【作者】 杨胜昌；

【导师】 马春玲；

【作者基本信息】 河北医科大学 ， 法医学， 2010， 硕士

【摘要】 目的:当前,毒品滥用日趋严重,严重地危害着社会进步和经济发展。阿片类(如吗啡、海洛因)是我国吸毒人员使用最多的成瘾物质,其特征是强迫性使用药物,但其成瘾机制尚不完全明确。吗啡成瘾涉及到神经系统多种神经递质、调质及神经肽的改变。胆囊收缩素(cholecystokinin,CCK)是一种存在于体内多个系统的神经肽,在中枢神经系统内有着广泛分布,参与机体多种功能的调控。其中八肽胆囊收缩素(CCK-8)是目前已知的作用最强的内源性抗阿片肽。有研究表明吗啡依赖时在多个脑区发现了CCK-8的上调,CCK-8通过其受体参与了吗啡成瘾过程。研究表明长期应用阿片类药物可导致多个脑区神经元的一系列适应性和可塑性变化,如尾壳核(caudate putamen,CPu)、伏隔核(nucleus accumbens,NAc)、杏仁核(amygdaloid nucleus,Amy)、腹侧被盖区(ventral tegmental area,VTA)、黒质(substantia nigra,SN)、导水管周围灰质(periaoueductal gray matter,PAG)、蓝斑(locus caeruleus, LC)等。吗啡的长期使用使特定脑区和核团的某些蛋白和基因表达的改变,如阿片受体、G蛋白、第二信使合成酶、蛋白激酶及转录因子等。转录因子cAMP反应元件结合蛋白(cAMP response element binding protein, CREB)在吗啡成瘾过程中至关重要。本研究在以剂量递增法建立吗啡依赖及戒断模型的基础上,采用腹腔和侧脑室局部注射的方式,观察CCK-8及其受体拮抗剂对吗啡戒断症状及多个脑区内CREB和pCREB的变化,以探讨CCK-8参与吗啡依赖和戒断的受体后机制。方法:研究采用成年雄性Wistar大鼠(由河北医科大学实验动物中心提供),体重200-220g,以吗啡剂量递增法(10、20、30、40、50mg·kg-1)皮下注射吗啡5天,每日2次(8:00, 20:00),第6天给予吗啡后两小时腹腔注射纳洛酮5mg·kg-1急性催促戒断,建立吗啡依赖及戒断模型。应用免疫组织化学方法观察与吗啡成瘾相关的多个脑区内CREB和pCREB的变化。研究采用腹腔注射及侧脑室注射两种给药途径,在每日给予吗啡前30min注射CCK-8(50μg·kg-1, i.p.; 0.1μg/rat,i.c.v)、CCK1受体拮抗剂L-364,718(1mg·kg-1, i.p.; 1μg/rat,i.c.v)、CCK2受体拮抗剂LY-288,513 (1mg·kg-1, i.p.;1μg/rat,i.c.v)进行干预,末次注射吗啡后2小时给予纳洛酮(5mg·kg-1)催促戒断,按照戒断症状评分表评价CCK-8及其受体拮抗剂对吗啡依赖大鼠戒断症状的影响,用免疫组化检测各脑区在依赖和戒断后CREB和pCREB的变化。结果:1 CCK-8及其受体拮抗剂对吗啡依赖形成及戒断症状的影响在成功建立吗啡依赖及催促戒断模型基础上,腹腔和侧脑室给予CCK-8及L-364,718、LY-288,415,均不同程度的抑制了吗啡依赖的形成,戒断症状明显减轻。2 CCK-8及其受体拮抗剂对吗啡戒断大鼠尾壳核、伏隔核、杏仁核、黒质、腹侧被盖区、导水管周围灰质和蓝斑CREB和pCREB的影响应用免疫组织化学方法观察了CCK-8及CCK受体拮抗剂对各组大鼠尾壳核、伏隔核、杏仁核、黒质、腹侧被盖区、导水管周围灰质和蓝斑内CREB和pCREB的影响。结果发现:在伏核内CREB和pCREB的趋势一致,即依赖组明显升高(P<0.01),戒断后进一步升高,与依赖组相比有显著差异(P<0.01),腹腔及侧脑室注射CCK-8、L-364,718、LY-288,513均使伏核内CREB和pCREB下降,与戒断组相比有显著差异(P<0.01);在隔核内,CREB和pCREB在依赖组明显升高(P<0.01),CREB在戒断后,进一步升高,与依赖组相比有显著差异(P<0.01),而pCREB在戒断组与依赖组相比无显著差异(P>0.05),腹腔注射L-364,718、LY-288,513,侧脑室注射CCK-8、L-364,718、LY-288,513均使隔核内CREB和pCREB下降,与戒断组相比有显著性差异(P<0.01),但是腹腔注射CCK-8对CREB和pCREB无明显影响,与戒断组相比无显著差异(P>0.05);在尾壳核CREB和pCREB的趋势一致,即依赖后明显升高(P<0.01),戒断后进一步升高,与依赖组相比具有显著差异(P<0.01),腹腔及侧脑室注射CCK-8、L-364,718、LY-288,513均使尾壳核内CREB和pCREB水平下降,与戒断组相比具有显著差异(P<0.01);在杏仁核内,CREB在依赖组相对于盐水组无明显差异(P>0.05),pCREB在依赖组明显升高,与盐水组相比具有显著差异(P<0.01),戒断后CREB和pCREB均明显升高,与依赖组相比具有显著差异(P<0.01),仅腹腔及侧脑室注射LY-288,513使CREB下降,与戒断组相比具有显著差异(P<0.01),其余干预对CREB无影响,与戒断组相比无显著差异(P>0.05),腹腔注射L-364,718、LY-288,513和侧脑室注射CCK-8、L-364,718、LY-288,513均使pCREB下降,与戒断组相比具有显著差异(P<0.01),但是腹腔注射CCK-8对pCREB无影响,与戒断组相比无显著差异(P>0.05);在腹侧被盖区内,CREB在各组之间无明显差异(P>0.05),pCREB在依赖组明显升高(P<0.01),戒断组相对于依赖组无明显变化(P>0.05),腹腔及侧脑室注射CCK-8、L-364,718、LY-288,513对pCREB无明显影响,与戒断组相比无明显差异(P>0.05);在黒质内,CREB在各组之间无明显差异(P>0.05),pCREB在依赖组明显升高(P<0.01),戒断组相对于依赖组无明显差异(P>0.05),仅侧脑室注射CCK-8使黒质内pCREB的下降,与戒断组相比有显著差异(P<0.01),其余干预均对pCREB无明显影响,与戒断组相比无显著差异(P>0.05);在中脑导水管周围灰质内,CREB在各组之间无明显差异(P>0.05),pCREB在依赖后明显升高(P<0.01),戒断后pCREB明显下降,与依赖组相比具有显著差异(P<0.01),腹腔及侧脑室注射CCK-8、L-364,718、LY-288,513均使中脑导水管周围灰质内pCREB水平升高,与戒断组相比具有显著差异(P<0.01);在蓝斑内,CREB在各组之间无显著差异(P>0.05),pCREB在依赖后明显升高,与盐水组相比具有显著差异(P<0.01),戒断后进一步升高,与依赖组相比具有显著差异(P<0.01),腹腔及侧脑室注射L-364,718、LY-288,513均使蓝斑内pCREB的水平下降,与戒断组相比具有显著差异(P<0.01),但是腹腔及侧脑室注射CCK-8对pCREB水平无明显影响,与戒断组相比无显著差异(P>0.05)。结论:本研究证实尾壳核、伏隔核、杏仁核、黒质、腹侧被盖区、导水管周围灰质和蓝斑参与了吗啡依赖和戒断过程,CCK-8及其受体拮抗剂均可减轻吗啡依赖大鼠的戒断症状,并通过不同方式调节上述脑区CREB发挥作用。更多还原

