【作者】 武文鹏；

【导师】 孙远征；

【作者基本信息】 黑龙江中医药大学 ， 针灸推拿学， 2016， 博士

【摘要】 目 的:观察电针对吗啡戒断大鼠空间学习记忆能力、VTA和NAc脑组织病理形态学、单胺类神经递质、Ca2+-CaM-CaMKⅡ-CREB信号通路的影响,探讨电针减轻吗啡戒断症状的作用机制,为吗啡戒断的临床治疗提供实验基础和理论依据。方 法:采用Morris水迷宫筛选出学习记忆能力正常的SD大鼠56只,将其随机分为对照组14只、造模组42只。采用剂量递增法颈背部皮下注射吗啡,连续5日,每日2次;末次吗啡注射3小时后,纳洛酮进行急性催促戒断。造模成功的 42只大鼠再随机分为模型组、针刺组、电针组,每组各 14只。其中对照组和模型组进行与针刺组相同时间、相同程度的捉抓刺激,不予任何治疗干预。针刺组取大鼠“神庭”穴、“百会”穴、双侧“肾俞”穴,常规消毒,以毫针分别刺入上述腧穴,进针2～3mm,快速捻转1min，留针15min,1次/日,连续干预6日。电针组取穴同针刺组,在针刺组操作的基础上将“神庭”穴与“百会”穴连接至导线的正负极;双侧“肾俞”穴连接至导线的正负极,接通电针治疗仪,选用疏密波(频率为2/100Hz),干预15min,1次/d,连续干预6d。采用Morris水迷宫检测大鼠的学习记忆能力;在光镜下观察VTA和NAc脑组织病理学变化;采用ELISA法进行测定血清单胺类神经递质(DA、5-HT、NE)含量;流式细胞术检测VTA和NAc神经细胞内[Ca2+]i和 CaM活性;免疫组化染色检测 CaMK Ⅱ和 CREB 阳性表达,及CaMK Ⅱ和CREB磷酸化;实时定量荧光PCR检测VTA和NAc神经元 CaMKⅡ α亚基 mRNA表达及 CaMK Ⅱ α亚基磷酸化水平;Western Blot 检测 VTA 和 NAc 神经元 CaMK Ⅱ α 蛋白表达及CaMK Ⅱ α亚基磷酸化水平。应用SPSS 1 8.0软件包进行统计分析,计量资料以-x±S表示,组间均数比较采用单因素方差分析,方差齐时用LSD法进行组间两两比较,方差不齐时用Tamhαne’s T2检验,P<0.05差异有统计学意义。结 果:1.体质量变化:针刺干预后,与对照组比较,模型组大鼠体质量下降明显,差异具有统计学意义(P<0.01)。与模型组比较,针刺组和电针组大鼠体质量具体显著性差异(P<0.05)。与针刺组比较,电针组大鼠体质量无显著性差异(P>0.05)。2.Morris水迷宫检测结果:在定向航行实验中,对照组、模型组、针刺组、电针组大鼠随着训练测试次数的增加,各组大鼠的逃避潜伏期均逐渐缩短。同一时间点与对照组比较,模型组大鼠逃避潜伏期明显延长(P<0.01)。同一时间点与模型组比较,针刺组和电针组大鼠逃避潜伏期明显缩短(P<0.01)。同一时间点与针刺组比较,电针组大鼠逃避潜伏期缩短(P<0.05)。在空间搜索实验中,与对照组比较,模型组大鼠跨越平台次数明显减少(P<0.05)。与模型组比较,针刺组和电针组大鼠跨越平台次数显著增加(P<0.05,P<0.0 1)。与针刺组比较,电针组大鼠跨越平台次数明显增加(P<0.05)。3.HE染色结果:对照组:脑组织染色清晰,神经细胞形态规整,胞质胞核界限清晰,无组织水肿,散在少量的炎细胞,血管无充血。模型组:脑组织结构疏松,神经细胞形态欠规整,部分神经元细胞核固缩，胶质细胞明显增多,炎细胞数目增多,毛细血管肿胀。针刺组和电针组:脑组织病理状况有明显改变,主要表现为脑组织疏松程度明显减轻,神经元细胞核固缩数目减少,胶质细胞和炎细胞数目也减少,毛细血管肿胀减轻。4.血清单胺神经递质含量:与对照组比较,模型组大鼠血清DA、5-HT、NE含量显著升高,差异具有统计学意义(P<0.01)。针刺组和电针组吗啡戒断大鼠血清DA、5-HT、NE含量显著减少,与模型组比较,差异具有统计学意义(P<0.01)。与针刺组比较,电针组血清DA、5-HT、NE含量减少,差异具有统计学意义(P<0.05)。5.流式细胞术结果:针刺组和电针组大鼠VTA和NAc神经细胞内[Ca2+]i和CaM活性比模型组显著降低(P<0.05,P<0.01)。与针刺组比较,电针干预抑制慢性吗啡依赖纳洛酮急性催促戒断所引起的神经细胞内[Ca2+]i 和 CaM 活性急剧升高更为显著(P<0.05)。6.免疫组化结果:与模型组比较,针刺和电针干预能够显著减少吗啡戒断大鼠 VTA和N Ac神经元 CaMKⅡ 阳性细胞表达(P<0.05,P<0.01),减少CREB 阳性细胞表达(P<0.05,P<0.01),抑制CREB磷酸化(P<0.05,P<0.01),且电针组比针刺组更为显著(P<0.05)。7.实时定量荧光 PCR 结果:针刺和电针干预能够显著下调CaMK Ⅱ α mRNA表达,抑制CaMK Ⅱ α亚基磷酸化,与模型组比较差异具有显著性(P<0.05,P<0.0 1),且电针组比针刺组更为显著(P<0.05)。8.Western Blot结果:针刺和电针干预能够显著下调CaMKⅡαmRNA蛋白表达,抑制CaMK Ⅱ α亚基磷酸化,与模型组比较差异具有显著性(P<0.05,P<0.01),且电针组比针刺组更为显著(P<0.05)。结 论:1.电针能够减缓慢性吗啡依赖纳洛酮急性催促戒断大鼠体质量丢失,能够缩短吗啡戒断大鼠平台逃避潜伏期,增加跨越平台次数,改善空间学习记忆能力。2.电针能够改善吗啡戒断大鼠神经元病理形态学改变。3.电针能够降低吗啡戒断大鼠血清单胺类神经递质DA、5-HT、NE含量。4.电针能够抑制慢性吗啡依赖纳洛酮急性催促戒断引起的细胞内[Ca2+]i和CaM活性的急剧升高,下调VTA和NAc神经元CaMKⅡ阳性表达,下调VTA和NAc神经元CaMKⅡα亚基mRNA表达和蛋白表达,抑制CaMKⅡα亚基磷酸化,下调CREB核转录因子蛋白表达,并抑制CREB磷酸化。5.电针抑制吗啡戒断反应、改善吗啡戒断大鼠学习记忆能力可通过Ca2+-CaM-CaMKⅡ-CREB信号途径发挥其生物学效应。更多还原

