【作者】 赵艳梅；

【导师】 丛斌； 李锦；

【作者基本信息】 河北医科大学 ， 法医学， 2008， 博士

【摘要】 阿片依赖是一种慢性复发性脑病,以强迫用药、不断增加药物摄入量和停药时出现戒断综合征为主要特征。目前阿片依赖发生的确切机制还不清楚。大量研究表明,突触可塑性的改变在阿片依赖过程中起了重要作用。这些变化包括突触前神经递质的释放和突触后受体的信号转导。以往研究发现,吗啡可以通过调节突触前影响神经递质释放的分子影响递质释放。在神经递质的释放过程中,囊泡膜和质膜的融合是影响递质释放的关键步骤。可溶性NSF附着蛋白(Soluble NSF attachment proteins,SNAPs)是一类在膜融合过程中发挥重要作用的蛋白质分子,介导了囊泡膜和质膜的融合,在哺乳动物中存在α-,β-,和γ- 3种亚型。鉴于SNAPs在膜融合过程中发挥的重要作用,SNAPs参与许多重要的生理进程,包括调节钙依赖的胞吐机制、刺激胰岛β细胞分泌胰岛素、增加肺泡II型细胞表面活性物质的分泌、参与有丝分裂过程中核膜的形成等。虽然大量研究表明SNAPs在神经递质释放的膜融合过程中起重要作用,但是SNAPs是否参与了阿片依赖过程中突触前神经可塑性的发生目前尚未见报道。为了揭示SNAPs与阿片依赖的关系,我们首先在大鼠吗啡依赖及戒断模型上观察了与神经可塑性关系密切的脑区伏隔核(nucleus accumbens,NAc)、尾壳核(caudate putamen,CPu)及海马(hippocampus,Hip)中SNAPs表达的变化,初步探讨SNAPs和吗啡依赖的关系。研究采用成年雄性Wistar大鼠,随机分为对照组、吗啡组和戒断不同时间组。吗啡组大鼠背部皮下注射吗啡8d,每日3次,剂量递增,对照组大鼠注射等体积生理盐水。吗啡组末次注射吗啡4 h后处死,断头取脑。自然戒断组分别在戒断不同时间处死动物。各组均设平行对照。利用RT-PCR和Western blot技术分别检测各脑区SNAPs的mRNA和蛋白表达水平。结果发现,与对照组相比,慢性吗啡依赖大鼠CPu内γ-SNAP的mRNA和蛋白表达水平均上调25%左右(P<0.01) (图1.4,图1.9),NAc及Hip脑区则无明显变化,自然戒断d2、d3、d7组亦未观察到γ-SNAP明显的表达改变(图1.8,图1.10,表1.1,表1.3)。α-SNAP和β-SNAP在吗啡依赖和自然戒断状态下3个被检脑区(NAc、CPu、Hip)均未检测到明显的表达变化(图1.2,图1.3,图1.5,图1.6,图1.7,表1.1,表1.2)。以上结果说明:慢性吗啡依赖可增加CPu内γ-SNAP的表达,对α-SNAP和β-SNAP的表达没有影响,提示SNAPs 3种亚型在慢性吗啡依赖过程中行使不同的功能,可能与特定神经递质的分泌有关。慢性吗啡依赖及戒断引起的突触前神经递质释放改变可能不是由α-SNAP和β-SNAP表达量变化介导的,其具体机制可能与其内在活性变化或蛋白在细胞内的分布有关。为了进一步研究SNAPs与阿片依赖的关系,我们选择了一种与神经递质释放关系最为明确的亚型—α-SNAP进行研究。已有大量研究报道α-SNAP参与钙依赖的胞吐作用,该作用在神经递质释放过程中的囊泡膜与质膜的融合步骤中是至关重要的。且相关研究报道α-SNAP通过刺激NSF的ATP酶活性来发挥其在递质释放中的作用,α-SNAP对NSF的ATP酶刺激作用明显强于γ-SNAP。因此,虽然我们在动物模型上未检测到α-SNAP的蛋白表达变化,但由于其在递质释放中的重要作用,我们依然选择这种亚型作为研究对象。为了进一步研究α-SNAP的功能与吗啡依赖的关系,选择一个良好的、调控模式相对简单和直接的细胞模型是十分必要的。该细胞模型必须能够满足以下条件:(1)表达阿片受体;(2)拥有类似成熟神经内分泌细胞的一些特性,能释放与阿片依赖有关的神经递质,如单胺类、谷氨酸(glutamate,Glu)、γ-氨基丁酸(γ-aminobutyric acid ,GABA)等。经过初步筛选,我们选择了分化的人神经母细胞瘤细胞株SH-SY5Y作为研究对象。据研究报道,SH-SY5Y细胞株经过维甲酸(retinoic acid,RA)分化后可表现出许多类似成熟神经元的特性,如神经突起变长、电刺激的兴奋性增加、神经元特异性烯醇化酶(neuron specific enolase,NSE)表达增加、神经分泌颗粒增多等。在研究中我们发现,RA分化6天的SH-SY5Y细胞可形成较长的神经突起(图2.1),免疫印迹分析检测到分化后的细胞内NSE表达增加(图2.2)。另外,在我们的研究中亦发现了RA分化后的细胞μ阿片受体(μopioid receptor,MOR)表达上调,与以往研究相一致(图2.3)。这些特性都为我们研究阿片依赖的分子机制提供了有力的条件,尤其是与突触前神经递质释放有关的分子机制。为了研究与阿片依赖有关的分子机制,我们首先需要在SH-SY5Y细胞上建立一个阿片依赖的细胞模型。阿片长期暴露后给予纳络酮催促戒断出现cAMP超射常常被作为评定阿片依赖模型建立是否成功的一个重要指标。本研究采用免疫竞争结合法,测定了吗啡处理前后细胞内cAMP含量的变化,发现100μM吗啡作用分化SH-SY5Y细胞24h后给予纳络酮催促戒断可形成明显的cAMP超射,cAMP含量约为催促前的2.36倍(图2.5),证实了分化SH-SY5Y细胞上吗啡依赖的细胞模型建立是成功的。为了进一步确定吗啡处理过程中神经递质的释放情况,我们采用高效液相-电化学检测法(high performance liquid chromatography- electrochemical detection,HPLC-ECD)测定了急性吗啡作用及慢性吗啡作用纳络酮催促戒断后不同时间SH-SY5Y细胞培养上清液中单胺类神经递质的含量,探讨吗啡处理与神经递质释放的关系。结果显示,在急性吗啡作用下,单胺类神经递质,包括去甲肾上腺素(norepinephrine,NE)、多巴胺(dopamine,DA)、5-羟色胺(5-hydroxytryptamine,5-HT)的释放均受到普遍抑制(表3.1)。100μM吗啡孵育SH-SY5Y细胞10min~1h,细胞培养上清内NE,DA和5-HT及其代谢产物3,4-二羟基苯乙酸(3,4-dihydroxy- phenylacetic acid,DOPAC) (DA代谢产物)和5-羟基吲哚乙酸(5-hydroxyindolacetic acid,5-HIAA )(5-HT代谢产物)均显著下降,其中NE含量从16.97 ng/g蛋白下降至5.34 ng/g蛋白,下降幅度以吗啡作用10min内下降最为明显(P <0.01),在吗啡继续作用的50min内一直保持在较低水平(图3.4)。DA的变化趋势与NE类似,在吗啡作用的最初10min亦出现明显下降,而且下降幅度在2/3以上(P <0.01) (图3.6)。虽然5-HT在吗啡急性作用的早期出现中等程度的下降,但在吗啡继续作用的50min内,5-HT含量继续降低,至吗啡作用后1h达到基础水平的1/3左右(P<0.01)(图3.8)。与吗啡处理前相比,代谢产物DOPAC和5-HIAA均出现不同程度的降低(P<0.01) (图3.5,图3.7)。