miR-21慢病毒表达载体的构建及稳定转染U266细胞系  

The constraction of miR-21 slow virus expression vector and stable transfection U266 cell line

作　　者：王金行[1] 周雯雯[1] 程仕彤[1] 刘柏新[1] 满冬亮[1] 杨志东[1] 宋鉴清[1] 

机构地区：[1]中国医科大学附属第一医院检验科,辽宁沈阳110001

出　　处：《现代肿瘤医学》2014年第4期776-779,共4页Journal of Modern Oncology

基　　金：辽宁省自然科学基金资助项目(编号:201102291)

摘　　要：目的:构建miR-21慢病毒表达载体,感染多发性骨髓瘤(MM)细胞U266,建立稳定表达miR-21的U266细胞系。方法:以has-miR-21前体序列pre-miR-21为模板,设计并合成引物,进行PCR扩增目的基因,并连接到慢病毒表达质粒LV-anti中。对重组质粒进行双酶切鉴定后,所构建载体命名为LV-anti-miR-21-MOI。再进行miR-21基因慢病毒LV-anti-miR-21的包装及病毒滴度测定,将慢病毒颗粒以最适滴度感染MM细胞株U266,通过Aldefluor流式分选出带有目的基因和空载病毒的细胞,用real-time PCR检测感染效率。结果:目的基因与慢病毒载体连接成功。慢病毒LV-anti-miR-21浓缩后的病毒滴度约为1.0×108TU/ml。慢病毒成功感染目标细胞U266,转染效率达90%以上。real-time PCR检测,U266/LV-anti-miR-21慢病毒MOI 20组和MOI 40组miR-21表达量分别为0.69±0.03和0.51±0.05较U266/un组(1.08±0.05)下降了38.9%和54.9%,差异有显著统计学意义(P<0.05)。结论:成功构建了miR-21慢病毒表达载体,并建立U266-miR-21稳定转染细胞系。为进一步研究miR-21的功能及作用机制奠定了基础。Objective：To constract the expression vector of miR -21 slow virus,infect U266 cells in muhiple mye- loma,establish U266 cell line expressing miR- 21 stably. Methods：According to the templates of pre- miR- 21 precursor sequence -miR -21, design and synthesis primers, for PCR amplification purpose gene and connect to plas- mid LV -anti virus expressed by slow virus, constructed carrier was named Louis vuitton -anti -miR -21 -MOI Af- ter the double enzyme identification of the recombinant plasmid. Then packaging and testing virus drops miR - 21 gene lentivirus LV - anti - miR -21, slow virus particles infect MM cell line U266 at the optimal drop degrees. Alde- fluor streaming sort out the cells with purpose gene and no - load virus, detecting efficiency of infection by real - time PCR. Results： Slow virus connect with gene carrier successfully. Lentivirus LV -anti -miR -21 virus drops was a- bout 1.0 ± l0s TU/ml after the enrichment degree. Slow virus infect target U266 cells, transfection efficiency was more than 90%. Real - time PCR detect U266/LV - anti - miR - 21 lentivirus MOI 20 group and MOI 40 miR - 21 that expression quantities were 0.69 ± 0.03 and 0.03 ±0.05 compared with U299/un group 1.08± 0.05 felling 38.9% and 54.9% ,respectively. There was significant statistical significance difference （P 〈 0.05 ）. Conclusion： Constract the miR- 21 slow virus expression vector successfully, and establish the U266 -miR -21 stable transfection cell line.

关 键 词：MIR-21 慢病毒 多发性骨髓瘤 

分 类 号：R73-34[医药卫生—肿瘤] R733.3[医药卫生—临床医学]