RNA干扰基因敲除MAGE-1在恶性胶质瘤U87细胞中的初步研究  

Knockdown MAGE-1 by RNA interference system in U87 of the human glioma cell line

作　　者：程光[1] 章翔[1] 鲍炜[2] 张赟[2] 张新海[2] 曹卫东[1] 高大宽[1] 宋蕾[1] 

机构地区：[1]第四军医大学西京医院解放军神经外科研究所 [2]第四军医大学西京医院免疫教研室,陕西西安710032

出　　处：《医学研究生学报》2006年第4期292-297,i0011,共7页Journal of Medical Postgraduates

基　　金：国家自然科学基金资助项目(批准号:39970752)

摘　　要：目的:通过构建抑制MAGE-1的短片段双链核糖核酸(siRNA)表达载体,鉴定其在人恶性胶质瘤细胞U87细胞中对MAGE1基因表达的干涉作用。方法:化学合成2对编码短发夹RNA序列的靶向MAGE1基因核苷酸链,克隆至经BglⅡ、HindⅢ双酶切的pSUPER载体上,重组构建核糖核酸干扰(RNAi)质粒载体。利RTPCR、流式细胞术和荧光显微镜,检测经稳定转染后胶质瘤U87细胞中MAGE-1的表达,以了解siRNA的干效果。结果:重组构建的pSUPERMAGE1载体经双酶切、电泳及插入基因片段序列分析,表明寡核苷酸链成地插入至预计位点,且序列与预期完全一致。稳定转染后G418筛选出的U87多克隆细胞MAGE-1的表达RTPCR、流式细胞术和荧光显微镜检测,2对siRNA均有较明显的干扰作用。结论:载体的成功构建并能对U细胞中的MAGE1分子进行RNAi,为进一步研究MAGE1在肿瘤中的作用,分析基因功能,展开肿瘤基因治疗奠了基础。Objective： To study the inhibition of expression of MAGE-1 by RNAi system in U87 of the human glioma cell line. Methods： 64 base-pair oligos for hairpin RNA expression which targeted MAGE-1 gene were chemically synthesized and annealed, pSUPER vector was linearized with Bgl Ⅱ and Hind Ⅲ. Annealed oligos were inserted into the treated pSUPER to construct RNAi plasmid （pSUPER- MAGE-1 ）. Expression of MAGE-1 was detected in U87 cell by RT-PCR and after the stable transfection. Results ： Recombinant pSUPER-MAGE-1 vector was identified and confirmed by sequencing analysis. The results demonstrated that 64 bp had been inserted the expected site and the insertion sequence was exactly correct. The expression of MAGE-1 in U87 detected by RT-PCR, flow cytometry and fluorescence microscope was greatly downregulated by these two siRNA respectively. Conclusion： pSUPER-MAGE-1 RNAi system can successfully inhibit the expression of MAGE-1 in U87 and will facilitate the study of the function of MAGE-1.

关 键 词：PSUPER 短片段双链核糖核酸 核糖核酸干扰 MAGE—1 胶质瘤 

分 类 号：R730.264[医药卫生—肿瘤]