这些结果表明,急性吗啡作用可以显著抑制SH-SY5Y细胞单胺类神经递质的释放。在给予100μM吗啡孵育细胞24h后用100μM纳络酮催促戒断测定SH-SY5Y细胞上清单胺类神经递质的含量,结果发现:NE、DA和5-HT及其代谢产物DOPAC和5-HIAA均有不同程度的增加(表3.2)。NE的含量在戒断20 min后由戒断前7.59 ng/g蛋白增加至12.74 ng/g蛋白,增加50%左右,与催促前相比有显著性差异(P <0.01),在戒断40min时恢复至戒断前水平(图3.9)。与NE相比,DA的变化趋势更加明显,DA在戒断的10min即有增加趋势,但与催促前相比无显著性差异(P> 0.05),在戒断20min时达最高值,约为催促前基础水平的2倍(P <0.05) (图3.11)。5-HT在戒断后40min出现明显增高,增加幅度约为催促前的2倍(P <0.05) (图3.13)。与吗啡处理前相比,代谢产物DOPAC和5-HIAA均出现不同程度的增加(P<0.05) (图3.10,图3.12)。这些结果说明:慢性吗啡作用后给予纳络酮催促戒断可明显刺激SH-SY5Y细胞内单胺类神经递质的释放,在给予纳络酮20~40min时培养上清中单胺类神经递质含量达到高峰,其后迅速下降。为了进一步探讨α-SNAP是否参与了吗啡处理引起的递质释放变化的调节,我们在SH-SY5Y细胞模型上又检测了吗啡作用不同时间α-SNAP的表达情况。结果发现,α-SNAP mRNA在吗啡作用的1h、6h、24h均未检测到明显的表达变化(图4.2),Western blot检测吗啡作用1h、8h、24hα-SNAP的蛋白表达亦未发现明显改变(图4.3),上述结果与动物模型上结果相一致。以上结果提示:吗啡作用不引起α-SNAP的表达变化。那么作为一个与神经递质释放关系密切的因子,它是否参与了吗啡处理引起的突触前可塑性的改变呢?研究发现,神经递质的胞吐过程有一系列分子事件的参与,包括囊泡移动、搭靠、融合、内吞等。现在公认的递质释放中膜融合的模式是“SNARE假说”。“SNARE假说”认为,在膜融合过程中,20S复合体的聚合和解聚是其中的关键步骤。复合体的聚合始于v-SNAREs和t-SNAREs的结合。v-SNAREs和t-SNAREs分别定位于囊泡膜和质膜上,t-SNAREs包括SNAP-25和Syntaxin。膜融合发生前,t-SNAREs与v-SNAREs结合形成一个沉降系数为7S的SNARE复合物。一个7S SNARE复合物可以吸引3个α-SNAP分子,然后招募NSF使其连接至膜上。这样,SNARE、NSF和α-SNAP就形成了一个20S复合体。20S复合体一旦形成,随即在NSF的ATP酶作用下水解,同时引起SNARE分子的变构,阻止其继续聚合。SANRE解体后,形成20S复合体的各个组分又被释放到胞浆中,参与下一轮的囊泡循环。由于膜融合过程中形成20S复合体的t-SNARE位于突触前膜,所以我们推测α-SNAP在参与膜融合发生的过程中其细胞内分布有可能发生了变化,如由胞浆分布转移至胞膜。因此,我们在SH-SY5Y细胞模型上采用免疫细胞化学结合激光共聚焦检测技术又继续观察了α-SNAP在吗啡处理后的亚细胞定位。结果发现,在吗啡处理24h后给予纳络酮催促戒断20min可观测到α-SNAP开始由胞浆向胞膜转位,在随后的40min内该现象一直持续存在。在纳络酮处理60min时,转位现象仍较明显(图4.5)。而急性吗啡作用1h内未检测到上述变化(图4.4)。以上结果说明:慢性吗啡处理后给予纳络酮催促戒断可引起α-SNAP由胞浆向胞膜的转位,其转位发生的时间与递质释放变化的时间大致吻合,提示其有可能与吗啡作用引起的神经递质释放变化有关。本研究通过测定吗啡处理及戒断不同状态下SNAPs表达、亚细胞定位及神经递质的释放情况,得出以下结论:(1)慢性吗啡处理及自然戒断对大鼠脑区NAc、CPu和Hip内α-SNAP的表达无明显影响;(2)慢性吗啡处理可上调大鼠CPu内γ-SNAP的表达,但自然戒断无上述改变,慢性吗啡处理及自然戒断对大鼠NAc和Hip内γ-SNAP的表达均无明显影响;(3)SH-SY5Y细胞可用于建立慢性吗啡依赖模型;(4)急性吗啡处理可抑制SH-SY5Y细胞单胺类神经递质的释放,慢性吗啡处理后给予纳络酮催促戒断可刺激SH-SY5Y细胞单胺类神经递质的释放;(5)急慢性吗啡处理不影响SH-SY5Y细胞内α-SNAP的表达;急性吗啡处理不引起α-SNAP亚细胞定位的改变;慢性吗啡处理后给予纳络酮催促戒断可引起α-SNAP由胞浆至胞膜的亚细胞定位发生改变,其转位可能与戒断过程中神经递质释放增加有关。更多还原

【Abstract】 Opioid dependence is a chronic relapsing disorder in the brain characterized by compulsive drug taking, inability to limit the intake of drugs, and the emergence of withdrawal syndrome after cessation of drug taking. Nevertheless, the exact mechanisms underlying opioid dependence are still unknown. Accumulating evidence suggests that synaptic plasticity plays an important role in opioid dependence. These changes include presynaptic neurotransmitter release and postsynaptic receptor signal transduction. Previous studies demonstrated that morphine could affect the synaptic transmission by regulating molecules related with neurotransmitter release in presynapse.Membrane fusion between synaptic vesicle membrane and presynaptic membrane has been generally received as an important step in the process of neurotransmitter release. NSF attachment proteins (SNAPs) play important roles in membrane fusion by mediating the fusion between synaptic vesicle membrane and presynaptic membrane. Three isoforms (α-,β-, andγ-) of SNAP are expressed in mammals. In view of its function in mediating membrane