【作者】 陈敏；

【导师】 田玉科；

【作者基本信息】 华中科技大学 ， 麻醉学， 2011， 博士

【摘要】 研究背景阿片类药物成瘾是由于长期、反复或失去控制地应用阿片类药物而引起的慢性、复发性脑病,如果不能很好地进行预防和治疗,就会引发较多的医疗、社会和经济问题。阿片类药物成瘾主要表现为生理性依赖和心理性依赖,生理性依赖又称躯体性依赖,主要表现为阿片类药物突然停用时出现的具有特征性的戒断症状,心理性依赖,又称精神性依赖,是指反复使用阿片类药物后产生的一种愉快满足的欣快感觉,这种心理上的欣快感觉会导致机体产生继续使用相关药物的强烈欲望,继而引发强迫用药行为。目前对于生理性依赖状态已经有了多种有效的控制办法,但对阿片类药物造成的心理性依赖还难以清除,因此很容易使患者产生复吸。强啡肽和K阿片受体(KOPr)存在于多巴胺能黑质纹状体系统和中脑边缘系统的多个区域,强啡肽和强啡肽原mRNA在伏核、尾状核、杏仁核、海马以及下丘脑中都有丰富的表达。强啡肽在阿片类药物成瘾中可能通过调制基础和药物诱导的多巴胺神经元功能而起着重要作用。与μ阿片受体配体不同,K阿片受体的特异性配体强啡肽能够降低多巴胺能黑质纹状体系统和中脑边缘系统中多个区域的基础和药物诱导的多巴胺水平。因此KOPr强啡肽系统被认为是阿片成瘾过程中,在直接或间接药物诱导的多巴胺能兴奋后大脑进行反向调节机制的一部分。有研究表明预先给予KOPr激动剂可以降低可卡因成瘾大鼠产生的精神兴奋效应和条件奖赏效应,并降低静脉内可卡因自动给药的速率。因此有假说认为作用于KOPr的配体在成瘾性疾病的特定阶段可能是潜在的药物治疗手段。强啡肽原基因的表达受到钙结合蛋白DREAM (downstream regulatory element antagonist modulator)的调控。DREAM是一种多功能蛋白,它以四聚体的形式结合到基因启动子TATA盒下游的DRE (downstream regulatory element)位点,能够阻断RNA聚合酶Ⅱ复合物的前行,从而抑制目的基因转录。目前已公认DREAM是前强啡肽原(preprodynorphin, PPD)基因表达的转录抑制子,如果对PPD基因的DRE位点进行定点突变后,其基因的表达会明显增强。RNA干扰(RNA interference, RNAi)是指在多种生物体细胞内,由双链RNA (double-stranded RNA, dsRNA)分子诱发、通过高效、特异性地阻断或者降低同源mRNA的表达而产生的基因沉默(gene silencing)的现象。因RNAi所诱发的基因沉默发生在转录后水平,因此也被称作转录后基因沉默(post-transcriptional gene silencing, PTGS)。在哺乳动物细胞中,进行RNAi目前主要有两种方法：第一种是采用直接制备siRNA的方法,制备的siRNA长度大约为19-23bp,然后再将其转入生物体细胞中；第二种方法是构建编码siRNA的小发夹结构RNA(shRNAs)表达载体,再将其转入生物体细胞中,首先表达产生shRNA,再经过细胞质中核酸酶Dicer识别切割后产生siRNA。通过这两种方法进入细胞内的siRNA与特定的酶结合形成RNA诱导沉默复合体(RNA-induced silencing complex, RISC),利用ATP提供的能量解开siRNA的双链,激活RISC,然后在siRNA反义链的指导下,通过碱基配对,RISC结合到与siRNA同源的目标mRNA上,并在接近中点的位置将目标mRNA切割,使其降解,进而使相应的蛋白质合成减少,目的基因表达沉默。siRNAs的专一性非常高,能有效地抑制特定基因的表达,但不会对非相关基因的表达产生特别的影响。这个结论使得siRNA成为鉴定基因功能的有力工具。进行RNAi治疗的另一关键步骤是要选用合适的递送系统将siRNA完整高效地传递至细胞内。递送系统大致可分为病毒载体递送和非病毒载体递送两大类。非病毒载体递送系统(Nonviral gene delivery systems)包括裸siRNA直接或共价修饰后递送、采用脂质体／脂质复合物递送、采用纳米多聚颗粒递送、采用蛋白和肽类物质递送、采用抗体缀合物递送；病毒载体递送系统包括腺病毒载体、腺相关病毒载体、逆转录病毒载体、慢病毒载体以及其他病毒载体。病毒载体转染效率和表达效率较高,是在整体动物和细胞水平进行基因转导的有效工具。其中腺相关病毒载体(AAV)是一种到目前为止没有发现与人类疾病有任何相关性的病毒。它的安全性高,因为它只有很低的细胞毒性；它的宿主细胞范围广,因为其有感染分裂细胞和非分裂细胞的能力；它的免疫源性低,因为它不会引起细胞强烈的免疫反应,此外,它还能特异性地整合到宿主细胞染色体,在体内长时间表达外源基因,这些特点使AAV广泛应用于基因治疗和疫苗研究中,成为最有希望的基因转移载体之一。基于以上理论基础,我们设想,如果采用RNA干扰技术,使中枢神经系统内DREAM基因表达沉默,从而解除对PPD基因的抑制,使中脑边缘多巴胺通路中多巴胺的释放量减少,是否会有助于改善与多巴胺水平升高有关的渴求行为呢?因此,我们设计了以下两部分的研究来评价RNAi介导海马、伏核DREAM基因表达下调对吗啡成瘾大鼠条件性位置偏好和戒断症状的抑制作用。1.构建表达DREAM基因的小分子干扰RNA (siRNA)重组腺相关病毒(rAAV)载体,并将其感染体外培养的PC12细胞,观察rAAV感染PC12细胞的效率,并采用Western Blot方法测定DREAM蛋白表达量以观察RNA干扰效应。2.应用大鼠脑立体定向技术通过重组腺相关病毒载体把编码DREAM siRNA的短发夹结构导入大鼠海马和伏核,在体内稳定表达后,建立吗啡成瘾模型,通过观察大鼠行为学表现和条件性位置偏爱(conditioned place preference, CPP),评价基因治疗对大鼠戒断症状和条件奖赏效应的抑制作用。研究方法与结果1.大鼠DREAM基因shRNA重组腺相关病毒载体的构建及其干扰效率的鉴定方法：根据大鼠DREAM基因,选择文献报道有效的19nt靶序列作为特异性干扰序列,设计并合成编码DREAM siRNA小发夹结构的正义链和反义链,退火后形成双链,同时在两端加入限制性内切酶BamHⅠ、HindⅢ的酶切位点。将经过BamHⅠ和HindⅢ双酶切的pDC316-EGFP-U6回收载体片段与上述短的双链连接,构建重组质粒pDC316-U6-Dream shRNA-EGFP。设计两条引物,一条与pDC316-U6-Dream shRNA-EGFP质粒的EGFP基因的读码框的5’端特异互补,引入EcoRI位点,另一条引物与pDC316-U6-Dream shRNA-EGFP质粒上U6启动子的5’端特异互补,引入SalI位点,利用已构建好的重组质粒pDC316-U6-Dream shRNA-EGFP,以其为模板,用高保真酶进行扩增。回收PCR产物,用EcoRI和SalI双酶切,再回收,与EcoRI、SalI双酶切pSNAV2.0后回收的载体片段连接,即得到重组载体质粒pSNAV2.0-EGFP-DREAM-shRNA-U6。经过酶切和测序鉴定后,用脂质体包裹携带DREAM shRNA的pSNAV2.0载体质粒,转染BHK-21细胞,利用载体上的新霉素抗性基因标记建立相对稳定的长期基因沉默AAV载体细胞株,然后用具有AAV rep、cap基因和包装功能的辅助病毒——HSV1-rc/ΔUL2对该细胞株进行感染,通过细胞裂解、病毒纯化等步骤,即可得到包含DREAM shRNA的高滴度的rAAV2/1颗粒rAAV2/1-DREAM shRNA-EGFP。