阿片受体真核表达系统的构建与表达及μ和δ受体的相互作用研究
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摘要
研究背景和目的
吗啡、海洛因等毒品的非法使用产生的毒品成瘾已经成为席卷全球的社会公害,成瘾性强,戒断症状严重,复吸率高,根治困难。毒品成瘾是一种慢性复发性脑病,毒品成瘾机理十分复杂,涉及中枢神经系统的许多脑区。随着药理学、神经解剖学和神经生物学等学科的发展,目前发现脑内有一个对毒品的使用产生奖赏效应的系统,涉及多巴胺(DA)、阿片肽、γ-氨基丁酸(GABA)能系统三个主要的调控毒品成瘾的神经机制。吗啡、海洛因等毒品使用后,可以直接作用于阿片肽神经元,使其释放的内啡肽增加,内啡肽再作用于多巴胺神经元上的阿片受体,促发多巴胺神经元的活性而发挥药物奖赏效应；阿片肽神经环路与多巴胺神经环路还存在着大量的交互作用,共同对毒品的成瘾起增强作用；也有人认为吗啡等毒品可以直接作用于中脑-边缘系统多巴胺神经通路和中脑-边缘系统-皮质系统多巴胺神经通路中的多巴胺能神经元,导致多巴胺释放增加,多巴胺通过作用于脑内多巴胺D1、D2受体而完成奖赏效应,产生毒品成瘾作用；另外吗啡还可以通过GABA能神经元上的μ阿片受体的介导,抑制GABA能神经元,解除GABA能神经元对VTA内的多巴胺神经元的抑制,使得多巴胺神经元的活性增加,从而产生吗啡的间接药物强化效应。
近年来,随着对药物依赖形成的神经解剖学、神经药理学及神经生物学机制的广泛深入研究,发现伏隔核在参与药物依赖的众多神经核(团)中起着关键作用。较多的动物实验及解剖学研究表明,伏隔核与药物成瘾关系密切,毁损双侧伏隔核会抑制大鼠海洛因自我给药行为的建立,能够消除成瘾大鼠的觅药行为,可以抑制吗啡诱导的复燃。
我们推测海洛因成瘾与伏隔核中μ和δ阿片受体间的蛋白-蛋白相互作用有重要关系。该推论的依据是：(1)基因敲除μ或δ受体,尤其是μ基因敲除的小鼠对于海洛因不能引起条件性位置偏爱效应、运动增多等表现,鼠失去海洛因奖赏效应,不出现成瘾性；(2)在体外培养的细胞中,已有多种方法证明共表达的μ和δ受体形成受体复合物,从而交互影响各自的配体结合、受体向胞内运动和细胞内信号传导；(3)μ和δ受体不仅共存于相同的感觉神经元,而且还共存于伏隔核内相同神经元；(4)Schmidt BL等在大鼠活体实验中,进行了伏隔核内选择性受体激动剂注射,发现DAMGO联合DPDPE(分别是μ、δ受体激动剂)注射,具有明显的镇痛效应,而单一注射则效应明显降低。这些结果提示μ和δ受体激动剂的镇痛效应需要μ和δ受体的共同活化,支持伏隔核内μ、δ受体以复合物形式存在的观点。(5)立体定向伏隔核毁损对实验动物和海洛因成瘾患者均取得了较好的戒毒效果。
Ji SP等首次证明特异性干扰肽能通过打断抑癌酶PTEN和5-HT2C受体间的蛋白-蛋白连接来阻断大麻奖赏效应。大鼠全身注射或向VTA直接注射Tat-3L4F干扰肽(Tat为源于HIV病毒分子启动子的11个氨基酸肽,3L4F为抑癌酶PTEN的第三胞内环的某一区段,Tat肽可携带3L4F穿过血脑屏障)能够显著抑制由大麻诱导的VTA多巴胺能神经元激活,可阻断由大麻或尼古丁诱导的条件性位置偏爱效应,并未产生常用阻断剂Ro600175容易引起的产生焦虑、阴茎勃起、进食减少和运动功能抑制等副作用。
如果能找到μ和δ受体之间相互作用的特异性多肽序列,可能也能有效的打断μ和δ受体之间的相互作用,从而对海洛因成瘾起到阻断作用。
使用药物脱毒后心理依赖不易消除,患者屡次复吸,海洛因脱毒后一个月内的复吸率达80%-85%,半年内的复吸率达95%,半年以上的复吸率高达97%,很多患者陷入“戒断-复吸-戒断”的恶性循环当中。寻找更有效的毒品成瘾治疗方法具有重要的意义。本课题研究就是基于这种需要,结合我们的推论,构建阿片受体μ、δ、κ的真核表达质粒,表达鉴定后,进行了免疫共沉淀研究,并采用GST pull down技术进行了μ和δ受体相互作用的domain区的寻找。
实验方法和结果
1、构建了大鼠的μ、δ、κ阿片受体的真核表达载体：本研究采用反转录巢式PCR方法,提取伏隔核为主的脑组织总RNA,随机引物逆转录为cDNA,通过外侧及内侧引物两次扩增,从大鼠脑组织总RNA中扩增出μ、δ、κ阿片受体cDNA基因,通过AT克隆,将目的基因构建到pMD20-T Vector中,测序鉴定均存在单点突变,使用Stratagene的定点突变试剂盒纠正点突变；然后再设计相应的引物,使用高保真酶,通过PCR扩增在μ、δ、κ阿片受体基因的C端分别引入蛋白抗原表位标签Myc、FLAG、HA,获得目的基因μMyc、δFLAG、κHA,AT克隆到pMD20-T Vector中,测序鉴定正确后,通过双酶切含目的基因(μMyc、δFLAG、κHA)的T载体及目的质粒pIRES2-EGFP,割胶回收,酶切片段连接,转化大肠杆菌,菌落PCR及酶切鉴定,最后测序确认,成功构建了如下三种阿片受体真核表达载体：μ-Myc-pIRES2-EGFP、δ-FLAG-pIRES2-EGFP、κ-HA-pIRES2-EGFP。
2、μ-Myc-pIRES2-EGFP、δ-FLAG-pIRES2-EGFP、κ-HA-pIRES2-EGFP在HEK293中的表达及鉴定：将质粒μ-Myc-pIRES2-EGFP、δ-FLAG-pIRES2-EGFP、κ-HA-pIRES2-EGFP转染到HEK293细胞中,荧光显微镜下观察到表达绿色荧光蛋白的HEK293细胞比例高,提示转染效率较高。使用细胞免疫荧光和蛋白印迹也分别证明转染了相应质粒的HEK293细胞中,目的基因μ、δ、κ获得了高效表达。其中μ和δ阿片受体分别使用了标签抗体Myc一抗、FLAG一抗和μ一抗、δ一抗进行鉴定,结果显示构建的质粒在HEK293中表达的目的蛋白μ-Myc、δ-FLAG很好的保持了抗原抗体的结合特性。Western blot分析结果提示三种阿片受体(μ、δ、κ)主要以糖基化的二聚体(分子量约为140kD)形式存在,同时也存在少量的糖基化的单体蛋白(分子量约为85 kD)和存在少量未糖基化的单体蛋白(分子量约为45 kD),而且形成的同源二聚体的作用力较强,可以有效的抵抗SDS的作用,在SDS存在下和100℃高温的处理下仍能保持二聚体形态。
3、μ和δ阿片受体的免疫共沉淀研究：我们在体外培养的HEK293细胞中,采用免疫共沉淀技术对μ和δ受体的相互作用进行了研究。将δ-FLAG-pIRES2-EGFP、μ-Myc-pIRES2-EGFP共转染到HEK293细胞,用Lysis buffer (Tris pH 7.4,50mmol/L,NaCl 120mmol/L,Triton-X100 1%,蛋白酶抑制剂混合物临用前加入)提取蛋白,采用免疫共沉淀技术(使用Myc一抗进行免疫沉淀,FLAG一抗进行检测；反过来用FLAG一抗进行免疫沉淀,Myc一抗进行检测)验证μ和δ受体之间的相互作用。免疫共沉淀结果显示使用Myc一抗(针对μ阿片受体)可以沉淀δ阿片受体,而使用FLAG一抗(针对δ阿片受体)可以沉淀μ阿片受体,Western blot的结果显示被沉淀下来的μ或是δ阿片受体均存在三种形式：同源二聚体形式、糖基化单体及未糖基化的单体形式。免疫共沉淀出现μ、δ的二聚体形式,提示μ和δ阿片受体的相互作用有部分发生在多聚体水平,即μ、δ各自以二聚体的形式再参与μ和δ受体的相互作用,而这种异源多聚体的μ、δ之间相互作用力比较弱,不能对抗SDS和高温的处理,经过SDS和100℃高温处理后,分离为各自的同源二聚体等形式存在。免疫共沉淀的结果出现未糖基化的受体单体杂交带,这也提示μ和δ受体间发生相互作用可能不一定需要受体糖基化。将μ-Myc-pIRES2-EGFP和δ-FLAG-pIRES2-EGFP分别转染HEK293细胞,单独表达后提取细胞裂解液,混合孵育后,未见到免疫共沉淀现象发生,这提示免疫共沉淀实验结果是特异的,μ和δ阿片受体的相互作用需要两者共表达为前提,这提示μ和δ阿片受体发生相互作用需要细胞内的加工环境或是需要其他第三者参与,μ、δ阿片受体单独表达后再混合并不能产生免疫共沉淀。
4、μ与δ阿片受体相互作用的具体domain的pull down研究：分析δ阿片受体的分子结构拓扑图,使用原核表达质粒pGEX2TK,设计相应的引物,PCR扩增相应domain,与pGEX2TK质粒通过EcoR I和BamH I酶切位点进行链接,分别构建了δ受体的第二胞内环Ⅱ、第三胞内环Ⅲ、C末端、第一跨膜区TM1、第三跨膜区TM3、第五跨膜区TM5、第六跨膜区TM6及带FLAG标签的δ全长的GST融合蛋白的表达质粒pGEX2TK,相应的质粒分别为δⅡ-pGEX2TK、δⅢ-pGEX2TK、δC-pGEX2TK、δTM1-pGEX2TK、δTM3-pGEX2TKδTM5-pGEX2TK、δTM6-pGEX2TK、δ-FLAG-pGEX2TK。δⅡ-pGEX2TK、δⅢ-pGEX2TK、δC-pGEX2TK在BL21(DE3)菌株经IPTG诱导后实现了有效表达,采用Glutathione Sepharose 4B的batch purification方法,纯化相应的GST融合蛋白,然后与带Myc标签的μ受体蛋白孵育,离心洗涤沉淀后,进行蛋白印记,Myc一抗检测。第二胞内环Ⅱ、第三胞内环Ⅲ、C末端表达成功,pull down实验表明它们不在μ和δ受体相互作用中发挥重要作用。
δTM1-pGEX2TK、δTM3-pGEX2TK、δTM5-pGEX2TK、δTM6-pGEX2TK、δ-FLAG-pGEX2TK分别采用了不同的IPTG诱导浓度(0.05、0.2、0.5、1、2mmol/L)、诱导温度(37、30、25℃)、诱导时间(4、7、16h)及不同的菌株(BL21(DE3)、BL21(DE3)plysS、Rosetta gami (DE3)、Rosetta (DE3))进行了大量表达诱导尝试。收集的细菌裂解液经SDS-PAGE电泳后,考马斯亮蓝染色鉴定,结果显示第三跨膜区和第五跨膜区获得了低浓度的表达,其他均未见到目的条带出现。第三跨膜区TM3、第五跨膜区TM5的GST-pull down结果也是阴性,显示它们也不是δ受体和μ受体的相互作用的主要domain。将TM1和δ-FLAG克隆到pET41a质粒,在BL21(DE3)、BL21(DE3)plysS、Rosetta gami(DE3)、Rosetta(DE3)菌株中也未见目的蛋白的表达。这一结果提示膜蛋白和疏水性的跨膜区在原核表达系统中表达存在相当大的困难。
结论
1、通过巢式PCR等技术成功构建了经典阿片受体μ、δ、κ的真核表达质粒,并在HEK293细胞中进行了表达鉴定。三种经典阿片受体在体外培养的HEK293中,主要以同源二聚体的形式存在。
2、膜蛋白和疏水性的跨膜区在原核表达系统中表达存在相当大的困难。
3、免疫共沉淀的结果提示μ和δ阿片受体的相互作用是特异的,需要以共表达为前提。两者的相互作用可以发生于同源二聚体、糖基化单体及未糖基化的单体的水平。
4、δ阿片受体的第二胞内环、第三胞内环及C末端可能不是μ和δ阿片受体相互作用的主要domain。
5、免疫共沉淀阳性而pull down阴性,提示μ和δ受体间的连接不排除通过其他蛋白介导的可能,即μ和δ之间的连接可能需要第三者介入,而不是普遍认为μ和δ阿片受体之间的直接作用。
本实验创新之处
1、通过巢式PCR扩增等,构建了μ-Myc-pIRES2-EGFP、δ-FLAG-pIRES2-EGFP、κ-HA-pIRES2-EGFP,pIRES2-EGFP表达质粒的使用及相应抗原表位标签的引入,可以方便转染效率观察和目的蛋白表达鉴定。
2、免疫共沉淀结果提示μ和δ阿片受体的相互作用可以发生在多聚体的水平,而不单是异源二聚体水平。
3、GST pull down结果提示μ和δ受体间的连接不排除通过其他蛋白介导的可能,即μ和δ之间的连接可能需要第三者介入,而不是普遍认为μ和δ阿片受体之间的直接作用。
BACKGROUND & OBJECTIVE
The illicit use of drugs such as morphine, heroin has become a social nuisance. It is difficult to cure drug addiction because of severe withdrawal symptoms and high rate of reuse.Drug addiction is a chronic relapsing encephalopathy. The mechanism of drug addiction is complex and involves many brain areas of the central nervous system.