fusion, SNAPs take part in many important physiological processes, including regulating Ca2+-dependent exocytosis, stimulating insulin secretion in pancreatic islandβcells, increasing surfactant secretion in alveolar type II cells, participating in the nuclear envelope formation, etc.Although considerable evidence suggests that SNAPs play key roles in the process of neurotransmitter release by mediating membrane fusion between plasma membrane and vesicle membrane, whether SNAPs are related to the presynaptic plasticity underlying opioid dependence has been not reported. To reveal the relationship between SNAPs and opioid dependence, we investigated the expression of SNAPs in different brain regions of rats with chronic morphine exposure and withdrawal, including nucleus accumbens (NAc), caudate putamen (CPu) and hippocampus (Hip), which were recognized to have close relationship with synaptic plasticity. Adult male Wistar rats were randomly assigned into control, morphine and spontaneous withdrawal group. The morphine dependent rat model was established by subcutaneous morphine injection with increasing doses for 8d, three times a day (8:00, 12:00, and 18:00). The control group was injected with the same volume of normal saline. Rats in morphine group were killed 4h after the last injection, rats in withdrawal group were killed at indicated time. Control groups were paralleled with all treatment groups. The NAc, CPu and Hip were isolated. The expression of SNAPs mRNA and protein were determined by RT-PCR and Western blot, respectively. Results showed that the expression ofγ-SNAP in CPu of morphine dependent rats was up-regulated about 25%(P<0.01) (Fig.1.4, Fig.1.9), and no alteration ofγ-SNAP in NAc and Hip was detected in morphine dependent and withdrawal groups(Fig.1.8, Fig.1.10, Table1.1, Table1.3).α-SNAP andβ-SNAP were unchanged in NAc, CPu and Hip of rats in morphine dependent and withdrawal groups. From these results above, we could draw a conclusion that morphine dependence leads to up-regulation ofγ-SNAP in CPu, but it did not have any effects on the expression ofα-SNAP andβ-SNAP in NAc, CPu and Hip(Fig1.2,Fig1.3,Fig1.5,Fig1.6, Fig1.7, Table1.1,Table1.2), which suggested that 3 isoforms of SNAPs functioned differently in morphine dependence and might be related with the secretion of specific neurotransmitter. The release of presynaptic neurotransmitter in morphine dependence and withdrawal rat was not mediated by the content ofα-SNAP andβ-SNAP, and the intracellular translocation and intrinsic activity ofα-SNAP andβ-SNAP may contribute to the molecular mechanisms underlying morphine dependence and withdrawal.To further confirm the relationship between SNAPs and opioid dependence,α-SNAP, an isoform considered to play a key role in neurotransmitter release, was selected in the following study. Accumulating evidence reported the function ofα-SNAP in membrane fusion between vesicle membrane and presynaptic membrane, which has been regarded as the most important step in calcium-dependent exocytosis during the process of