CAJ | 학술논문

目的以小鼠白血病L1210细胞系为基础，构建稳定高表达miR-17-92的细胞系。方法采用核转法，将miR-17-92的过表达质粒MSCV-LMP-miR-17-92、对照质粒MSCV-LMP转导入L1210细胞系；利用含嘌呤霉素选择、荧光倒置显微镜下观察、流式细胞术等技术筛选阳性克隆并检测转染效率；并用qPCR技术验证构建的阳性细胞系中的靶标miRNA表达水平，验证miR-17-92相对过表达比例。结果成功将MSCV-LMP-miR-17-92、MSCV-LMP转入L1210细胞系，转染效率较高，利用含嘌呤霉素的培养基筛选核转后的细胞，阳性率达到99％以上，miR-17-92簇中的靶标miRNA表达量持续。结论通过核转仪转染法成功将MSCV-LMP-miR-17-92、MSCV-LMP（对照质粒）转入L1210细胞系，并通过长期药物筛选获得了稳定表达细胞系，为进一步研究该基因在肿瘤中的作用提供了实验平台。
목적이소서백혈병L1210세포계위기출，구건은정고표체miR-17-92적세포계。방법채용핵전법，장miR-17-92적과표체질립MSCV-LMP-miR-17-92、대조질립MSCV-LMP전도입L1210세포계；이용함표령매소선택、형광도치현미경하관찰、류식세포술등기술사선양성극륭병검측전염효솔；병용qPCR기술험증구건적양성세포계중적파표miRNA표체수평，험증miR-17-92상대과표체비례。결과성공장MSCV-LMP-miR-17-92、MSCV-LMP전입L1210세포계，전염효솔교고，이용함표령매소적배양기사선핵전후적세포，양성솔체도99％이상，miR-17-92족중적파표miRNA표체량지속。결론통과핵전의전염법성공장MSCV-LMP-miR-17-92、MSCV-LMP（대조질립）전입L1210세포계，병통과장기약물사선획득료은정표체세포계，위진일보연구해기인재종류중적작용제공료실험평태。
Objective To construct the mouse L1210 leukemia cell line with a stable high-level expression vector miR-17-92 .Methods The high expression vector MSCV-LMP-miR-17-92 and control vector MSCV-LMP were transfected into the L1210 leukemia cell line by using the nucleofection , the cells were then selected with puromycin ,and detected by fluorescence microscopy and flow cytometry .The level of target miRNA in positive clones were verified by real time PCR .Results The results showed a high efficiency of L 1210 cell line with MSCV-LMP-miR-17-92 ,MSCV-LMP by nucleofection .The expression among of target miRNA was stable which was up to 99%after the selection of puromycin .Conclusions The nucleofection could construct the L 1210 cell lines with a stable high expression vector MSCV-LMP-miR-17-92 and control vector MSCV-LMP, which would be helpful for further study in oncology about miR-17-92 and provided more researching spaces on these subjects .