Opioid receptors have distinct pharmacological profiles and discrete but overlapping distributions in brain. The endogenous opioid peptide receptor systems mediate important physiological functions related to pain perception, locomotion, motivation, reward, autonomic function, immunomodulation and neuroendocrine function. Pharmacological and molecular cloning studies have identified three opioid receptor types,μ(MOR),δ(DOR)and k(KOR) that mediate these diverse effects.
In recent years, with the development of neuroanatomy, neuropharmacology and neural mechanisms of drug dependence, more and more facts show that nucleus accumbens plays a key role in drug dependence. Many animal experiments and anatomical studies have shown that nucleus accumbens is closely related with drug addiction. Bilateral destruction of rat nucleus accumbens can inhibit the establishment of heroin self-administration, eliminate drug-seeking behavior and recrudescence in rats addicted.
We speculate that heroin addiction has a significant bearing on protein-protein interaction ofμandδopioid receptors in nucleus accumbens. The inference is based on several facts. Firstly, a lack ofμorδreceptors abolishes the analgesic effect of morphine, as well as place-preference activity, hypolocomotion and physical dependence. Mice withμorδreceptor gene knock out do not develop analgesic tolerance to morphine. Secondly, many experiments have proved that there is a physical interaction betweenμandδreceptors and thus have the potential to create novel signaling units. Thirdly, theδopioid receptor andμopioid receptor are abundantly distributed in the dorsal horn of the spinal cord. Simultaneous activation of each receptor by selective opiate agonists has been shown to result in synergistic analgesic effects. Immunoperoxidase and immunogold-silver labeling methods showed 8 opioid receptors are often in cooperation withμopioid receptors in striatal patches. Fourthly, Schmidt BL showed thatμandδreceptors contributed to capsaicin-induced antinociception in nucleus accumbens; selective activation of individual receptor subtypes was insufficient, but coactivation ofμandδopioid receptors induced antinociception. Fifthly, stereotactic destruction of nucleus accumbens in experimental animals and patients with heroin addiction have achieved good results.
Shao-Ping Ji synthesized the interfering peptide Tat-3L4F, which is able to disrupt PTEN coupling with 5-HT2cR. Systemic application of Tat-3L4F suppressed the increased firing rate of VTA dopaminergic neurons induced by Δ9-tetrahydrocannabinol (THC), the psychoactive ingredient of marijuana. Tat-3L4F blocks conditioned place preference of THC or nicotine, and does not produce side effects such as anxiogenic effects, penile erection, hypophagia and motor functional suppression. These results suggested a potential strategy for treating drug addiction with the Tat-3L4F peptide.
If we can find the specific peptide sequence for interaction betweenμandδopioid receptors, and thus using the peptide as interfering peptide, which maybe be able to disruptμreceptor coupling withδreceptor, may suppress behavioral responses induced by drugs of abuse such as heroin.
METHODS & RESULTS
The total RNA was isolated from nucleus accubems using Trizol according to its instructions. Full length cDNAs of ratμ,δ,κopioid receptor were amplified from the rat brain tissue through reverse transcription and nested PCR, respectively. The full length cDNAs ofμ,δ,κopioid receptor were ligated with pMD20 T vector through A-T ligation, respectively. There was a single site mutation inμ,δ,κopioid receptor, respectively. Site mutation ofμ,δ,κopioid receptor was corrected using Stratagene mutation kit. Full length DNA of ratμopioid receptor tagged with a 10-residue Myc epitope in carboxyl terminal was amplified fromμ-pMD20 T vector by PCR with high fidelity DNA polymerase. Full length DNA of rat 8 opioid receptor tagged with a 8-residue FLAG epitope in carboxyl terminal was amplified from 8-pMD20 T vector by PCR with high fidelity DNA polymerase. Full length DNA of ratκopioid receptor tagged with a 9-residue HA epitope in carboxyl terminal was amplified from K-pMD20 T vector by PCR with high fidelity DNA polymerase.
The PCR amplified DNAμ-Myc was purified by PCR clean up kit and attached a 3'dA nucleotide overhang and ligated with pMD20 T vector to formμ-Myc-pMD20 T vector. The PCR amplified DNAδ-FLAG was purified by PCR clean up kit and attached a 3'dA nucleotide overhang and ligated with pMD20 T vector to formδ-FLAG-pMD20 T vector. The PCR amplified DNAκ-HA was purified by PCR clean up kit and attached a 3'dA nucleotide overhang and ligated with pMD20 T vector to formκ-HA-pMD20 T vector.
δ-FLAG-pMD20 T vector and pIRES2-EGFP plasmid were digested with EcoRl and BamHI, respectively. DNA fragmentδ-FLAG and pIRES2-EGFP digested by EcoRI and BamHI was isolated using agarose gel DNA recycling kit, respectively. Then they were ligated to formδ-FLAG-pIRES2-EGFP plasmid with T4 DNA ligase. The DNA sequence ofδopioid receptor with FLAG epitope was inserted into pIRES2-EGFP and confirmed by sequencing on both strands and restriction enzyme digestion. At the same time,μ-Myc-pMD20 T vector and pIRES2-EGFP plasmid was digested with EcoRI and NheⅠ, respectively. By the same method of restriction enzyme digestion, isolation using agarose gel DNA recycling kit and ligation using T4 DNA ligase,μ-Myc-pIRES2-EGFP andκ-HA-pIRES2-EGFP were constructed and confirmed by sequencing on both strands and restriction enzyme digestion.
μ-Myc-pIRES2-EGFP,δ-FLAG-pIRES2-EGFP,κ-HA-pIRES2-EGFP recombinant plasmid was transfected into HEK293 using Lipofectamine2000 according to its instructions, respectively. The expression ofμ-Myc-pIRES2-EGFP,δ-FLAG-pIRES2-EGFP,κ-HA-pIRES2-EGFP in HEK293 was examined through immunofluorescence and western blot. The EGFP expression was observed through fluorescence microscopy using blue light, which showed the transfection efficiency of recombinant eukaryotic plasmid in HEK293 was high. Expression level ofδgene with FLAG epitope was detected in HEK293 afterδ-FLAG-pIRES2-EGFP plasmid transfection. We examined lysates from cells expressingδopioid receptors tagged with a FLAG epitope using FLAG primary antibody and corresponding secondary antibody tagged with horseradish peroxidase. The result was showed through enhanced chemiluminescence. We found that mostδreceptors exist as dimers of relative molecular mass (Mr) 140,000. The dimers are stable in 10% SDS buffer and 100℃water bath. At the same time, there were also being monomer regardless of glycosylation. Homotetramers and higher molecular mass oligomers were not observed. The result ofμ-Myc-pIRES2-EGFP andκ-HA-pIRES2-EGFP in HEK293 were similar to that ofδ-FLAG-pIRES2-EGFP in HEK293 using western blot. These results showed opioid receptors exist as homodimers in HEK293 using recombinant eukaryotic plasmid and the homodimers are stable in SDS detergents and 100℃water bath.
We examined the ability ofμopioid receptors to heterodimerize withδopioid receptors by coexpressing Myc-taggedμreceptors with Flag-taggedδreceptors. The interaction betweenμandδopioid receptors was examined by immunoprecipitation experiments using HEK293 transfectedμ-Myc-pIRES2-EGFP andδ-FLAG-pIRES2-EGFP recombinant plasmid. Collecting the cells 48h after transient expression, immunoprecipitation and western blotting were carried out with lysates of whole cells using lysis buffer(Tris pH 7.4,50mmol/L, NaCl 120mmol/L, Triton-X100 1%, protease inhibitor cocktail(10 pg/ml leupeptin,10 pg/ml aprotinin,10 mM EDTA,1 mM EGTA,10 pg/ml bacitracin,1 mM pepstatin A and 0.5 mM phenylmethylsulphonyl fluoride)). We found that an antibody to the Myc-taggedμreceptor can co-precipitate Flag-taggedδreceptors from cells expressing both Myc-taggedμopioid receptor and Flag-taggedδopioid receptors, vice versa. The receptors could be co-precipitated only from cells cotransfectedμ-Myc-pIRES2-EGFP andδ-FLAG-pIRES2-EGFP, and not from a mixture of cells individually expressing the receptors. These results indicate that the heterodimerization betweenμandδopioid receptors is specific and selective. Coexppression ofμandδopioid receptor is necessary forμandδreceptor heterodimization.