neurotransmitter release. It was reported thatα-SNAP functioned in stimulating NSF ATPase, and the effect ofα-SNAP significantly surpassed that ofγ-SNAP. Therefore, although no alteration ofα-SNAP expression was detected in animal model, this isoform was still focused on. To further study the function ofα-SNAP in morphine treatment, a well defined experimental cell model was necessarily needed owing to the relative simple and direct regulation mechanisms. The experimental model must be characterized by following traits: (1) expressing opioid receptor (2) processing similar features to mature neuroendocrine cell and secreting neurotransmitters related to opioid dependence, such as monoamine transmitters, glutamate, GABA, etc. After preliminary screening, the differentiated human neuroblastoma SH-SY5Y cell line was singled out to be applied for this study. After differentiation with retinoic acid(RA), SH-SY5Y cells were reported to show many characteristics of mature neurons, such as the appearance of extensive neurite outgrowth and electrical excitability, enhancement of neuron specific enolase(NSE) activity and increase of neurosecretory granules, etc.In this study, we observed the formation of long neuritic processes in SH-SY5Y cells 6 days after treated with RA (Fig.2.1). The expression of NSE was also measured by Western blot. Results indicated that NSE andμopioid receptor(MOR) were up-regulated in differentiated SH-SY5Y cells compared with that in undifferentiated cells (Fig.2.2 , Fig.2.3), which was in accordance with previous research. All these properties of differentiated SH-SY5Y cells facilitated research in the molecular mechanisms of opioid dependence, especially in the aspect of neurotransmitter release related with synaptic plasticity. To elucidate the molecular mechanisms of opioid dependence, a cell model for opioid dependence was established. The cAMP overshoot after morphine withdrawal was generally applied to evaluate whether the cell model was successful. In our study, we assessed the content of intracellular cAMP by the LANCE cAMP assay and discovered cAMP overshoot in SH-SY5Y cells after naloxone-precipitated withdrawal. The content of cAMP markedly increased to a level as 2.36 fold as that in untreated cells (Fig.2.5). This confirmed that the cell model of opioid dependence in our study was successfully established.In order to confirm the neurotransmitter changes, the contents of monoamine neurotransmitters and their metabolites were investigated in SH-SY5Y cell culture supernatant by HPLC-ECD under acute morphine treatment and naloxone-precipitated withdrawal after long-term morphine treatment. Data showed that the monoamine neurotransmitters including NE, DA and 5-HT were generally inhibited with acute morphine administration (Table3.1). After incubation with 100μM morphine for 10 min ~ 1h in SH-SY5Y cells, NE, DA and 5-HT as well as their metabolites DOPAC (DA metabolite) and 5-HIAA (5-HT metabolite) were significantly reduced, the content of NE was significantly decresed from 16.97 ng/g to 5.34 ng/g