【Abstract】 Objective: For the moment, drug abuse become more and more severe which restrict social and economic development. Opioid(such as morphine and heroin) is the most commonly abused drug in our country, characterized by compulsive drug use, but the mechanism is not indentified yet. Morphine addiction involve many kinds of neurotransmitters, neuro-modulators and neuropeptide. Cholecystokinin (CCK) is a neuropeptide found in varieties systems, which has a borad distribution in the central system and takes part in regulation of mang physiological functions. Study reported that CCK-8 is the most potent endogenous anti-opioid peptides and there is a CCK-8 up-regulation in muti-brain region with morphine dependence, therefore CCK-8 participates in the morphine dependence by combining with its receptors.Long-term application of opium results in neural adaptive and plasticity change in many diffirent brain regions, such as caudate putamen, nucleus accumbens, amygdaloid nucleus, ventral tegmental area, substantia nigra, periaou- eductal gray matter, locus caeruleus and so on. Morphine addiction involves temporary protein and long-term gene expression changes in these regions, such as opioid receptor, G-protein, second messenger synthetase, protein kinase, transcription factor. CREB, as a nuclear factor, plays a important role in this process.In our study, morphine dependent and withdrawal models were established by subcutaneous injection of morphine in gradually increased dose. CCK-8 and its recepter antagoists were injected by i.p and i.c.v to observe their effect on morphine withdrawal. Then, immunohistochemical method was used to measure the change of CREB and pCREB in the brain region concerned to explore the cellular signal transduction mechanism of CCK on morphine dependent and withdrawal.Method: Adult male Wistar rats were used(200-220g) which were provided by Experimental Animal Center of Hebei Province.The morphine dependent model was established by subcutaneous injectiong of morphine. The daily dose gradually increased as following: 10, 20, 30, 40, 50mg·kg-1 twice a day, at 8:00 and 20:00, for 5 days. On the 6th day, 50mg·kg-1 morphine was given at 8:00, withdrawal syndrome was precipitated by intraperitoneal injection of naloxone 5 mg·kg-1 two hours later.Two ways, i.p and i.c.v, were used in our research to observe the effect of CCK on morphine withdrawal syndrome, CCK-8(50μg·kg-1, i.p.; 0.1μg/rat, i.c.v), L-364,718(1mg·kg-1, i.p.; 1μg/rat, i.c.v), LY-288,513(1mg·kg-1, i.p.; 1μg/rat,i.c.v) were given 30min before morphine injection every day. At the last day, naloxone was used two hours after morphine given. Then, the morphine withdrawal syndrome was observed and estimated by Gellert-Holtzman scale. The change of CREB and pCREB were measured by immunohistochemical method in the related brain regionsResult:1 CCK-8 and its antagosists on morphine dependent and morphine withdrawal syndrome. Morphine dependent and withdrawal models were successfully established. After chronic treatment with CCK-8 and its receptor antagosists by i.p or i.v.c, development of morphine dependence was inhibited and morphine withdrawal syndrome was alleviated.2 Effect of CCk-8 and its receptor antagosists on CREB and pCREB in caudate putamen, nucleus accumbens, amygdaloid nucleus, ventral tegmental area, substantia nigra, periaoueductal gray