According to molecular structure topology map of ratδopioid receptor, we constructed prokaryotic expression plasmid of some domains ofδreceptor with pGEX2TK. These domains are the second intercellular loop, the third intercellular loop, the carboxyl terminal, the first transmembrane, the third transmenbrane, the fifth transmembrane, the sixth transmembrane, the full length ofδreceptor tagged with FLAG. These constructed prokaryotic expression plasmids are as follows:δⅡ-pGEX2TK,δⅢ-pGEX2TK,8C-pGEX2TK,δTM1-pGEX2TK,δTM3-pGEX2TK,δTM5-pGEX2TK,δTM6-pGEX2TK,δ-FLAG-pGEX2TK. ForδⅡ-pGEX2TK,δⅢ-pGEX2TK,δC-pGEX2TK, after transforming the recombinant prokaryotic expression plasmid into expression bacteria BL21 (DE3), we used IPTG to introduce the expression of GST fusion protein. Coomassie brilliant blue staining showed theδⅡ-pGEX2TK,δⅢ-pGEX2TK,δC-pGEX2TK successfully expressed GST fusion protein of target domain in BL21 (DE3). According to batch purification protocol of Glutathione Sepharose 4B, the GST-δⅡ,GST-δⅢ,GST-δC were collected and incubated with cell lysate form HEK293 transfected withμ-Myc-pIRES2-EGFP plasmid. The precipitation was analysed by western blot with Myc primary antibody. The results showed that the second intercellular loop, the third intercellular loop, the carboxyl terminal don't play an important role in interaction betweenμandδopioid receptor. Expression ofδTM1-pGEX2TK,δTM3-pGEX2TK,δTM5-pGEX2TK,δTM6-pGEX2TK,δ-FLAG-pGEX2TK failed in BL21 (DE3). We tried different concentration of IPTG (0.05,0.2,0.5,1.2mmol/L) different temperature (37,30, 25℃) different time (4,7,16h) different expression bacteria(BL21(DE3)plysS, Rosetta gami (DE3),Rosetta (DE3)),but still couldn't get the GST fusion protein. These results showed that it is difficult to express the membrane protein and hydrophobic transmembrane in prokaryotic expression bacteria.
Conclusions
1.μ-Myc-pIRES2-EGFP,δ-FLAG-pIRES2-EGFP,κ-HA-pIRES2-EGFP eukaryotic recombinant plasmid were constructed correctly and expressed in high level in HEK293 cells. These opioid receptors are mainly in the form of homodimer when expressed in HEK293 cells.
2. It is difficult to express the membrane protein and hydrophobic transmembrane in prokaryotic expression system.
3. Heterodimerization betweenμandδopioid receptor is specific and selective. Coexppression ofμandδopioid receptor is necessary forμandδreceptor heterodimization.
4. The second intercellular loop, the third intercellular loop and the carboxyl terminal ofδopioid receptor don't play an important role in interaction betweenμandδopioid receptor.
5. Our data indicated that the interaction betweenμandδopioid receptor may require other protein participation.
吗啡、海洛因等毒品的非法使用产生的毒品成瘾已经成为席卷全球的社会公害,成瘾性强,戒断症状严重,复吸率高,根治困难。毒品成瘾是一种慢性复发性脑病,毒品成瘾机理十分复杂,涉及中枢神经系统的许多脑区。随着药理学、神经解剖学和神经生物学等学科的发展,目前发现脑内有一个对毒品的使用产生奖赏效应的系统,涉及多巴胺(DA)、阿片肽、γ-氨基丁酸(GABA)能系统三个主要的调控毒品成瘾的神经机制。吗啡、海洛因等毒品使用后,可以直接作用于阿片肽神经元,使其释放的内啡肽增加,内啡肽再作用于多巴胺神经元上的阿片受体,促发多巴胺神经元的活性而发挥药物奖赏效应；阿片肽神经环路与多巴胺神经环路还存在着大量的交互作用,共同对毒品的成瘾起增强作用；也有人认为吗啡等毒品可以直接作用于中脑-边缘系统多巴胺神经通路和中脑-边缘系统-皮质系统多巴胺神经通路中的多巴胺能神经元,导致多巴胺释放增加,多巴胺通过作用于脑内多巴胺D1、D2受体而完成奖赏效应,产生毒品成瘾作用；另外吗啡还可以通过GABA能神经元上的μ阿片受体的介导,抑制GABA能神经元,解除GABA能神经元对VTA内的多巴胺神经元的抑制,使得多巴胺神经元的活性增加,从而产生吗啡的间接药物强化效应。
近年来,随着对药物依赖形成的神经解剖学、神经药理学及神经生物学机制的广泛深入研究,发现伏隔核在参与药物依赖的众多神经核(团)中起着关键作用。较多的动物实验及解剖学研究表明,伏隔核与药物成瘾关系密切,毁损双侧伏隔核会抑制大鼠海洛因自我给药行为的建立,能够消除成瘾大鼠的觅药行为,可以抑制吗啡诱导的复燃。
我们推测海洛因成瘾与伏隔核中μ和δ阿片受体间的蛋白-蛋白相互作用有重要关系。该推论的依据是：(1)基因敲除μ或δ受体,尤其是μ基因敲除的小鼠对于海洛因不能引起条件性位置偏爱效应、运动增多等表现,鼠失去海洛因奖赏效应,不出现成瘾性；(2)在体外培养的细胞中,已有多种方法证明共表达的μ和δ受体形成受体复合物,从而交互影响各自的配体结合、受体向胞内运动和细胞内信号传导；(3)μ和δ受体不仅共存于相同的感觉神经元,而且还共存于伏隔核内相同神经元；(4)Schmidt BL等在大鼠活体实验中,进行了伏隔核内选择性受体激动剂注射,发现DAMGO联合DPDPE(分别是μ、δ受体激动剂)注射,具有明显的镇痛效应,而单一注射则效应明显降低。这些结果提示μ和δ受体激动剂的镇痛效应需要μ和δ受体的共同活化,支持伏隔核内μ、δ受体以复合物形式存在的观点。(5)立体定向伏隔核毁损对实验动物和海洛因成瘾患者均取得了较好的戒毒效果。
Ji SP等首次证明特异性干扰肽能通过打断抑癌酶PTEN和5-HT2C受体间的蛋白-蛋白连接来阻断大麻奖赏效应。大鼠全身注射或向VTA直接注射Tat-3L4F干扰肽(Tat为源于HIV病毒分子启动子的11个氨基酸肽,3L4F为抑癌酶PTEN的第三胞内环的某一区段,Tat肽可携带3L4F穿过血脑屏障)能够显著抑制由大麻诱导的VTA多巴胺能神经元激活,可阻断由大麻或尼古丁诱导的条件性位置偏爱效应,并未产生常用阻断剂Ro600175容易引起的产生焦虑、阴茎勃起、进食减少和运动功能抑制等副作用。
如果能找到μ和δ受体之间相互作用的特异性多肽序列,可能也能有效的打断μ和δ受体之间的相互作用,从而对海洛因成瘾起到阻断作用。
使用药物脱毒后心理依赖不易消除,患者屡次复吸,海洛因脱毒后一个月内的复吸率达80%-85%,半年内的复吸率达95%,半年以上的复吸率高达97%,很多患者陷入“戒断-复吸-戒断”的恶性循环当中。寻找更有效的毒品成瘾治疗方法具有重要的意义。本课题研究就是基于这种需要,结合我们的推论,构建阿片受体μ、δ、κ的真核表达质粒,表达鉴定后,进行了免疫共沉淀研究,并采用GST pull down技术进行了μ和δ受体相互作用的domain区的寻找。
实验方法和结果
1、构建了大鼠的μ、δ、κ阿片受体的真核表达载体：本研究采用反转录巢式PCR方法,提取伏隔核为主的脑组织总RNA,随机引物逆转录为cDNA,通过外侧及内侧引物两次扩增,从大鼠脑组织总RNA中扩增出μ、δ、κ阿片受体cDNA基因,通过AT克隆,将目的基因构建到pMD20-T Vector中,测序鉴定均存在单点突变,使用Stratagene的定点突变试剂盒纠正点突变；然后再设计相应的引物,使用高保真酶,通过PCR扩增在μ、δ、κ阿片受体基因的C端分别引入蛋白抗原表位标签Myc、FLAG、HA,获得目的基因μMyc、δFLAG、κHA,AT克隆到pMD20-T Vector中,测序鉴定正确后,通过双酶切含目的基因(μMyc、δFLAG、κHA)的T载体及目的质粒pIRES2-EGFP,割胶回收,酶切片段连接,转化大肠杆菌,菌落PCR及酶切鉴定,最后测序确认,成功构建了如下三种阿片受体真核表达载体：μ-Myc-pIRES2-EGFP、δ-FLAG-pIRES2-EGFP、κ-HA-pIRES2-EGFP。
2、μ-Myc-pIRES2-EGFP、δ-FLAG-pIRES2-EGFP、κ-HA-pIRES2-EGFP在HEK293中的表达及鉴定：将质粒μ-Myc-pIRES2-EGFP、δ-FLAG-pIRES2-EGFP、κ-HA-pIRES2-EGFP转染到HEK293细胞中,荧光显微镜下观察到表达绿色荧光蛋白的HEK293细胞比例高,提示转染效率较高。使用细胞免疫荧光和蛋白印迹也分别证明转染了相应质粒的HEK293细胞中,目的基因μ、δ、κ获得了高效表达。其中μ和δ阿片受体分别使用了标签抗体Myc一抗、FLAG一抗和μ一抗、δ一抗进行鉴定,结果显示构建的质粒在HEK293中表达的目的蛋白μ-Myc、δ-FLAG很好的保持了抗原抗体的结合特性。Western blot分析结果提示三种阿片受体(μ、δ、κ)主要以糖基化的二聚体(分子量约为140kD)形式存在,同时也存在少量的糖基化的单体蛋白(分子量约为85 kD)和存在少量未糖基化的单体蛋白(分子量约为45 kD),而且形成的同源二聚体的作用力较强,可以有效的抵抗SDS的作用,在SDS存在下和100℃高温的处理下仍能保持二聚体形态。
3、μ和δ阿片受体的免疫共沉淀研究：我们在体外培养的HEK293细胞中,采用免疫共沉淀技术对μ和δ受体的相互作用进行了研究。将δ-FLAG-pIRES2-EGFP、μ-Myc-pIRES2-EGFP共转染到HEK293细胞,用Lysis buffer (Tris pH 7.4,50mmol/L,NaCl 120mmol/L,Triton-X100 1%,蛋白酶抑制剂混合物临用前加入)提取蛋白,采用免疫共沉淀技术(使用Myc一抗进行免疫沉淀,FLAG一抗进行检测；反过来用FLAG一抗进行免疫沉淀,Myc一抗进行检测)验证μ和δ受体之间的相互作用。免疫共沉淀结果显示使用Myc一抗(针对μ阿片受体)可以沉淀δ阿片受体,而使用FLAG一抗(针对δ阿片受体)可以沉淀μ阿片受体,Western blot的结果显示被沉淀下来的μ或是δ阿片受体均存在三种形式：同源二聚体形式、糖基化单体及未糖基化的单体形式。免疫共沉淀出现μ、δ的二聚体形式,提示μ和δ阿片受体的相互作用有部分发生在多聚体水平,即μ、δ各自以二聚体的形式再参与μ和δ受体的相互作用,而这种异源多聚体的μ、δ之间相互作用力比较弱,不能对抗SDS和高温的处理,经过SDS和100℃高温处理后,分离为各自的同源二聚体等形式存在。免疫共沉淀的结果出现未糖基化的受体单体杂交带,这也提示μ和δ受体间发生相互作用可能不一定需要受体糖基化。将μ-Myc-pIRES2-EGFP和δ-FLAG-pIRES2-EGFP分别转染HEK293细胞,单独表达后提取细胞裂解液,混合孵育后,未见到免疫共沉淀现象发生,这提示免疫共沉淀实验结果是特异的,μ和δ阿片受体的相互作用需要两者共表达为前提,这提示μ和δ阿片受体发生相互作用需要细胞内的加工环境或是需要其他第三者参与,μ、δ阿片受体单独表达后再混合并不能产生免疫共沉淀。
4、μ与δ阿片受体相互作用的具体domain的pull down研究：分析δ阿片受体的分子结构拓扑图,使用原核表达质粒pGEX2TK,设计相应的引物,PCR扩增相应domain,与pGEX2TK质粒通过EcoR I和BamH I酶切位点进行链接,分别构建了δ受体的第二胞内环Ⅱ、第三胞内环Ⅲ、C末端、第一跨膜区TM1、第三跨膜区TM3、第五跨膜区TM5、第六跨膜区TM6及带FLAG标签的δ全长的GST融合蛋白的表达质粒pGEX2TK,相应的质粒分别为δⅡ-pGEX2TK、δⅢ-pGEX2TK、δC-pGEX2TK、δTM1-pGEX2TK、δTM3-pGEX2TKδTM5-pGEX2TK、δTM6-pGEX2TK、δ-FLAG-pGEX2TK。δⅡ-pGEX2TK、δⅢ-pGEX2TK、δC-pGEX2TK在BL21(DE3)菌株经IPTG诱导后实现了有效表达,采用Glutathione Sepharose 4B的batch purification方法,纯化相应的GST融合蛋白,然后与带Myc标签的μ受体蛋白孵育,离心洗涤沉淀后,进行蛋白印记,Myc一抗检测。第二胞内环Ⅱ、第三胞内环Ⅲ、C末端表达成功,pull down实验表明它们不在μ和δ受体相互作用中发挥重要作用。
