1 and 3.35μmol.L-1, respectively. The interference of HL-60 cells for 48 h decreased the cell density and aggregation, The size of HL-60 cells treated by shRNA-NS-2 changed from circular to fusiform and had obvious pseudopodia. Compared with the control group, the inhibitory rates of NS-mRNA reached 37.82% and 71.88%, respectively. Conclusions Two shRNAs targeting Nu-c leostem in gene were successfully constructed and synthesized in vitro. The shRNA-NS-2 shRNA inhibited the gene expression of HL-60 cells. The synthesized shR-NA-NS-2 can be used as a tool to further study the gene and to explore the gene therapy of leukemia.