matterand locus caeruleus of morphine withdrawal rats.The change of CREB and pCREB were measured in caudate putamen, nucleus accumbens,amygdaloid nucleus, ventral tegmental area, substantia nigra,periaoueductal gray matter,and locus caeruleus to observe the effect of CCK-8 and its receptor antagosists on morphine dependent and withdrawal by immunohistochemical method. The results showed that in the AcbSH there was a similar tendency of CREB and pCREB. Chornic morphine treatment increased CREB and pCREB expression(P<0.01) and they further increased after naloxone precipitation(P<0.01). CCK-8(i.p.;i.c.v), L-364,718(i.p.; i.c.v), LY-288,513(i.p.; i.c.v)could decrease their expression(P<0.01). In the LSI, chornic morphine treatment incressed CREB and pCREB (P<0.01). Naloxone treatment increased CREB (P<0.01) but had no effect on pCREB (P>0.05). CCK-8 (i.c.v), L-364,718(i.p.; i.c.v), LY-288,513(i.p.;i.c.v)treatment decreased the CREB and pCREB expression(P<0.01), but CCK-8 (i.p.) had no effect (P>0.05). In the CPu , there was a similar tendency of CREB and pCREB. Chornic morphine treatment increased CREB and pCREB expression(P<0.01) and they further increased after naloxone precipitation(P<0.01). CCK-8(i.p.;i.c.v), L-364,718 (i.p.; i.c.v), LY-288,513(i.p.; i.c.v)could decrease their expression(P<0.01). In amygdaloid nucleus, chornic morphine treatment increased pCREB(P<0.01)but had no effect on CREB. Naloxone treatment increased CREB and pCREB(P<0.01). LY-288,513(i.p.; i.c.v)decreased CREB expression(P<0.01), but CCK-8 (i.p.; i.c.v),L-364,718(i.p.; i.c.v)had no effect on CREB(P>0.05). CCK-8 (i.c.v),L-364,718(i.p.;i.c.v),LY-288,513(i.p. ;i.c.v)decreased pCREB(P<0.01), but CCK-8 (i.p.)had no effect on pCREB(P>0.05).In the VTA, there was no distinct difference of CREB among each group.Chornic morphine treatment increased pCREB(P<0.01). Naloxone treatment had no effect on pCREB (P>0.05). CCK-8 (i.p; i.c.v), L-364,718(i.p.;i.c.v), LY-288,513(i.p.;i.c.v) had no effect on pCREB (P>0.05).In the substantia nigra, there was no obvious difference of CREB among each group. Chornic morphine treatment increased pCREB (P<0.01).Naloxone treatment has no effect on pCREB(P>0.05).CCK-8 (i.c.v)decreased pCREB (P<0.01), but CCK-8 (i.p.),L-364,718(i.p.;i.c.v), LY-288,513(i.p.;i.c.v)had no effect on pCREB (P>0.05). In PAG, there was no distinct difference of CREB among each group. Chornic morphine treatment increased pCREB (P<0.01). Naloxone treatment decreased pCREB (P < 0.01), CCK-8 (i.p.;i.c.v ) , L-364,718(i.p.;i.c.v), LY-288,513(i.p.;i.c.v)increased pCREB(P<0.01).In LC, there was no obvious difference of CREB among each group. Chornic morphine treatment increased pCREB ( P < 0.01 ) .Naloxone treatment increased pCREB (P<0.01), L-364,718(i.p.;i.c.v), LY-288,513(i.p.;i.c.v)decreased pCREB (P<0.01), but CCK-8 (i.p. ;i.c.v)had no effect on pCREB.Conclusion:Our findings suggested that caudate putamen, nucleus accumbens, amygdaloid nucleus, ventral tegmental area, substantia nigra, periaoueductal gray matter and locus caeruleus were involved in the morphine dependent and withdrawal process. CCK-8、L-364,718、LY-288,513 could alleviate morphine withdrawal syndrome through regulating CREB and/or pCREB expression in different brain regions.更多还原