δTM1-pGEX2TK、δTM3-pGEX2TK、δTM5-pGEX2TK、δTM6-pGEX2TK、δ-FLAG-pGEX2TK分别采用了不同的IPTG诱导浓度(0.05、0.2、0.5、1、2mmol/L)、诱导温度(37、30、25℃)、诱导时间(4、7、16h)及不同的菌株(BL21(DE3)、BL21(DE3)plysS、Rosetta gami (DE3)、Rosetta (DE3))进行了大量表达诱导尝试。收集的细菌裂解液经SDS-PAGE电泳后,考马斯亮蓝染色鉴定,结果显示第三跨膜区和第五跨膜区获得了低浓度的表达,其他均未见到目的条带出现。第三跨膜区TM3、第五跨膜区TM5的GST-pull down结果也是阴性,显示它们也不是δ受体和μ受体的相互作用的主要domain。将TM1和δ-FLAG克隆到pET41a质粒,在BL21(DE3)、BL21(DE3)plysS、Rosetta gami(DE3)、Rosetta(DE3)菌株中也未见目的蛋白的表达。这一结果提示膜蛋白和疏水性的跨膜区在原核表达系统中表达存在相当大的困难。
结论
1、通过巢式PCR等技术成功构建了经典阿片受体μ、δ、κ的真核表达质粒,并在HEK293细胞中进行了表达鉴定。三种经典阿片受体在体外培养的HEK293中,主要以同源二聚体的形式存在。
2、膜蛋白和疏水性的跨膜区在原核表达系统中表达存在相当大的困难。
3、免疫共沉淀的结果提示μ和δ阿片受体的相互作用是特异的,需要以共表达为前提。两者的相互作用可以发生于同源二聚体、糖基化单体及未糖基化的单体的水平。
4、δ阿片受体的第二胞内环、第三胞内环及C末端可能不是μ和δ阿片受体相互作用的主要domain。
5、免疫共沉淀阳性而pull down阴性,提示μ和δ受体间的连接不排除通过其他蛋白介导的可能,即μ和δ之间的连接可能需要第三者介入,而不是普遍认为μ和δ阿片受体之间的直接作用。
本实验创新之处
1、通过巢式PCR扩增等,构建了μ-Myc-pIRES2-EGFP、δ-FLAG-pIRES2-EGFP、κ-HA-pIRES2-EGFP,pIRES2-EGFP表达质粒的使用及相应抗原表位标签的引入,可以方便转染效率观察和目的蛋白表达鉴定。
2、免疫共沉淀结果提示μ和δ阿片受体的相互作用可以发生在多聚体的水平,而不单是异源二聚体水平。
3、GST pull down结果提示μ和δ受体间的连接不排除通过其他蛋白介导的可能,即μ和δ之间的连接可能需要第三者介入,而不是普遍认为μ和δ阿片受体之间的直接作用。
BACKGROUND & OBJECTIVE
The illicit use of drugs such as morphine, heroin has become a social nuisance. It is difficult to cure drug addiction because of severe withdrawal symptoms and high rate of reuse.Drug addiction is a chronic relapsing encephalopathy. The mechanism of drug addiction is complex and involves many brain areas of the central nervous system.
Opioid receptors have distinct pharmacological profiles and discrete but overlapping distributions in brain. The endogenous opioid peptide receptor systems mediate important physiological functions related to pain perception, locomotion, motivation, reward, autonomic function, immunomodulation and neuroendocrine function. Pharmacological and molecular cloning studies have identified three opioid receptor types,μ(MOR),δ(DOR)and k(KOR) that mediate these diverse effects.
In recent years, with the development of neuroanatomy, neuropharmacology and neural mechanisms of drug dependence, more and more facts show that nucleus accumbens plays a key role in drug dependence. Many animal experiments and anatomical studies have shown that nucleus accumbens is closely related with drug addiction. Bilateral destruction of rat nucleus accumbens can inhibit the establishment of heroin self-administration, eliminate drug-seeking behavior and recrudescence in rats addicted.
We speculate that heroin addiction has a significant bearing on protein-protein interaction ofμandδopioid receptors in nucleus accumbens. The inference is based on several facts. Firstly, a lack ofμorδreceptors abolishes the analgesic effect of morphine, as well as place-preference activity, hypolocomotion and physical dependence. Mice withμorδreceptor gene knock out do not develop analgesic tolerance to morphine. Secondly, many experiments have proved that there is a physical interaction betweenμandδreceptors and thus have the potential to create novel signaling units. Thirdly, theδopioid receptor andμopioid receptor are abundantly distributed in the dorsal horn of the spinal cord. Simultaneous activation of each receptor by selective opiate agonists has been shown to result in synergistic analgesic effects. Immunoperoxidase and immunogold-silver labeling methods showed 8 opioid receptors are often in cooperation withμopioid receptors in striatal patches. Fourthly, Schmidt BL showed thatμandδreceptors contributed to capsaicin-induced antinociception in nucleus accumbens; selective activation of individual receptor subtypes was insufficient, but coactivation ofμandδopioid receptors induced antinociception. Fifthly, stereotactic destruction of nucleus accumbens in experimental animals and patients with heroin addiction have achieved good results.
Shao-Ping Ji synthesized the interfering peptide Tat-3L4F, which is able to disrupt PTEN coupling with 5-HT2cR. Systemic application of Tat-3L4F suppressed the increased firing rate of VTA dopaminergic neurons induced by Δ9-tetrahydrocannabinol (THC), the psychoactive ingredient of marijuana. Tat-3L4F blocks conditioned place preference of THC or nicotine, and does not produce side effects such as anxiogenic effects, penile erection, hypophagia and motor functional suppression. These results suggested a potential strategy for treating drug addiction with the Tat-3L4F peptide.
If we can find the specific peptide sequence for interaction betweenμandδopioid receptors, and thus using the peptide as interfering peptide, which maybe be able to disruptμreceptor coupling withδreceptor, may suppress behavioral responses induced by drugs of abuse such as heroin.
METHODS & RESULTS
The total RNA was isolated from nucleus accubems using Trizol according to its instructions. Full length cDNAs of ratμ,δ,κopioid receptor were amplified from the rat brain tissue through reverse transcription and nested PCR, respectively. The full length cDNAs ofμ,δ,κopioid receptor were ligated with pMD20 T vector through A-T ligation, respectively. There was a single site mutation inμ,δ,κopioid receptor, respectively. Site mutation ofμ,δ,κopioid receptor was corrected using Stratagene mutation kit. Full length DNA of ratμopioid receptor tagged with a 10-residue Myc epitope in carboxyl terminal was amplified fromμ-pMD20 T vector by PCR with high fidelity DNA polymerase. Full length DNA of rat 8 opioid receptor tagged with a 8-residue FLAG epitope in carboxyl terminal was amplified from 8-pMD20 T vector by PCR with high fidelity DNA polymerase. Full length DNA of ratκopioid receptor tagged with a 9-residue HA epitope in carboxyl terminal was amplified from K-pMD20 T vector by PCR with high fidelity DNA polymerase.
The PCR amplified DNAμ-Myc was purified by PCR clean up kit and attached a 3'dA nucleotide overhang and ligated with pMD20 T vector to formμ-Myc-pMD20 T vector. The PCR amplified DNAδ-FLAG was purified by PCR clean up kit and attached a 3'dA nucleotide overhang and ligated with pMD20 T vector to formδ-FLAG-pMD20 T vector. The PCR amplified DNAκ-HA was purified by PCR clean up kit and attached a 3'dA nucleotide overhang and ligated with pMD20 T vector to formκ-HA-pMD20 T vector.
δ-FLAG-pMD20 T vector and pIRES2-EGFP plasmid were digested with EcoRl and BamHI, respectively. DNA fragmentδ-FLAG and pIRES2-EGFP digested by EcoRI and BamHI was isolated using agarose gel DNA recycling kit, respectively. Then they were ligated to formδ-FLAG-pIRES2-EGFP plasmid with T4 DNA ligase. The DNA sequence ofδopioid receptor with FLAG epitope was inserted into pIRES2-EGFP and confirmed by sequencing on both strands and restriction enzyme digestion. At the same time,μ-Myc-pMD20 T vector and pIRES2-EGFP plasmid was digested with EcoRI and NheⅠ, respectively. By the same method of restriction enzyme digestion, isolation using agarose gel DNA recycling kit and ligation using T4 DNA ligase,μ-Myc-pIRES2-EGFP andκ-HA-pIRES2-EGFP were constructed and confirmed by sequencing on both strands and restriction enzyme digestion.
