siRNA特异抑制树突状细胞BAK基因表达的研究  

Study on Expression of BAK Gene on Human Dendritic Cells Inhibited by Special siRNA

作　　者：余森辉[1] 曹以诚[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006

出　　处：《现代食品科技》2010年第7期693-696,共4页Modern Food Science and Technology

基　　金：广州市科技局siRNA重大专项资助(项目编号:G02-B2060030)

摘　　要：本文探讨了小干扰RNA(siRNA)对人树突状细胞(DC)中促凋亡基因BAK表达的影响。针对BAK基因区设计了2条高效特异的siRNA,合成相应的oligoDNA;构建重组质粒psi-BAK-1与psi-BAK-2,并对这些质粒进行了限制性酶切、测序等鉴定工作;在脂质体的介导下转染树突状细胞,转染后72h收集细胞,用蛋白印迹法检测Bak蛋白的表达量;并通过流式细胞仪检测转染后72h树突状细胞的存活率。结果表明,经过酶切鉴定和测序证实两个重组质粒psi-BAK-1、psi-BAK-2分别构建成功;WesternBlot结果显示转染后72h,si-BAK-1对Bak蛋白表达的抑制率为(51.5±3.54)%,si-BAK-2对Bak蛋白表达的抑制率为(26.5±6.36)%;转染重组质粒后72h树突状细胞存活率分别为93.95%、89.66%,均高过对照组的88.00%。si-BAK-1、si-BAK-2都能有效降低树突状细胞中BAK基因的表达,为延长树突状细胞的寿命,增强抗原递呈能力提供了新的思路和方法。The effect of specific small interfering RNA on expression of proapoptotic gene BAK on human dendritic cells （DCs） were investigated in this paper. Two highly specific siRNA candidates targeting BAK gene were designed, and their recording sequences were constructed into the recombinant plasmids psi-BAK-1 and psi-BAK-2. After validation through DNA restriction enzyme digestion and specific DNA fragment sequencing, both recombinant plamids and control plasmid were transfected into DCs with liposome. After transfection for 72 hours, the expression of BAK gene in DCs was detected with Western-blot, and the viablel rate of DCs were detected with flow cytomertry. The results of Western Blot showed that two special siRNA could inhibit the expression of Bak up to （51.5±3.54）% and （26.5±6.36）% respectively at 72 hours after transfection; The viable rate of DC at 72 hours after transfection with recombinant plasmids were 93.95% and 89.66%, both which higher than 88.00% of control. It was concluded that si-BAK-1 and si-BAK-2 can reduce the expression of proapoptotic gene BAK in human dendritic cell. This work supplied an experiment base for prolonging the life of dendritic cells, and also provided a new strategy and approach for enhancing antigen presenting capability.

关 键 词：树突状细胞 BAK 小干扰RNA 

分 类 号：Q343[生物学—遗传学]