再将rAAV2/1病毒颗粒感染体外培养的PC12细胞,经免疫印迹技术(Western blot)检测DREAM基因的蛋白表达水平,以评价rAAV2/1-DREAM- shRNA-EGF的RNA干扰效率。没有包含DREAM shRNA的rAAV2/1-EGFP设立为阴性对照。结果：PCR鉴定和测序证实了大鼠DREAM基因shRNA的重组腺相关病毒载体构建成功,在感染体外培养的PC12细胞后,rAAV2/1-DREAM shRNA-EGFP可以明显抑制DREAM蛋白的表达水平。2.海马、伏核立体定向注射DREAM-shRNA rAAV2/1载体抑制吗啡成瘾大鼠条件性位置偏爱和戒断症状方法：选取符合标准的24只SD大鼠随机分为四组,rAAV2/1-DREAM组、rAAV2/1组、PBS组、对照组。每组6只。rAAV2/1-DREAM组向大鼠双侧海马、伏核各靶点注射rAAV2/1-DREAM shRNA-EGFP, rAAV2/1组注射rAAV2/1-EGFP, PBS组注射PBS；对照组进行立体定向手术,不进行任何注射。2周后rAAV2/1-DREAM组、rAAV2/1组、PBS组大鼠建立吗啡成瘾和位置偏爱模型。三组大鼠按逐日递增原则,腹腔注射(ip)盐酸吗啡,每隔12h注射一次,起始剂量为每次5mg/kg,每日递增5-10mg/kg,连续10d,10d后剂量达到每次90mg/kg。每次注射完吗啡后放下隔板1h,让大鼠停留在伴药侧。1h后提起隔板,使大鼠自由活动。每次注射吗啡前或后3h腹腔注射与吗啡等体积的生理盐水,注射完后关入非伴药侧,时间也是lh。对照组按同样的时间程序进行注射,只是不用注射吗啡,而代之以等体积的生理盐水。最后一次注射吗啡3h后,腹腔给予纳洛酮2mg/kg催瘾,观察并记录戒断症状,共30min,包括跳跃、湿狗样抖、体重减轻、咀嚼。药物注射完成3d后进行条件性位置偏爱测试,直接以大鼠15min(共900s)内在偏爱箱伴药侧停留的时间(秒)表示。各组大鼠在观察结束后立即处死,快速分离大脑海马、伏核组织,采用Western blot方法；检测各组大鼠海马、伏核DREAM蛋白的表达变化。结果：1.成瘾大鼠在纳洛酮作用下诱发戒断症状,rAAV2/1、PBS组大鼠与对照组相比,出现明显的跳跃、湿狗样抖动、体重减轻、咀嚼等戒断症状,而rAAV2/1-DREAM组大鼠与rAAV2/1、PBS组相比,这些成瘾体征明显减少,差异有统计学意义(P<0.05)；2.CPP训练后,rAAV2/1、PBS组大鼠在伴药箱的停留时间(575.17±66.69s、555.50±49.79s)与对照组(315.33±31.95s)相比明显延长(P<0.01),表明在吗啡作用下rAAV2/1、PBS组大鼠均对伴药箱产生CPP效应,而rAAV2/1-DREAM组大鼠在伴药箱的停留时间(366.00±26.38s)与对照组(315.33±31.95s)相比,差异无统计学意义(P>0.05)。3. Western blot方法检测结果显示,与PBS组、rAAV2/1组相比,rAAV2/1-DREAM组大鼠海马、伏核DREAM蛋白表达量显著降低,差异有统计学意义(P<0.01)。3.统计学分析计量资料以均数±标准差(x±s)表示,采用SPSS11.0软件进行数据处理,组内比较采用配对t检验,组间比较采用单因素方差分析,以P<0.05为差异有统计学意义。研究结论本研究成功地构建了包含大鼠DREAM基因shRNA的重组腺相关病毒载体rAAV2/1-DREAM shRNA-EGFP,感染体外培养的具有内源性DREAM表达的细胞,可以在蛋白水平特异、高效地抑制DREAM的表达；应用大鼠脑立体定向技术通过重组腺相关病毒载体把编码DREAM siRNA的短发夹结构导入大鼠海马和伏核,在体内稳定表达后,明显降低海马、伏核中DREAM蛋白的表达量,并改善吗啡成瘾大鼠的戒断症状,抑制条件性位置偏爱的形成,说明siRNA介导的DREAM基因沉默可以有效地抑制吗啡引起的条件奖赏效应。研究总结本研究成功地构建了大鼠DREAM基因的重组腺相关病毒沉默表达载体,并在体外培养细胞和大鼠体内获得了稳定的表达,同时通过目的基因相应蛋白质的表达水平证实了siRNA的干扰作用是有效的,克服了以往RNA干扰技术研究基因功能瞬时性的缺陷。重组腺相关病毒沉默载体导入大鼠脑内,可明显抑制吗啡成瘾大鼠的戒断症状和条件性位置偏爱的形成,是一种有效、可行的转基因治疗药物成瘾的技术,这是对阿片类药物成瘾治疗所进行的一次探索性研究。同时表明DREAM可能是减轻阿片成瘾奖赏效应的潜在治疗靶点。更多还原

【Abstract】 Addiction to opiates is a chronic, relapsing brain disease induced by long-term use of morphine repeated and uncontrolled. If left untreated, it can cause major medical, social, and economic problems. The clinical manifestations included physiological dependence and psychological dependence. Physiological dependence, which is also called physical, is defined by the appearance of characteristic withdrawal symptoms when opiates are suddenly discontinued. Psychological dependence, which is also called psychic dependence, refers to a sence of well-being and contentment cheerfulness induced by repeated use of opiates. This pleasure effects produce an overwhelming desire to continue with the drug experience followed by a craving or compulsive to use the drug. At present, various effective methods have been devised to improve physiological dependent status. However, it is difficult to get rid of psychological dependence induced by opiates, which tends to relapse.Dynorphin and the kappa opioid receptor (KOPr) are localized in several areas of the dopaminergic nigrostriatal and mesolimbic-mesocortical systems. Dynorphin peptides and pro- dynorphin mRNA are particularly abundant in the nucleus