μ-Myc-pIRES2-EGFP,δ-FLAG-pIRES2-EGFP,κ-HA-pIRES2-EGFP recombinant plasmid was transfected into HEK293 using Lipofectamine2000 according to its instructions, respectively. The expression ofμ-Myc-pIRES2-EGFP,δ-FLAG-pIRES2-EGFP,κ-HA-pIRES2-EGFP in HEK293 was examined through immunofluorescence and western blot. The EGFP expression was observed through fluorescence microscopy using blue light, which showed the transfection efficiency of recombinant eukaryotic plasmid in HEK293 was high. Expression level ofδgene with FLAG epitope was detected in HEK293 afterδ-FLAG-pIRES2-EGFP plasmid transfection. We examined lysates from cells expressingδopioid receptors tagged with a FLAG epitope using FLAG primary antibody and corresponding secondary antibody tagged with horseradish peroxidase. The result was showed through enhanced chemiluminescence. We found that mostδreceptors exist as dimers of relative molecular mass (Mr) 140,000. The dimers are stable in 10% SDS buffer and 100℃water bath. At the same time, there were also being monomer regardless of glycosylation. Homotetramers and higher molecular mass oligomers were not observed. The result ofμ-Myc-pIRES2-EGFP andκ-HA-pIRES2-EGFP in HEK293 were similar to that ofδ-FLAG-pIRES2-EGFP in HEK293 using western blot. These results showed opioid receptors exist as homodimers in HEK293 using recombinant eukaryotic plasmid and the homodimers are stable in SDS detergents and 100℃water bath.
We examined the ability ofμopioid receptors to heterodimerize withδopioid receptors by coexpressing Myc-taggedμreceptors with Flag-taggedδreceptors. The interaction betweenμandδopioid receptors was examined by immunoprecipitation experiments using HEK293 transfectedμ-Myc-pIRES2-EGFP andδ-FLAG-pIRES2-EGFP recombinant plasmid. Collecting the cells 48h after transient expression, immunoprecipitation and western blotting were carried out with lysates of whole cells using lysis buffer(Tris pH 7.4,50mmol/L, NaCl 120mmol/L, Triton-X100 1%, protease inhibitor cocktail(10 pg/ml leupeptin,10 pg/ml aprotinin,10 mM EDTA,1 mM EGTA,10 pg/ml bacitracin,1 mM pepstatin A and 0.5 mM phenylmethylsulphonyl fluoride)). We found that an antibody to the Myc-taggedμreceptor can co-precipitate Flag-taggedδreceptors from cells expressing both Myc-taggedμopioid receptor and Flag-taggedδopioid receptors, vice versa. The receptors could be co-precipitated only from cells cotransfectedμ-Myc-pIRES2-EGFP andδ-FLAG-pIRES2-EGFP, and not from a mixture of cells individually expressing the receptors. These results indicate that the heterodimerization betweenμandδopioid receptors is specific and selective. Coexppression ofμandδopioid receptor is necessary forμandδreceptor heterodimization.
According to molecular structure topology map of ratδopioid receptor, we constructed prokaryotic expression plasmid of some domains ofδreceptor with pGEX2TK. These domains are the second intercellular loop, the third intercellular loop, the carboxyl terminal, the first transmembrane, the third transmenbrane, the fifth transmembrane, the sixth transmembrane, the full length ofδreceptor tagged with FLAG. These constructed prokaryotic expression plasmids are as follows:δⅡ-pGEX2TK,δⅢ-pGEX2TK,8C-pGEX2TK,δTM1-pGEX2TK,δTM3-pGEX2TK,δTM5-pGEX2TK,δTM6-pGEX2TK,δ-FLAG-pGEX2TK. ForδⅡ-pGEX2TK,δⅢ-pGEX2TK,δC-pGEX2TK, after transforming the recombinant prokaryotic expression plasmid into expression bacteria BL21 (DE3), we used IPTG to introduce the expression of GST fusion protein. Coomassie brilliant blue staining showed theδⅡ-pGEX2TK,δⅢ-pGEX2TK,δC-pGEX2TK successfully expressed GST fusion protein of target domain in BL21 (DE3). According to batch purification protocol of Glutathione Sepharose 4B, the GST-δⅡ,GST-δⅢ,GST-δC were collected and incubated with cell lysate form HEK293 transfected withμ-Myc-pIRES2-EGFP plasmid. The precipitation was analysed by western blot with Myc primary antibody. The results showed that the second intercellular loop, the third intercellular loop, the carboxyl terminal don't play an important role in interaction betweenμandδopioid receptor. Expression ofδTM1-pGEX2TK,δTM3-pGEX2TK,δTM5-pGEX2TK,δTM6-pGEX2TK,δ-FLAG-pGEX2TK failed in BL21 (DE3). We tried different concentration of IPTG (0.05,0.2,0.5,1.2mmol/L) different temperature (37,30, 25℃) different time (4,7,16h) different expression bacteria(BL21(DE3)plysS, Rosetta gami (DE3),Rosetta (DE3)),but still couldn't get the GST fusion protein. These results showed that it is difficult to express the membrane protein and hydrophobic transmembrane in prokaryotic expression bacteria.
Conclusions
1.μ-Myc-pIRES2-EGFP,δ-FLAG-pIRES2-EGFP,κ-HA-pIRES2-EGFP eukaryotic recombinant plasmid were constructed correctly and expressed in high level in HEK293 cells. These opioid receptors are mainly in the form of homodimer when expressed in HEK293 cells.
2. It is difficult to express the membrane protein and hydrophobic transmembrane in prokaryotic expression system.
3. Heterodimerization betweenμandδopioid receptor is specific and selective. Coexppression ofμandδopioid receptor is necessary forμandδreceptor heterodimization.
4. The second intercellular loop, the third intercellular loop and the carboxyl terminal ofδopioid receptor don't play an important role in interaction betweenμandδopioid receptor.
5. Our data indicated that the interaction betweenμandδopioid receptor may require other protein participation.
引文
1、Hutcheson DM, Parkinson JA, Robbins TW, Everitt BJ. The effects of nucleus accumbens core and shell lesions on intravenous heroin self-administration and the acquisition of drug-seeking behaviour under a second-order schedule of heroin reinforcement.Psychopharmacology (Berl),2001,153(4):464-72.
2、Alderson HL, Parkinson JA, Robbins TW, Everitt BJ. The effects of excitotoxic lesions of the nucleus accumbens core or shell regions on intravenous heroin self-administration in rats.Psychopharmacology (Berl).2001,153(4):455-63.
3、王庆丰,高国栋.伏隔核的外壳部和核心部毁损对大鼠吗啡觅药行为的影响.第四军医大学学报,2003,24(9)：773-775.
4、杨文进,胡小吾.药物成瘾心理依赖的外科治疗进展.立体定向和功能神经外科杂志,2006,19(5)：318-320.
5、Medvedev SV,Anichkow AD,Polyakow Iul. Physiological mechanisms of the effectiveness of bilaterial stereotactic cingulotomy in t reatment of st rong psychological dependence in drug addictions. Fiziol Cheloveka,2003,29 (4):117-123.
6、王咸昌,郭沈昌,杨理荣,成良正,任廷文,杨和增,谭家亮,温金峰,宁连才,徐海燕.立体定向手术治疗221例阿片类药物依赖的近期疗效与不良反应分析.立体定向和功能性神经外科杂志,2005,18(3)：129-134.
7、杨开军,漆松涛,王克万,刘承勇,周永春.伏隔核毁损术治疗阿片类药物精神依赖的初步临床报告.中国微侵袭神经外科杂志,2005,10(2)：69-74.
8、唐运林,刘伟钦,周连银,张永,张奕生,邝培桂,汪鲁刚,罗学文.外科手术治疗海洛因依赖185例临床总结.中华神经外科杂志,2005,21(10)：600-602.
9、陈礼刚,李定君,夏祥国,明扬,刘洛同,刘亮,包长顺,白克镇,兰永树,韩福刚,陈跃.脑立体定向多靶点毁损术治疗海洛因依赖的临床分析.中华神经外科杂志,2005,21(10)：594-599.
10、王学廉,贺世明,衡立君,梁秦川,李江,王举磊,高国栋.定向伏隔核射频毁损戒毒的毁损情况与疗效关系探讨.中国微侵袭神经外科杂志,2006,11(3)：108-111.
11、Kuhn J, Lenartz D, Huff W, Lee S, Koulousakis A, Klosterkoetter J, Sturm V.Remission of alcohol dependency following deep brain stimulation of the nucleus accumbens:valuable therapeutic implications?J Neurol Neurosurg Psychiatry.2007,78(10):1152-1163.
12、Liu HY, Jin J, Tang JS, Sun WX, Jia H, Yang XP, Cui JM, Wang CG.Addict Biol. Chronic deep brain stimulation in the rat nucleus accumbens and its effect on morphine reinforcement.2008,13(1):40-46.
13、徐纪文,王桂松,周洪语,田鑫,王祥瑞,应隽,朱玫娟,张新凯,虞一萍,江基尧,罗其中.深部脑刺激戒断阿片类药物精神依赖1例临床报道.立体定向和功能性神经外科杂志,2005,18(3)：140-144.
14、Matthes HW, Maldonado R, Simonin F, Valverde 0, Slowe S, Kitchen I, Befort K, Dierich A, Le Meur M, Dolle P, Tzavara E, Hanoune J, Roques BP, Kieffer BL.Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene.Nature. 1996,383(6603):819-823.
15、Contarino A, Picetti R, Matthes HW, Koob GF, Kieffer BL, Gold LH.Lack of reward and locomotor stimulation induced by heroin in mu-opioid receptor-deficient mice.Eur J Pharmacol.2002,446(1-3):103-109.
16、Zhu Y, King MA, Schuller AG, Nitsche JF, Reidl M, Elde RP, Unterwald E, Pasternak GW, Pintar JE.Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice.Neuron. 1999,24(1):243-252.
17、George SR, Fan T, Xie Z, Tse R, Tam V, Varghese G, O'Dowd BF.Oligomerization of mu-and delta-opioid receptors. Generation of novel functional properties.J Biol Chem.2000,275(34):26128-26135.
18、Gomes I, Gupta A, Filipovska J,Szeto HH, Pintar JE, Devi LA.A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia.Proc Natl Acad Sci U S A,2004,101(14):5135-5139.
19、Cheng PY, Liu-Chen LY, Pickel VM.Dual ultrastructural immunocytochemical labeling of mu and delta opioid receptors in the superficial layers of the rat cervical spinal cord.Brain Res.1997,778(2):367-380.
20、Wang H, Pickel VM.Preferential cytoplasmic localization of delta-opioid receptors in rat striatal patches:comparison with plasmalemmal mu-opioid receptors.J Neurosci.2001,21(9):3242-3250.
21、Schmidt BL, Tambeli CH, Levine JD, Gear RW. mu/delta Cooperativity and opposing kappa-opioid effects in nucleus accumbens-mediated antinociception in the rat. Eur J Neurosci,2002,15(5):861-868.
22、Rozenfeld R, Abul-Husn NS, Gomez I, Devi LA. An emerging role for the delta opioid receptor in the regulation of mu opioid receptor function. ScientificWorldJournal,2007,7:64-73.
23、Ji SP, Zhang Y, Van Cleemput J, Jiang W, Liao M, Li L, Wan Q, Backstrom JR, Zhang X.Disruption of PTEN coupling with 5-HT2C receptors suppresses behavioral responses induced by drugs of abuse.Nat Med,2006,12(3):324-329.
