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*,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,RNA,干扰技术,董莹 董翠玲 陈崇波,1,2006年诺贝尔生理学或医学奖：,美国斯坦福大学,Andrew ZFire,美国马萨诸塞大学,Craig CMello,，,以表彰他们发现了RNAi现象,2,什么是,RNAi,？,一、,RNAi,的发现,二、,RNAi,的分子机制,三、,RNAi,的生物学功能与意义,四、,RNAi,试验方法,3,一、,RNAi,的发现,4,WT,Plant with a pigment transgene,Post Transcriptional Gene Silencing in Petunia,RNAi的发现,5,1995年，,Guo S,等试图阻断秀丽线虫（,C.elegans,）中的par-1基因的表达,设计：,反义,RNA,特异性地阻断,par-1,基因的表达,正义,RNA,以期观察到基因表达的增强,结果:,二者都同样地切断了,par-1,基因的表达途径。这是与传统上对反义RNA技术的解释不相符合。该研究小组一直没能给这个意外以合理解释,6,直到1998年2月，,Fire A,和,Mello C,才首次揭开这个悬疑之谜。,他们将体外转录得到的,单链,RNA,纯化后,注射线虫时发现，基因抑制效应变得十分微弱；而经过,纯化,的双链,RNA,却正好相反，能够高效特异性阻断相应基因的表达。,他们证实，,Guo S,博士遇到的正义,RNA,抑制基因表达的现象，以及过去的反义RNA技术对基因表达的阻断，都是由于体外转录所得,RNA,中,污染了微量双链,RNA,而引起。,该小组将这一现象称为,RNA干扰,（简称RNAi）。,7,RNAi,概念,RNA,干扰,(,RNAi,),是指,dsRNA,在细胞内特异性地诱导与之同源互补的,mRNA,的降解，使相应基因的表达关闭，从而引发基因转录后水平沉默的现象。,8,RNAi,特点,转录后水平的基因沉默机制,较高的特异性,抑制基因表达具有较高的效率,有浓度、时间双重依赖性,基因表达的效应可以突破细胞界限,9,二、,RNAi,的分子机制,1.,起始阶段,(,the initiation phase,),2.,效应阶段,(,the subsequent effector phase,),10,dsRNA通过外源导入、转基因或者病毒感染等方式进入细胞后，专一性的双链RNA内切酶Dicer识别dsRNA，在ATP的参与下逐步把dsRNA切割成长约2123 nt的片段。,Dicer,11,siRNA参与形成RNA诱导的沉默复合物(RISC)，ATP激活的RISC在双链siRNA的反义链指导下，寻找与siRNA具有同源序列的内源靶mRNA，并在距离siRNA3端12个碱基的位置切割靶mRNA，导致转录后基因沉默。,12,13,三、,RNAi,的生物学功能与意义,14,RNAi,的生物学功能,抵抗病毒入侵,抑制转座子活动，保护基因组 的完整性,调控基因表达,清除畸变的,RNA,参与基因组重排,15,RNAi的生物学意义,1. 研究功能基因组学的新工具,2. 研究信号传导通路的新途径,3. 研究发育过程中起作用的基因,4. 开展基因治疗的新策略,16,四、RNAi试验方法,17,1.筛选RNAi探针,2.制备,siRNA,化学合成、体外转录 、用,RNase,消化长片断双链,RNA、siRNA,表达载体、,siRNA,表达框架,3.导入细胞,电穿孔法、磷酸钙共沉淀、DEAE一葡聚糖、,阳离子脂质体试剂,4.RNAi转染效率,荧光蛋白(如GFP)或荧光素酶等报告基因与其共表达法,18,19,Abstract：,In vitro, RNAi is an almost-standard method to knockdown any target gene. In vivo, its effcient delivery and specificity to the target gene challenge its therapeutic application. Now, many efforts have made to enhance siRNA delivery and target organ specificity.,20,In vitro,several transfection reagents allow the delivery of siRNAs in mammalian cells in the presence or absence of serum,In,vivo,require the development of more sophisticated formulations and/or the identification of optimal modes of administration,21,Therapeutic siRNA molecules,Cancer :,Target molecules usually represent genes that have been shown previously to be relevant or rate-limiting for tumor growth,Antiviral treatment,:,novel RNAi-based approaches to battle viral infections rely on the specific siRNA-mediated knockdown of virus-specific genes,22,This studies provide valuable insights into the delivery and efficacy of for the induction of RNAi.,23,Clinical trials,ALN-RSV01:,target the human RSV after viral infection, which is the first example of an antivirus siRNA-based therapeutic in a phaseclinical study.,Sirna-027:,To treat AMD,after 24-month phase II study,It has already used for recruiting patients with AMD.,Cand5:,which is now named,Bevasiranib,was the first siRNA to enter both phase I and II clinical trials.,24,Strategies for in vivo siRNA delivery,The,advantages,of the direct application of siRNAs:,the lower probability on specific side effects,the safety of siRNA delivery is based on nonviral transfer strategies,siRNAs can not integrate into the genome,25,Successful siRNA based gene targeting relies on several preconditions:,Protect the instable siRNA molecules,Efficient cellular uptake and intracellular release into the cytoplasm,The absence of intracellular immune responses,26,The formulation of siRNAs in carrier systems：,Liposomal formulations,Nanoparticles,polyethylenimine (PEI),It can be considered as examples of nonviral envelopes that protect siRNAs, thus increasing serum stability, reducing renal excretion and mediating siRNA uptake into the cells through endocytosis,nanoparticles with positively charged macromolecules. Based on electrostatic interactions, complexes are formed with atelocollagen chitosan or PEI,PEI/siRNA complexes are good for enhancing tissue specificity and in vivo biocompatibility, reducing immunogenicity and toxicity and increasing siRNA delivery,27,28,Conclusion and outlook,A very efficient and specific method for the knockdown,The success of siRNAs will depend on their,efficient,and,safe,in vivo delivery,siRNAs for gene knockdown will be their “double specificity”,optimal siRNA sequences,and,increased target organ specificity,29,Thank you,for your attention！,30,
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