RNA干扰技术-生物化学与分子生物学课件.ppt

1、 RNA干扰技术干扰技术 董莹董莹 董翠玲董翠玲 陈崇波陈崇波 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试验方法一、RNAi的发现WTPlant with a pigment transgenePost Transcriptional Gene Silencing in PetuniaRNAi的发现n1995年，Guo S等试图阻断秀丽线虫（C.elegans）中的par-1基因的表达n设计：反义

2、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n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n直到1998年2月，Fire A和Mello C才首次揭开这个悬疑之谜。n他们将体外转录得到的单链单链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。n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

3、。n 该小组将这一现象称为RNA干扰（简称RNAi）。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。RNAi 特点n转录后水平的基因沉默机制n较高的特异性n抑制基因表达具有较高的效率n有浓度、时间双重依赖性n基因表达的效应可以突破细胞界限二、RNAi的分子机制1.起始阶段(the initiation phase)2.效应阶段(the subsequent effector phase)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d

4、sRNA，在ATP的参与下逐步把dsRNA切割成长约2123 nt的片段。Dicer 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。三、RNAi的生物学功能与意义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四、RNAi试验方

5、法1.筛选RNAi探针2.制备 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 电穿孔法、磷酸钙共沉淀、DEAE一葡聚糖、阳离子脂质体试剂 荧光蛋白(如GFP)或荧光素酶等报告基因与其共表达法 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 applicat

6、ion.Now,many efforts have made to enhance siRNA delivery and target organ specificity.n In vitro several transfection reagents allow the delivery of siRNAs in mammalian cells in the presence or absence of serumn In vivo require the development of more sophisticated formulations and/or the identifica

7、tion of optimal modes of administrationTherapeutic siRNA moleculesnCancer:Target molecules usually represent genes that have been shown previously to be relevant or rate-limiting for tumor growthnAntiviral treatment:novel RNAi-based approaches to battle viral infections rely on the specific siRNA-me

8、diated knockdown of virus-specific genes This studies provide valuable insights into the delivery and efficacy of for the induction of RNAi.Clinical trialsnALN-RSV01:target the human RSV after viral infection,which is the first example of an antivirus siRNA-based therapeutic in a phaseclinical study

9、.n Sirna-027:To treat AMD,after 24-month phase II study,It has already used for recruiting patients with AMD.nCand5:which is now named Bevasiranib,was the first siRNA to enter both phase I and II clinical trials.Strategies for in vivo siRNA deliveryThe advantages of the direct application of siRNAs:

10、nthe lower probability on specific side effects nthe safety of siRNA delivery is based on nonviral transfer strategies nsiRNAs can not integrate into the genomeSuccessful siRNA based gene targeting relies on several preconditions:nProtect the instable siRNA moleculesnEfficient cellular uptake and in

11、tracellular release into the cytoplasmnThe absence of intracellular immune responsesThe formulation of siRNAs in carrier systems：nLiposomal formulationsnNanoparticlesnpolyethylenimine(PEI)It can be considered as examples of nonviral envelopes that protect siRNAs,thus increasing serum stability,reduc

12、ing renal excretion and mediating siRNA uptake into the cells through endocytosisnanoparticles with positively charged macromolecules.Based on electrostatic interactions,complexes are formed with atelocollagen chitosan or PEIPEI/siRNA complexes are good for enhancing tissue specificity and in vivo b

13、iocompatibility,reducing immunogenicity and toxicity and increasing siRNA deliveryConclusion and outlooknA very efficient and specific method for the knockdownnThe success of siRNAs will depend on their efficient and safe in vivo deliverynsiRNAs for gene knockdown will be their“double specificity”,optimal siRNA sequences and increased target organ specificity Thank you for your attention！