[1]Evans CJ, Keith DE Jr, Morrison H, et al. Cloning of a delta opioid receptor by functional expression[J]. Science,1992,258(5090):1952-5.
[2]George Paxinos, Charles Watson.诸葛启钏译.大鼠脑立体定位图谱[M].第3版,北京：人民卫生出版社.2005,12：图9-16.
[3]姜泊,张亚历,周殿元主编.分子生物学常用实验方法.北京：人民军医出版社,3.
[4]金冬雁,黎孟枫等译.分子克隆实验指南.第二版.北京：科学出版社,49-55.
[5]Jordan BA, Devi LA. G-protein-coupled receptor heterodimerization modulates receptor function. Nature,1999,399(6737):697.
[6]George SR, Fan T, Xie Z, et al. Oligomerization of mu-and delta-opioid receptors. Generation of novel functional properties. J Biol Chem,2000,275(34):26128.
[7]Milligan G. G protein-coupled receptor dimerisation:molecular basis and relevance to function. Biochim Biophys Acta,2007,1768(4):825.
[8]McVey M, Ramsay D, Kellett E, Rees S, Wilson S, Pope AJ, Milligan G. Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. J Biol Chem, 2001,276(17):14092.
[9]Mountford PS, Smith AG. Internal ribosome entry sites and dicistronic RNAs in mammalian transgenesis [J]. Trends Genet,1995,11(5):179-84.
[10]Jang SK, Krausslich HG, Nicklin MJ, et al. A segment of the 5'nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation [J]. J Virol,1988,62(8):2636-43.
[11]Gomes I, Gupta A, Filipovska J, et al. A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia. Proc Natl Acad Sci USA, 2004,101(14):5135.
[12]Abul-Husn NS, Sutak M, Milne B, et al. Augmentation of spinal morphine analgesia and inhibition of tolerance by low doses of mu-and delta-opioid receptor antagonists. Br J Pharmacol,2007,151(6):877.
[13]Schmidt BL, Tambeli CH, Levine JD, et al. mu/delta Cooperativity and opposing kappa-opioid effects in nucleus accumbens-mediated antinociception in the rat. Eur J Neurosci,2002,15(5):861-868.
[14]Gendron L, Pintar JE, Chavkin C. Essential role of mu opioid receptor in the regulation of delta opioid receptor-mediated antihyperalgesia. Neuroscience, 2007,150(4):807.
[1]Kieffer B L, Gaveriaux R C.Exploring the opioid system by gene knockout[J].Prog Neurobiol,2002,66(5):285-306.
[2]Parnot C, Miserey L S, Bardin S, et al.Lessons from constitutively active mutants of G protein-coupled receptors[J].Trends Endocrinol Metab, 2002,13(8):336-343.
[3]Fuchs P N, Roza C, Sora I, et al.Characterization of mechanical withdrawal responses and effects of mu-,delta-and kappa-opioid agonists in normal and mu-opioid receptor knockout mice[J].Brain Res,1999,821(2):480-486.
[4]Mountford P S,Smith A G. Internal ribosome entry sites and dicistronic RNAs in mammalian transgenesis[J].Trends Genet,1995,11(5):179-184.
[5]Abul N S, Sutak M, Milne B, et al.Augmentation of spinal morphine analgesia and inhibition of tolerance by low doses of mu-and delta-opioid receptor antagonists[J].Br J Pharmacol,2007,151(6):877-887.
[6]Gomes I, Gupta A, Filipovska J, et al.A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia[J].Proc Natl Acad Sci U S A,2004,101(14):5135-5139.
[7]Etienne C L, Law P Y, Roerig S C, et al. Opioid-induced tolerance and dependence in mice is modulated by the distance between pharmacophores in a bivalent ligand series[J]. Proc Natl Acad Sci U.S.A,2005,102:19208-19213.
[8]Gendron L, Pintar JE, Chavkin C. Essential role of mu opioid receptor in the regulation of delta opioid receptor-mediated antihyperalgesia. Neuroscience, 2007,150(4):807.
[9]Dietis N, Guerrini R, Calo G, Salvadori S, Rowbotham DJ, Lambert DG.Simultaneous targeting of multiple opioid receptors:a strategy to improve side-effect profile.Br J Anaesth,2009,103(1):38-49.
[10]Lenard N R, Daniels D J, Portoghese P S, et al. Absence of conditioned place preference or reinstatement with bivalent ligands containing mu-opioid receptor agonist and delta-opioid receptor antagonist pharmacophores[J].Eur J Pharmacol,2007,566(1-3):75-82.
[1]Simonin F, Gaveriaux-Ruff C, Kieffer BL, et al. Kappa-opioid receptor in humans:cDNA and genomic cloning,chromosomal assignment, functional expression, pharmacology, and expression pattern in the central nervous system[J].Proc Natl Acad Sci USA,1995,92(15):7006-7010.
[2]Mountford PS, Smith AG.Internal ribosome entry sites and dicistronic RNAs in mammalian transgenesis[J].Trends Genet,1995,11(5):179-184.
[3]Jang SK, Krausslich HG, Nicklin MJ, et al.A segment of the 5'nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation[J].J Virol,1988,62(8):2636-2643.
[4]Gomes I, Gupta A, Filipovska J, et al.A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia[J].Proc Natl Acad Sci USA,2004,101(14):5135-5139.
[5]Abul-Husn NS, Sutak M, Milne B, et al.Augmentation of spinal morphine analgesia and inhibition of tolerance by low doses of mu-and delta-opioid receptor antagonists[J].Br J Pharmacol,2007,151(6):877-887.
[6]Schmidt BL, Tambeli CH, Levine JD, Gear RW.mu/delta Cooperativity and opposing kappa-opioid effects in nucleus accumbens-mediated antinociception in the rat[J].Eur J Neurosci,2002,15(5):861-868.
[7]Milligan G. A day in the life of a G protein-coupled receptor:the contribution to function of G protein-coupled receptor dimerization[J].Br J Pharmacol,2008, 153:216-229.
[1]George SR, Fan T, Xie Z, et al. Oligomerization of mu-and delta-opioid receptors. Generation of novel functional properties[J]. J Biol Chem, 275(34):26128.
[2]McVey M, Ramsay D, Kellett E, et al. Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. The human delta-opioid receptor displays constitutive oligomerization at the cell surface, which is not regulated by receptor occupancy[J]. J Biol Chem.2001,276(17):14092.
[3]Li-Wei C, Can G, De-He Z, Qiang W, Xue-Jun X, Jie C, Zhi-Qiang C.Homodimerization of human mu-opioid receptor overexpressed in Sf9 insect cells.Protein Pept Lett.2002,9(2):145-52.
[4]Garzon J, Castro M, Sanchez-Blazquez P.Influence of Gz and Gi2 transducer proteins in the affinity of opioid agonists to mu receptors[J]. Eur J Neurosci. 1998,10(8):2557.
[5]Schmidt BL, Tambeli CH, Levine JD, et al. mu/delta Cooperativity and opposing kappa-opioid effects in nucleus accumbens-mediated antinociception in the rat[J]. Eur J Neurosci,2002,15(5):861-868.
[6]Rozenfeld R, Abul-Husn NS, Gomez I, Devi LA. An emerging role for the delta opioid receptor in the regulation of mu opioid receptor function. ScientificWorldJournal,2007,7:64-73.
[7]Matthes HW, Maldonado R, Simonin F, Valverde O, Slowe S, Kitchen I, Befort K, Dierich A, Le Meur M, Dolle P, Tzavara E, Hanoune J, Roques BP, Kieffer BL.Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene.Nature.1996,383(6603):819-823.
[8]Contarino A, Picetti R, Matthes HW, Koob GF, Kieffer BL, Gold LH.Lack of reward and locomotor stimulation induced by heroin in mu-opioid receptor-deficient mice.Eur J Pharmacol.2002,446(1-3):103-109.
[9]Zhu Y, King MA, Schuller AG, Nitsche JF, Reidl M, Elde RP, Unterwald E, Pasternak GW, Pintar JE.Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice.Neuron. 1999,24(1):243-252.
[10]Gomes I, Gupta A, Filipovska J, Szeto HH, Pintar JE, Devi LA.A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia.Proc Natl Acad Sci U S A,2004,101 (14):5135-5139.
[11]Cheng PY, Liu-Chen LY, Pickel VM.Dual ultrastructural immunocytochemical labeling of mu and delta opioid receptors in the superficial layers of the rat cervical spinal cord.Brain Res.1997,778(2):367-380.
[12]Wang H, Pickel VM.Preferential cytoplasmic localization of delta-opioid receptors in rat striatal patches:comparison with plasmalemmal mu-opioid receptors.J Neurosci.2001,21(9):3242-3250.
[13]Ji SP, Zhang Y, Van Cleemput J, Jiang W, Liao M, Li L, Wan Q, Backstrom JR, Zhang X.Disruption of PTEN coupling with 5-HT2C receptors suppresses behavioral responses induced by drugs of abuse.Nat Med,2006,12(3):324-329.
[14]Angers S, Salahpour A, Bouvier M.Dimerization:an emerging concept for G protein-coupled receptor ontogeny and function.Annu Rev Pharmacol Toxicol. 2002;42:409-35.
[15]Milligan G.G protein-coupled receptor dimerization:function and ligand pharmacology.Mol Pharmacol.2004,66(1):1-7.
[16]Milligan G.A day in the life of a G protein-coupled receptor:the contribution to function of G protein-coupled receptor dimerization.Br J Pharmacol.2008,153 Suppl1:S216-29.
1. Evans CJ, Keith DE Jr, Morrison H, et al. Cloning of a delta opioid receptor by functional expression[J]. Science.1992,258(5090):1952.
2. Wang WW, Shahrestanifar M, Jin J, et al. Studies on mu and delta opioid receptor selectivity utilizing chimeric and site-mutagenized receptors [J].Proc Natl Acad SciUSA.1995,92(26):12436.
3. Varga EV, Li X, Stropova D, et al. The third extracellular loop of the human delta-opioid receptor determines the selectivity of delta-opioid agonists[J]. Mol Pharmacol.1996,50(6):1619.
4. Kong H, Raynor K, Yasuda K, et al. A single residue, aspartic acid 95, in the delta opioid receptor specifies selective high affinity agonist binding[J]. J Biol Chem. 1993,268(31):23055.
5. Befort K, Tabbara L, Bausch S, et al. The conserved aspartate residue in the third putative transmembrane domain of the delta-opioid receptor is not the anionic counterpart for cationic opiate binding but is a constituent of the receptor binding site[J]. Mol Pharmacol.1996,49(2):216.
6. Claude PA, Wotta DR, Zhang XH, et al. Mutation of a conserved serine in TM4 of opioid receptors confers full agonistic properties to classical antagonists[J]. Proc Natl Acad Sci U S A.1996,93(12):5715.
7. Claude-Geppert PA, Liu J, Solberg J, et al. Antagonist efficacy in MORS196L mutant is affected by the interaction between transmembrane domains of the opioid receptor[J]. J Pharmacol Exp Ther.2005,313(1):216.