protein at 10min after morphine exposure (P<0.01). In the following 50 min, it did not significantly decline compared to that in 10min after morphine treatment and kept at a relatively low level (Fig.3.4). The changes in DA showed a similar trend to NE , that was, an obvious decline was detected in the first 10 min, lower than the basal level (P<0.01)(Fig.3.6). The content of 5-HT was significantly decreased (P<0.05) in the initial 10 min and continuted decline till 1/3 of the original level at 1h after morphine treatment (P<0.01) (Fig.3.8). Their metabolites, DOPAC and 5-HIAA were also down-regulated compared with cells without exposure to morphine (P<0.01) (Fig.3.5, Fig.3.7). These results showed that acute morphine treatment significantly attenuated the release of monoamine neurotransmitters.When exposed to 100μM naloxone after incubation of morphine for 24h, monoamine neurotransmitter release was investigated in SH-SY5Y cell supernatants. Results showed that NE, DA and 5-HT and their metabolites, DOPAC and 5-HIAA increased (Table3.2). The content of NE rose from 7.59 ng/g to 12.74 ng/g, higher nearly 0.5 fold, at 20 min after the onset of withdrawal(P<0.01), then dropped to the basal level at 40 min after the onset of withdrawal (Fig.3.9). Compared with NE, the change in DA was more obvious, and the DA content reached a peak at 20 min after withdrawal, approximately twice the original level(P<0.05)(Fig.3.11). 5-HT was also detected a significant increase at 40 min, nearly twice the level of untreated ones (P<0.05) (Fig.3.13). Their metabolites, DOPAC and 5-HIAA, also increased, compared with that of cells before naloxone treatment(P<0.05) (Fig.3.10, Fig.3.12). These results showed that naloxone-precipitated withdrawal after morphine dependence significantly stimulated the release of monoamine neurotransmitters. The content of monoamine neurotransmitters reached peaks at 20~40min after the onset of naloxone withdrawal, then dropped rapidly in the following time.To determine whetherα-SNAP participated in the changes of neurotransmitters induced by morphine, theα-SNAP expression was detected in SH-SY5Y cells after morphine administration. Data showed that no alteration ofα-SNAP mRNA expression was detected at 1h, 6h, 24h in SH-SY5Y cells after morphine administration (Fig.4.2), and there was no change inα-SNAP protein expression at 1h, 8h, 24h after morphine treatment by Western blot analysis (Fig.4.3). The results obtained from cells were in accordance with that obtained from animal model. Data suggested that morphine treatment did not result in any change inα-SNAP expression. However, as a key role in neurotransmitter release, whetherα-SNAP takes part in presynaptic plasticity underlying morphine dependence has not been elucidated.Many evidences showed that a series of molecular events take place in the process of neurotransmitter exocytosis, including vesicle trafficking, docking, fusion and internalization.