accumbens, caudate, amygdala, hippocampus, and hypothalamus. Dynophin and KOPr play an important role in a modulation of opioid, cocaine, and other rewarding stimuli, presumably through modulation of basal and drug-induced dopaminergic tone. In contrast to the ligand of mu opioid receptor, dynorphin peptides, the ligand of kappa opioid receptor, decrease basal and drug-induced dopamine levels in several areas of the dopaminergic nigrostriatal and mesolimbic-mesocortical system. The KOPr-dynorphin system may therefore be considered to be a part of the countermodulatory mechanisms of the brain after direct or indirect drug-induced dopaminergic stimulation. Earlier studies showed that pretreatment with KOPr agonists decreases the psychostimulant and conditioned rewarding effects of cocaine in rats and decreases the rate of intravenous cocaine self-administration. Thus it has been hypothesized that ligands acting at KOPr may be potential pharmacotherapeutic agents for specific stages in the treatment of addictive diseases.Expression of the human PDYN gene is regulated by the calcium-binding protein downstream regulatory element (DRE) antagonist modulator (DREAM). DREAM, a multifunctional protein, binds as a tetramer downstream to the promoter via an intragenic sequence termed the downstream regulatory element (DRE), blocking transcription. DREAM as a PPD gene transcriptional inhibitor is commonly agreed upon now. If point mutations occur in the DRE site, PPD gene expression will be increased.RNA interference is a gene silencing phenomenon, induced by double-stranded RNA(dsRNA) efficient and special blocking or down-regulating homologous gene expression in various biological cells. Gene silencing induced by RNAi is also known as post-transcriptional gene silencing (PTGS). For mammal animals, transduction of long dsRNA above 30bp into cells often induces unexpected antiviral response. So the common way for mammal animals is to prepare siRNAs (19-23bp) and then transduct it into cells; or make DNA expression vector containing shot hairpin RNAs(shRNAs) and then transduct it into cells to express shRNA, which is cleaved into siRNAs by an enzyme called Dicer. The siRNAs in cells recruit additional components to form an RNA-induced Silencing Complex (RISC). siRNA strands guide RISCs to target mRNA molecules homologous with siRNAs by base pairing, then cleave the target mRNA at the midpoint and result in degradation of it. Finally the corresponding proteins synthesis reduced and thus the target gene expression silencing. siRNAs are characterized in high specificity and can inhibit target gene expression effectively without prejudice to irrelevant genes. This conclusion makes siRNA a powerful tool identifying gene function.The key step in siRNA therapy is using a suitable delivery system to facilitate siRNA access to its intracellular site of action. Delivery system roughly divided into two types:viral gene vectors delivery systems and nonviral gene delivery systems. Nonviral gene delivery systems included bare siRNA, covalent modificated siRNAs, liposomes and lipid complex, nanoparticle, proteins and polypeptide and antibody conjugate. Viral gene vectors included