8. Fukuda K, Kato S, Mori K. Location of regions of the opioid receptor involved in selective agonist binding[J]. J Biol Chem.1995,270(12):6702.
9. Meng F, Ueda Y, Hoversten MT, et al. Mapping the receptor domains critical for the binding selectivity of delta-opioid receptor ligands[J]. Eur J Pharmacol. 1996,311(2-3):285
10. Wang CH, Zhou GH, Chen J, et al. Binding properities of C-truncated delta opioid receptor[J]. Acta Pharmacol Sin,1997,18(4):337.
11. Zhu X, Wang C, Cheng Z, et al. The carboxyl term inus of mouse delta-opioid receptor is not required for agonist-dependent activation[J]. Biochem Biophys Res Commun,1997,232 (2):513.
12. Minami M, Nakagawa T, Seki T, et al. A single residue, Lys108, of the delta-opioid receptor prevents the mu-opioid-selective ligand [D-Ala2,N-MePhe4,Gly-ol5]enkephalin from binding to the delta-opioid receptor[J]. Mol Pharmacol.1996,50(5):1413.
13. George SR, Fan T, Xie Z, et al. Oligomerization of mu-and delta-opioid receptors. Generation of novel functional properties[J]. J Biol Chem,275(34):26128.
14. McVey M, Ramsay D, Kellett E, et al. Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. The human delta-opioid receptor displays constitutive oligomerization at the cell surface, which is not regulated by receptor occupancy[J]. J Biol Chem.2001,276(17):14092.
15.Garzon J, Castro M, Sanchez-Blazquez P.Influence of Gz and Gi2 transducer proteins in the affinity of opioid agonists to mu receptors[J]. Eur J Neurosci. 1998,10(8):2557.
16. Schmidt BL, Tambeli CH, Levine JD, et al. mu/delta Cooperativity and opposing kappa-opioid effects in nucleus accumbens-mediated antinociception in the rat[J]. Eur J Neurosci,2002,15(5):861.
17. Rozenfeld R, Abul-Husn NS, Gomez I, et al. An emerging role for the delta opioid receptor in the regulation of mu opioid receptor function[J]. ScientificWorldJournal,2007,7:64.
18. Gomes I, Gupta A, Filipovska J, et al. A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia[J]. Proc Natl Acad Sci U S A.2004,101(14):5135.
19. Lenard NR, Daniels DJ, Portoghese PS, et al. Absence of conditioned place preference or reinstatement with bivalent ligands containing mu-opioid receptor agonist and delta-opioid receptor antagonist pharmacophores[J]. Eur J Pharmacol. 2007,566(1-3):75.
20. Etienne, C.L., Law, P.Y., Roerig, S.C., et al. Opioid-induced tolerance and dependence in mice is modulated by the distance between pharmacophores in a bivalent ligand series[J]. Proc.Natl. Acad. Sci. U.S.A.2005,102:19208.
21. Jordan BA, Devi LA. G-protein-coupled receptor heterodimerization modulates receptor function[J]. Nature,1999,399(6737):697.
2、Alderson HL, Parkinson JA, Robbins TW, Everitt BJ. The effects of excitotoxic lesions of the nucleus accumbens core or shell regions on intravenous heroin self-administration in rats.Psychopharmacology (Berl).2001,153(4):455-63.
3、王庆丰,高国栋.伏隔核的外壳部和核心部毁损对大鼠吗啡觅药行为的影响.第四军医大学学报,2003,24(9)：773-775.
4、杨文进,胡小吾.药物成瘾心理依赖的外科治疗进展.立体定向和功能神经外科杂志,2006,19(5)：318-320.
5、Medvedev SV,Anichkow AD,Polyakow Iul. Physiological mechanisms of the effectiveness of bilaterial stereotactic cingulotomy in t reatment of st rong psychological dependence in drug addictions. Fiziol Cheloveka,2003,29 (4):117-123.
6、王咸昌,郭沈昌,杨理荣,成良正,任廷文,杨和增,谭家亮,温金峰,宁连才,徐海燕.立体定向手术治疗221例阿片类药物依赖的近期疗效与不良反应分析.立体定向和功能性神经外科杂志,2005,18(3)：129-134.
7、杨开军,漆松涛,王克万,刘承勇,周永春.伏隔核毁损术治疗阿片类药物精神依赖的初步临床报告.中国微侵袭神经外科杂志,2005,10(2)：69-74.
8、唐运林,刘伟钦,周连银,张永,张奕生,邝培桂,汪鲁刚,罗学文.外科手术治疗海洛因依赖185例临床总结.中华神经外科杂志,2005,21(10)：600-602.
9、陈礼刚,李定君,夏祥国,明扬,刘洛同,刘亮,包长顺,白克镇,兰永树,韩福刚,陈跃.脑立体定向多靶点毁损术治疗海洛因依赖的临床分析.中华神经外科杂志,2005,21(10)：594-599.
10、王学廉,贺世明,衡立君,梁秦川,李江,王举磊,高国栋.定向伏隔核射频毁损戒毒的毁损情况与疗效关系探讨.中国微侵袭神经外科杂志,2006,11(3)：108-111.
11、Kuhn J, Lenartz D, Huff W, Lee S, Koulousakis A, Klosterkoetter J, Sturm V.Remission of alcohol dependency following deep brain stimulation of the nucleus accumbens:valuable therapeutic implications?J Neurol Neurosurg Psychiatry.2007,78(10):1152-1163.
12、Liu HY, Jin J, Tang JS, Sun WX, Jia H, Yang XP, Cui JM, Wang CG.Addict Biol. Chronic deep brain stimulation in the rat nucleus accumbens and its effect on morphine reinforcement.2008,13(1):40-46.
13、徐纪文,王桂松,周洪语,田鑫,王祥瑞,应隽,朱玫娟,张新凯,虞一萍,江基尧,罗其中.深部脑刺激戒断阿片类药物精神依赖1例临床报道.立体定向和功能性神经外科杂志,2005,18(3)：140-144.
14、Matthes HW, Maldonado R, Simonin F, Valverde 0, Slowe S, Kitchen I, Befort K, Dierich A, Le Meur M, Dolle P, Tzavara E, Hanoune J, Roques BP, Kieffer BL.Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene.Nature. 1996,383(6603):819-823.
15、Contarino A, Picetti R, Matthes HW, Koob GF, Kieffer BL, Gold LH.Lack of reward and locomotor stimulation induced by heroin in mu-opioid receptor-deficient mice.Eur J Pharmacol.2002,446(1-3):103-109.
16、Zhu Y, King MA, Schuller AG, Nitsche JF, Reidl M, Elde RP, Unterwald E, Pasternak GW, Pintar JE.Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice.Neuron. 1999,24(1):243-252.
17、George SR, Fan T, Xie Z, Tse R, Tam V, Varghese G, O'Dowd BF.Oligomerization of mu-and delta-opioid receptors. Generation of novel functional properties.J Biol Chem.2000,275(34):26128-26135.
18、Gomes I, Gupta A, Filipovska J,Szeto HH, Pintar JE, Devi LA.A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia.Proc Natl Acad Sci U S A,2004,101(14):5135-5139.
19、Cheng PY, Liu-Chen LY, Pickel VM.Dual ultrastructural immunocytochemical labeling of mu and delta opioid receptors in the superficial layers of the rat cervical spinal cord.Brain Res.1997,778(2):367-380.
20、Wang H, Pickel VM.Preferential cytoplasmic localization of delta-opioid receptors in rat striatal patches:comparison with plasmalemmal mu-opioid receptors.J Neurosci.2001,21(9):3242-3250.
21、Schmidt BL, Tambeli CH, Levine JD, Gear RW. mu/delta Cooperativity and opposing kappa-opioid effects in nucleus accumbens-mediated antinociception in the rat. Eur J Neurosci,2002,15(5):861-868.
22、Rozenfeld R, Abul-Husn NS, Gomez I, Devi LA. An emerging role for the delta opioid receptor in the regulation of mu opioid receptor function. ScientificWorldJournal,2007,7:64-73.
23、Ji SP, Zhang Y, Van Cleemput J, Jiang W, Liao M, Li L, Wan Q, Backstrom JR, Zhang X.Disruption of PTEN coupling with 5-HT2C receptors suppresses behavioral responses induced by drugs of abuse.Nat Med,2006,12(3):324-329.
[1]Evans CJ, Keith DE Jr, Morrison H, et al. Cloning of a delta opioid receptor by functional expression[J]. Science,1992,258(5090):1952-5.
[2]George Paxinos, Charles Watson.诸葛启钏译.大鼠脑立体定位图谱[M].第3版,北京：人民卫生出版社.2005,12：图9-16.
[3]姜泊,张亚历,周殿元主编.分子生物学常用实验方法.北京：人民军医出版社,3.
[4]金冬雁,黎孟枫等译.分子克隆实验指南.第二版.北京：科学出版社,49-55.
[5]Jordan BA, Devi LA. G-protein-coupled receptor heterodimerization modulates receptor function. Nature,1999,399(6737):697.
[6]George SR, Fan T, Xie Z, et al. Oligomerization of mu-and delta-opioid receptors. Generation of novel functional properties. J Biol Chem,2000,275(34):26128.
[7]Milligan G. G protein-coupled receptor dimerisation:molecular basis and relevance to function. Biochim Biophys Acta,2007,1768(4):825.
[8]McVey M, Ramsay D, Kellett E, Rees S, Wilson S, Pope AJ, Milligan G. Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. J Biol Chem, 2001,276(17):14092.
[9]Mountford PS, Smith AG. Internal ribosome entry sites and dicistronic RNAs in mammalian transgenesis [J]. Trends Genet,1995,11(5):179-84.
[10]Jang SK, Krausslich HG, Nicklin MJ, et al. A segment of the 5'nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation [J]. J Virol,1988,62(8):2636-43.
[11]Gomes I, Gupta A, Filipovska J, et al. A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia. Proc Natl Acad Sci USA, 2004,101(14):5135.
[12]Abul-Husn NS, Sutak M, Milne B, et al. Augmentation of spinal morphine analgesia and inhibition of tolerance by low doses of mu-and delta-opioid receptor antagonists. Br J Pharmacol,2007,151(6):877.
[13]Schmidt BL, Tambeli CH, Levine JD, et al. mu/delta Cooperativity and opposing kappa-opioid effects in nucleus accumbens-mediated antinociception in the rat. Eur J Neurosci,2002,15(5):861-868.
[14]Gendron L, Pintar JE, Chavkin C. Essential role of mu opioid receptor in the regulation of delta opioid receptor-mediated antihyperalgesia. Neuroscience, 2007,150(4):807.
[1]Kieffer B L, Gaveriaux R C.Exploring the opioid system by gene knockout[J].Prog Neurobiol,2002,66(5):285-306.
[2]Parnot C, Miserey L S, Bardin S, et al.Lessons from constitutively active mutants of G protein-coupled receptors[J].Trends Endocrinol Metab, 2002,13(8):336-343.
[3]Fuchs P N, Roza C, Sora I, et al.Characterization of mechanical withdrawal responses and effects of mu-,delta-and kappa-opioid agonists in normal and mu-opioid receptor knockout mice[J].Brain Res,1999,821(2):480-486.
