CAJ | 학술논문

目的构建核干细胞因子基因RNA干扰（RNA interference，RNAi）慢病毒表达载体并对其在白血病细胞株中的干扰效果进行鉴定，为后期研究奠定基础。方法针对核干细胞因子基因的序列设计RNAi有效靶点，合成含RNA干扰序列、Loop环、AgeⅠ和EcoRⅠ酶切位点，以及终止信号的单链DNA oligo，经退火形成双链DNA，与双酶切线性化的带有GFP荧光标记和嘌呤霉素抗性标记的GV248慢病毒空载体进行连接产生重组慢病毒载体，转化感受态细菌，挑取重组阳性转化子进行菌落PCR反应，并对PCR阳性克隆进行测序。将重组慢病毒载体及其两种辅助包装原件载体质粒pHelper1.0和pHelper2.0共转染293 T细胞进行病毒的包装，收集、浓缩病毒液后测定其滴度，并转染HL-60、NB4以及K562等三种人白血病细胞株，倒置荧光显微镜下观察其转染效率，real-time PCR检测核干细胞因子基因的敲减效率。结果经测序证实正确构建出了核干细胞因子基因的RNAi重组慢病毒载体，包装、浓缩后其滴度为4×108 TU/ml，荧光显微镜下显示其能有效转染进入HL-60、NB4及K562等白血病细胞株中，转染效率均在80％以上；real-time PCR显示转染后核干细胞因子基因mRNA表达水平较阴性对照组显著下降（ P＜0.05），在HL-60、NB4和K562细胞中核干细胞因子基因的抑制效率分别为52.3％、80.5％、62.3％。结论成功构建出了核干细胞因子基因的RNAi慢病毒载体，其能有效干扰人白血病细胞株HL-60、NB4及K562中的核干细胞因子基因表达。
목적구건핵간세포인자기인RNA간우（RNA interference，RNAi）만병독표체재체병대기재백혈병세포주중적간우효과진행감정，위후기연구전정기출。방법침대핵간세포인자기인적서렬설계RNAi유효파점，합성함RNA간우서렬、Loop배、AgeⅠ화EcoRⅠ매절위점，이급종지신호적단련DNA oligo，경퇴화형성쌍련DNA，여쌍매절선성화적대유GFP형광표기화표령매소항성표기적GV248만병독공재체진행련접산생중조만병독재체，전화감수태세균，도취중조양성전화자진행균락PCR반응，병대PCR양성극륭진행측서。장중조만병독재체급기량충보조포장원건재체질립pHelper1.0화pHelper2.0공전염293 T세포진행병독적포장，수집、농축병독액후측정기적도，병전염HL-60、NB4이급K562등삼충인백혈병세포주，도치형광현미경하관찰기전염효솔，real-time PCR검측핵간세포인자기인적고감효솔。결과경측서증실정학구건출료핵간세포인자기인적RNAi중조만병독재체，포장、농축후기적도위4×108 TU/ml，형광현미경하현시기능유효전염진입HL-60、NB4급K562등백혈병세포주중，전염효솔균재80％이상；real-time PCR현시전염후핵간세포인자기인mRNA표체수평교음성대조조현저하강（ P＜0.05），재HL-60、NB4화K562세포중핵간세포인자기인적억제효솔분별위52.3％、80.5％、62.3％。결론성공구건출료핵간세포인자기인적RNAi만병독재체，기능유효간우인백혈병세포주HL-60、NB4급K562중적핵간세포인자기인표체。
Objective To construct a lentiviral expression vector for RNA interference ( RNAi ) of nucleostemin(NS) and detect its interference efficiency in three leukemia cell lines ( HL-60, NB4 and K562 ). Methods Effective RNAi target sequence was designed and screened towards NS gene sequence .Single-stranded DNA oligo containing RNAi sequence , Loop circle, AgeⅠ/EcoRⅠenzyme cutting site , and termination signal sequence was synthesized and annealed to double-stranded DNA,which was subsequently connected to the AgeⅠ/EcoRⅠ-digested GV248 lentiviral vector with GFP and puromycin resistance marker to form a recombinant lentiviral vector.Then it was transformed into competent E .coli cells,and the positive clones were confirmed by colony PCR and sequencing.The recombinant vector and two lentivirus packing plasmids (pHelper1.0 and pHelper2.0)were co-transfected into 293T cells to obtain packaged lentivirus particles .Viral titer was then determined.Subsequently,the recombinant lentivirus were transfected into three human leukemia cell lines (HL-60,NB4 and K562),then the transfection efficiency were observed under inverted fluorescence microscope ,and the inhibition rates were detected by real-time PCR.Results DNA sequencing showed that RNAi sequence was inserted into the GV 248 vector and the recombinant lentiviral vector was constructed successfully .The recombinant lentivirus harvested from 293 T cells had a titer of 4 ×108 TU/ml.Observation under inverted fluorescence microscope showed the lentiviral transfection efficiency was higher than 80% in all the three cell lines .Real-time PCR showed NS mRNA expression level of experimental group was significantly lower than negative control group (P<0.05).The interference efficiency in HL-60,NB4 and K562 were 52.3%,80.5%,62.3%,respectively.Conclusion The lentiviral vector for RNAi of NS has been successfully constructed with high titer ,and it could inhibit NS mRNA expression effectively in three human leukemia cell lines HL-60 ,NB4 and K562 ,which laid the foundation for follow-up studies .