adenovirus vector, adeno-associated virus vectors, retroviral vectors and lentiviral vectors and other viral vectors. Virus vector, is characterized by high transfection efficiency and high expression efficiency, is a powerful tool for gene transfer on the whole and cells level. Adeno-associated virus(AAV), a virus no evidence exist to suggest be related to human disease until now, have great advantages over other viral vectors. It is high safety due to very low cytotoxicity. It has a boarder host range due to its ability of infecting dividing and nondividing cells. It has low immunogenicity because it cannot induce the intensive immunologic reaction. Furthermore, it can integrate into chromosome of host cell and stably express foreign gene in vivo for a long time. These features made AAV a most hopeful gene delivery vector, which is widely used for gene therapy and Vaccines Research.On the basis of the above theory, we hypothesized that if the DREAM in central nervous system silenced by RNAi, the craving related to increased dopamine level will be improved through removing the inhibition of PPD gene expression and thus decreasing the release of dopamine in mesolimbic-mesocortical system. We designed two parts experiments to evaluate the inhibition effect of RNAi-mediated down-regulation of DREAM gene expression in hippocampus and nucleus accumbens on the conditioned placed preference and withdrawl syndromes in morphine-addicted rats.1. To construct recombined adeno-associated viral vector containing DREAM short hairpin RNA and then infect PC 12 cells in vitro. To observe efficiency of rAAV infecting PC 12 cells. Western blotting was used to detect the DREAM protein expression in order to evaluate the RNA interfering effect.2. To transduct DREAM shRNA structure enconding siRNA into hippocampus and nucleus accumbens of rats by means of rAAV using brain stereotaxic technology. After DREAM siRNA stabilize its expression in vivo, we build morphine addict model in rats and to evaluate the inhibition effect of gene therapy on the conditioned rewarding effect and withdrawl syndromes by observing behavior and conditioned placed preference. Methods and results 1. Construction of recombined adeno-associated viral vectors containing DREAM shRNA and identification of its interfering efficiency.Methods:We designed siRNA targeting the rat DREAM coding sequence based on recommendations described by previous literature. The targets of the siRNA comprised of 19 nucleotides of the rat DREAM gene. We designed and synthesized two DNA model-strands encoding DREAM shRNA. After annealing a double strand was resembled and BamHⅠand HindⅢsites was added at both ends simultaneously. Then we ligated the double strand into the plasmid pDC316-EGFP-U6 had been digested by the restriction enzyme BamHⅠand HindⅢ. The recombinant plasmid pDC316- U6-Dream shRNA-EGFP was construct successfully. We designed two primers, one of which complement with 5’(five prime) end of reading frame of EGFP gene on plasmid pDC316- U6-Dream shRNA-EGFP in order to induce EcoRI site, the other of which complement with 5’end of U6 promoter in order to induce SalI site. 