[4]Mountford P S,Smith A G. Internal ribosome entry sites and dicistronic RNAs in mammalian transgenesis[J].Trends Genet,1995,11(5):179-184.
[5]Abul N S, Sutak M, Milne B, et al.Augmentation of spinal morphine analgesia and inhibition of tolerance by low doses of mu-and delta-opioid receptor antagonists[J].Br J Pharmacol,2007,151(6):877-887.
[6]Gomes I, Gupta A, Filipovska J, et al.A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia[J].Proc Natl Acad Sci U S A,2004,101(14):5135-5139.
[7]Etienne C L, Law P Y, Roerig S C, et al. Opioid-induced tolerance and dependence in mice is modulated by the distance between pharmacophores in a bivalent ligand series[J]. Proc Natl Acad Sci U.S.A,2005,102:19208-19213.
[8]Gendron L, Pintar JE, Chavkin C. Essential role of mu opioid receptor in the regulation of delta opioid receptor-mediated antihyperalgesia. Neuroscience, 2007,150(4):807.
[9]Dietis N, Guerrini R, Calo G, Salvadori S, Rowbotham DJ, Lambert DG.Simultaneous targeting of multiple opioid receptors:a strategy to improve side-effect profile.Br J Anaesth,2009,103(1):38-49.
[10]Lenard N R, Daniels D J, Portoghese P S, et al. Absence of conditioned place preference or reinstatement with bivalent ligands containing mu-opioid receptor agonist and delta-opioid receptor antagonist pharmacophores[J].Eur J Pharmacol,2007,566(1-3):75-82.
[1]Simonin F, Gaveriaux-Ruff C, Kieffer BL, et al. Kappa-opioid receptor in humans:cDNA and genomic cloning,chromosomal assignment, functional expression, pharmacology, and expression pattern in the central nervous system[J].Proc Natl Acad Sci USA,1995,92(15):7006-7010.
[2]Mountford PS, Smith AG.Internal ribosome entry sites and dicistronic RNAs in mammalian transgenesis[J].Trends Genet,1995,11(5):179-184.
[3]Jang SK, Krausslich HG, Nicklin MJ, et al.A segment of the 5'nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation[J].J Virol,1988,62(8):2636-2643.
[4]Gomes I, Gupta A, Filipovska J, et al.A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia[J].Proc Natl Acad Sci USA,2004,101(14):5135-5139.
[5]Abul-Husn NS, Sutak M, Milne B, et al.Augmentation of spinal morphine analgesia and inhibition of tolerance by low doses of mu-and delta-opioid receptor antagonists[J].Br J Pharmacol,2007,151(6):877-887.
[6]Schmidt BL, Tambeli CH, Levine JD, Gear RW.mu/delta Cooperativity and opposing kappa-opioid effects in nucleus accumbens-mediated antinociception in the rat[J].Eur J Neurosci,2002,15(5):861-868.
[7]Milligan G. A day in the life of a G protein-coupled receptor:the contribution to function of G protein-coupled receptor dimerization[J].Br J Pharmacol,2008, 153:216-229.
[1]George SR, Fan T, Xie Z, et al. Oligomerization of mu-and delta-opioid receptors. Generation of novel functional properties[J]. J Biol Chem, 275(34):26128.
[2]McVey M, Ramsay D, Kellett E, et al. Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. The human delta-opioid receptor displays constitutive oligomerization at the cell surface, which is not regulated by receptor occupancy[J]. J Biol Chem.2001,276(17):14092.
[3]Li-Wei C, Can G, De-He Z, Qiang W, Xue-Jun X, Jie C, Zhi-Qiang C.Homodimerization of human mu-opioid receptor overexpressed in Sf9 insect cells.Protein Pept Lett.2002,9(2):145-52.
[4]Garzon J, Castro M, Sanchez-Blazquez P.Influence of Gz and Gi2 transducer proteins in the affinity of opioid agonists to mu receptors[J]. Eur J Neurosci. 1998,10(8):2557.
[5]Schmidt BL, Tambeli CH, Levine JD, et al. mu/delta Cooperativity and opposing kappa-opioid effects in nucleus accumbens-mediated antinociception in the rat[J]. Eur J Neurosci,2002,15(5):861-868.
[6]Rozenfeld R, Abul-Husn NS, Gomez I, Devi LA. An emerging role for the delta opioid receptor in the regulation of mu opioid receptor function. ScientificWorldJournal,2007,7:64-73.
[7]Matthes HW, Maldonado R, Simonin F, Valverde O, Slowe S, Kitchen I, Befort K, Dierich A, Le Meur M, Dolle P, Tzavara E, Hanoune J, Roques BP, Kieffer BL.Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene.Nature.1996,383(6603):819-823.
[8]Contarino A, Picetti R, Matthes HW, Koob GF, Kieffer BL, Gold LH.Lack of reward and locomotor stimulation induced by heroin in mu-opioid receptor-deficient mice.Eur J Pharmacol.2002,446(1-3):103-109.
[9]Zhu Y, King MA, Schuller AG, Nitsche JF, Reidl M, Elde RP, Unterwald E, Pasternak GW, Pintar JE.Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice.Neuron. 1999,24(1):243-252.
[10]Gomes I, Gupta A, Filipovska J, Szeto HH, Pintar JE, Devi LA.A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia.Proc Natl Acad Sci U S A,2004,101 (14):5135-5139.
[11]Cheng PY, Liu-Chen LY, Pickel VM.Dual ultrastructural immunocytochemical labeling of mu and delta opioid receptors in the superficial layers of the rat cervical spinal cord.Brain Res.1997,778(2):367-380.
[12]Wang H, Pickel VM.Preferential cytoplasmic localization of delta-opioid receptors in rat striatal patches:comparison with plasmalemmal mu-opioid receptors.J Neurosci.2001,21(9):3242-3250.
[13]Ji SP, Zhang Y, Van Cleemput J, Jiang W, Liao M, Li L, Wan Q, Backstrom JR, Zhang X.Disruption of PTEN coupling with 5-HT2C receptors suppresses behavioral responses induced by drugs of abuse.Nat Med,2006,12(3):324-329.
[14]Angers S, Salahpour A, Bouvier M.Dimerization:an emerging concept for G protein-coupled receptor ontogeny and function.Annu Rev Pharmacol Toxicol. 2002;42:409-35.
[15]Milligan G.G protein-coupled receptor dimerization:function and ligand pharmacology.Mol Pharmacol.2004,66(1):1-7.
[16]Milligan G.A day in the life of a G protein-coupled receptor:the contribution to function of G protein-coupled receptor dimerization.Br J Pharmacol.2008,153 Suppl1:S216-29.
1. Evans CJ, Keith DE Jr, Morrison H, et al. Cloning of a delta opioid receptor by functional expression[J]. Science.1992,258(5090):1952.
2. Wang WW, Shahrestanifar M, Jin J, et al. Studies on mu and delta opioid receptor selectivity utilizing chimeric and site-mutagenized receptors [J].Proc Natl Acad SciUSA.1995,92(26):12436.
3. Varga EV, Li X, Stropova D, et al. The third extracellular loop of the human delta-opioid receptor determines the selectivity of delta-opioid agonists[J]. Mol Pharmacol.1996,50(6):1619.
4. Kong H, Raynor K, Yasuda K, et al. A single residue, aspartic acid 95, in the delta opioid receptor specifies selective high affinity agonist binding[J]. J Biol Chem. 1993,268(31):23055.
5. Befort K, Tabbara L, Bausch S, et al. The conserved aspartate residue in the third putative transmembrane domain of the delta-opioid receptor is not the anionic counterpart for cationic opiate binding but is a constituent of the receptor binding site[J]. Mol Pharmacol.1996,49(2):216.
6. Claude PA, Wotta DR, Zhang XH, et al. Mutation of a conserved serine in TM4 of opioid receptors confers full agonistic properties to classical antagonists[J]. Proc Natl Acad Sci U S A.1996,93(12):5715.
7. Claude-Geppert PA, Liu J, Solberg J, et al. Antagonist efficacy in MORS196L mutant is affected by the interaction between transmembrane domains of the opioid receptor[J]. J Pharmacol Exp Ther.2005,313(1):216.
8. Fukuda K, Kato S, Mori K. Location of regions of the opioid receptor involved in selective agonist binding[J]. J Biol Chem.1995,270(12):6702.
9. Meng F, Ueda Y, Hoversten MT, et al. Mapping the receptor domains critical for the binding selectivity of delta-opioid receptor ligands[J]. Eur J Pharmacol. 1996,311(2-3):285
10. Wang CH, Zhou GH, Chen J, et al. Binding properities of C-truncated delta opioid receptor[J]. Acta Pharmacol Sin,1997,18(4):337.
11. Zhu X, Wang C, Cheng Z, et al. The carboxyl term inus of mouse delta-opioid receptor is not required for agonist-dependent activation[J]. Biochem Biophys Res Commun,1997,232 (2):513.
12. Minami M, Nakagawa T, Seki T, et al. A single residue, Lys108, of the delta-opioid receptor prevents the mu-opioid-selective ligand [D-Ala2,N-MePhe4,Gly-ol5]enkephalin from binding to the delta-opioid receptor[J]. Mol Pharmacol.1996,50(5):1413.
13. George SR, Fan T, Xie Z, et al. Oligomerization of mu-and delta-opioid receptors. Generation of novel functional properties[J]. J Biol Chem,275(34):26128.
14. McVey M, Ramsay D, Kellett E, et al. Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. The human delta-opioid receptor displays constitutive oligomerization at the cell surface, which is not regulated by receptor occupancy[J]. J Biol Chem.2001,276(17):14092.
15.Garzon J, Castro M, Sanchez-Blazquez P.Influence of Gz and Gi2 transducer proteins in the affinity of opioid agonists to mu receptors[J]. Eur J Neurosci. 1998,10(8):2557.
16. Schmidt BL, Tambeli CH, Levine JD, et al. mu/delta Cooperativity and opposing kappa-opioid effects in nucleus accumbens-mediated antinociception in the rat[J]. Eur J Neurosci,2002,15(5):861.
17. Rozenfeld R, Abul-Husn NS, Gomez I, et al. An emerging role for the delta opioid receptor in the regulation of mu opioid receptor function[J]. ScientificWorldJournal,2007,7:64.
18. Gomes I, Gupta A, Filipovska J, et al. A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia[J]. Proc Natl Acad Sci U S A.2004,101(14):5135.
19. Lenard NR, Daniels DJ, Portoghese PS, et al. Absence of conditioned place preference or reinstatement with bivalent ligands containing mu-opioid receptor agonist and delta-opioid receptor antagonist pharmacophores[J]. Eur J Pharmacol. 2007,566(1-3):75.
20. Etienne, C.L., Law, P.Y., Roerig, S.C., et al. Opioid-induced tolerance and dependence in mice is modulated by the distance between pharmacophores in a bivalent ligand series[J]. Proc.Natl. Acad. Sci. U.S.A.2005,102:19208.
21. Jordan BA, Devi LA. G-protein-coupled receptor heterodimerization modulates receptor function[J]. Nature,1999,399(6737):697.