“SNARE hypothesis”is widely accepted in membrane fusion during the neurotransmitter release. According to“SNARE hypothesis”, in the process of membrane fusion, 20S complex assembly and disassembly are the key steps. Complex assembly begins with binding between v-SNAREs and t-SNAREs. v-SNAREs and t-SNAREs are located in vesicle membrane and target membrane, respectively. t-SNAREs include SNAP-25 and Syntaxin. At the beginning of membrane fusion, t-SNAREs bound to v-SNAREs, and 7S SNARE complex is formed. One 7S SNARE complex can recruit 3α-SNAP molecules, this is followed by the binding of NSF. Thus, SNAREs,α-SNAP and NSF assembled a 20S complex. Then, subsequent ATP hydrolysis of NSF leads to disassembly of 20S complex and a conformational change in SNARE molecules, which prevented from its reassembly. Thus, all components of SNAREs enter into next cycle of vesicle transport. Since t-SNARE, a key element in the formation of 20S complex, located at presynaptic membrane, we supposed that the translocation ofα-SNAP might take place in the process of membrane fusion, for instance, from cytoplasm to cell membrane. Therefore, we further examined the subcellular location ofα-SNAP by confocal laser scanning microscopy in SH-SY5Y cells with morphine treatment. Results showed thatα-SNAP began to migrate from cytoplasm to cell membrane at 20min after naloxone withdrawal and lasted in the following 40 minutes. An obvious translocation was still observed at 60min after withdrawal (Fig.4.5). Nevertheless, no change was detected in subcellular location ofα-SNAP with acute morphine treatment (Fig.4.4). It showed that naloxone-precipitated withdrawal after chronic morphine treatment altered the subcellular localization ofα-SNAP in SH-SY5Y cells, and the time courses of changes in monoamine neurotransmitters approximately coincided with the time ofα-SNAP translocation, which might indicate a close relationship between neurotransmitter release andα-SNAP translocation.In this study, we drew the following conclusions through determination of SNAPs expression and subcellular location as well as neurotransmitters release after morphine treatment: (1) Chronic morphine administration and spontaneous withdrawal did not affectα-SNAP expression in NAc, CPu and Hip of rats. (2) Chronic morphine administration up-regulatedγ-SNAP expression in CPu of rats, but did not changeγ-SNAP expression in NAc and Hip of rats. Spontaneous withdrawal had no effect onγ-SNAP expression in NAc, CPu and Hip. (3) SH-SY5Y cells could be used to establish cell model of morphine dependence. (4) Acute morphine treatment significantly attenuated the release of monoamine neurotransmitters, while naloxone-precipitated withdrawal significantly stimulated the release of monoamine neurotransmitters. (5) Acute and chronic morphine treatment did not affectα-SNAP expression in SH-SY5Y cells. Acute morphine treatment did not alter the subcellular localization ofα-SNAP in SH-SY5Y cells. Naloxone-precipitated withdrawal after morphine dependence resulted inα-SNAP translocation from cytoplasm to cell membrane, which might be related with the neurotransmitter release after morphine withdrawal.更多还原