华中科技大学博士学位论文Then polymerase chain reaction(PCR) amplification taking pDC316-U6-Dream shRNA-EGFP as model was carried using the two primers. PCR products and plamid pSNAV2.0 were digested by the restriction enzyme EcoRI and SalI and set up ligation reaction to get recombinant vector plasmid pSNAV2.0-EGFP-DREAM-shRNA-U6. Had been identified by enzyme cutting and sequencing, pSNAV2.0 vector carrying foreign gene transfected BHK-21 cells and AAV vector cell strains were developed. Using helper virus HSV1-rc/ΔUL2 carrying rep and cap gene of AAV virus to infect the AAV vector cell strains, we get rAAV2/1 vector rAAV2/1-DREAM-shRNA-EGFP containing foreign gene by a series of steps of cell lysis and purification. Finally, rAAV2/1 vector containing DREAM shRNA infected PC 12 cells in vitro and western blot was used to detect DREAM protein expression in order to evaluate interfering efficiency of rAAV2/1-DREAM shRNA-EGFP. rAAV2/1-EGFP not containing DREAM shRNA were regarded as negative control.Results:PCR identification and sequencing confirmed the successful construction of recombinant adeno-associate viral vector containing DREAM gene shRNA, which can inhibit DREAM protein expression markedly after infected PC 12 cell in vitro.2. stereotaxic injection of rAAV2/1 vector containing DREAM shRNA into hippocampus and nucleus accumbens inhibit the conditioned placed preference and withdrawl syndromes in morphine-addicted rats.Methods:36 SD rats confirm to a certain standard were randomly divided into four groups:rAAV2/1-DREAM group, rAAV2/1 group, PBS group and control group. Bilateral stereotaxic injection of rAAV2/1-DREAM shRNA-EGFP into hippocampus and nucleus accumbens of rats was carry out in rAAV2/1-DREAM group, rAAV2/1-EGFP in rAAV2/1 group and PBS in PBS group. Control group had no injection. Two weeks later morphine-addicted model and conditioned placed preference model of rats were established. The rats in rAAV2/1-DREAM group, rAAV2/1 group and PBS group were administered intraperitoneally with gradually increasing doses of morphine twice daily(interval between two dose is 12 hours) for 10 consecutive days(day1:5 and 5mg/kg; day 2:10 and 10mg/kg; day 3:20 and 20mg/kg; day4:30 and 30mg/kg; day 5:40 and 40mg/kg; day 6:50 and 50 mg/kg; day 7:60 and 60mg/kg; day 8:70 and 70mg/kg; day 9:80 and 80mg/kg; day 10:90 and 90mg/kg). The animals were confined for 1 h to the drug-paired chamber when received the injection of morphine and then move freely. The rats were injected equivalent volume physiological saline intraperitoneally 3h before or after every dose of morphine and then confined for 1h to the non-drug-paired chamber. The rats in control group received physiological saline instead of morphine and the same injection procedure. 3h after the last dose of morphine was given, the rats in every group were administered with nalxone (2mg/kg, i.p.) to induce withdrawal behaviors. The behavioral signs of withdrawal syndrome were measured by the numbers of jumping, wet-dog shaking, mastication and the body weight loss within 30min.3d after the last dose of morphine was given, conditioned placed preference test was carry on. Time spent in each compartment was recorded separately for each animal for a total of 15min. Animals were sacrificed immediately following behavioral testing and their brains rapidly extracted and different brain regions dissected out. Immunohistochemical and western blot were used to detect the DREAM protein expression in hippocampus and nucleus accumbens of rats.Results:1. morphine-addicted rats that had received naloxone injection showed obvious signs of withdrawal. The rAAV2/1 and PBS groups showed marked body weight loss and dramatically increased signs of withdrawal, which were manifested by jumping, wet-dog shaking and mastication, compared to the rAAV2/1-DREAM group (P<0.05).2. The time in drug-paired chamber in rAAV2/1 and PBS group (575.17±66.69、555.50±49.79) are significantly longer than that in the control group (315.33±31.95) (P<0.01). There was no difference in the time change in the drug-paired side in rAAV2/1-DREAM group (366.00±26.38) and control group (315.33± 31.95) (P>0.05).3. Immunohistochemistry and western blotting results show that the DREAM protein expression in hippocampus and nucleus accumbens of rats significantly decreased in rAAV2/1-DREAM group, compared with PBS and rAAV2/1 group(P<0.01).3. Statistical analysisAll of the Measurement data were shown as mean±standard deviation. Statistical analyses were performed with SPSS11.0. Comparison among groups was made using a One-way ANOVA. Values of P<0.05 was considered statistically significant.ConclusionIn our study we successfully constructed Recombinant adeno-associated viral vector containing DREAM gene shRAN, which would targetedly reduce dream protein expression after it infected cells with endogenous expression of DREAM. When DREAM siRNA stabilized its expression in vivo after we transducted DREAM shRNA structure enconding siRNA into hippocampus and nucleus accumbens of rats by means of rAAV delivery system under the guidance of steric orientation technique, it can significantly reduced the DREAM protein expression in hippocampus and nucleus accumbens of rats and dramatically inhibit morphine induced withdrawal symptoms and the formation of conditioned placed preference. These results indicate that siRNA-mediated DREAM gene silencing can inhibit effectively morphine-induced conditioned rewarding effect.SummaryWe successfully constructed recombinant adeno-associated viral silence expression vector for DREAM and obtained stable expression in cultured cells in vitro and in vivo. The interfering effect of DREAM siRNA was confirmed by corresponding protein expression level. The recombinant adeno-associated viral silence expression vector overcome defects of transient expression in previous research on studying gene function using RNAi. The recombinant adeno-associated viral silence expression vector transferred into brain of rats can dramatically inhibit morphine induced withdrawal symptoms and the formation of conditioned placed preference. It’s a effective genetically modified therapy methods to treat the syndrome of addiction to opiates with great feasibility and a pilot-study for the therapy of addiction. Furthermore, we indicated that DREAM may be a potential therapy target to alleviate rewarding effect induced by opiates.更多还原