【Abstract】 Objective:Observing electricity on morphine withdrawal rats brain tissue spatial learning and memory ability,and different brain regions pathomorphism,single amine neurotransmitter,Ca2+-CaM-CaMK-after CREB signaling pathway and the effect of study will reduce the mechanism of action of morphine withdrawal symptoms,for morphine withdrawal provide experimental foundation and theoretical basis for clinical treatment.Method:Using Morris water maze select learning and memory ability of normal SD rat 56,only will be randomly divided into control group 14,module of 42.Dose escalation method is used to the back of the neck subcutaneous injection of morphine,5 in a row,2 times a day;Three hours after the last morphine injection and naloxone on acute urged withdrawal.Building successful 42 rats were randomly divided into model group,acupuncture group,the electric acupuncture group,each group of 14 only.The control group and model group and acupuncture group the same time,the same degree of catch catch a stimulus,will not be any treatment intervention.Acupuncture group rats "god court","the will","shen shu" point on both sides,regular disinfection,with filiform needle Pierce the acupoints,respectively,into the needle 2-3mm,fast turning 1 min,15min,retaining needle 1/d,6 d continuous intervention.Electric acupuncture group with acupuncture group and find out in the acupuncture group operation will be the basis of "god court" and "best" connect to lead is negative;Bilateral"shen shu" point connected to wires are negative,turn on the electric acupuncture therapeutic apparatus,selection of dilatational wave(frequency for 2/100hz),intervention in 15 min,1/d,6 d continuous intervention.Using Morris water maze test rats learning and memory ability;Under light microscopy to observe the VTA and NAc brain tissue pathology change;Using the ELISA method for determination of blood listing amine neurotransmitter(DA and 5-HT,NE)content;Flow cytometry to detect the VTA and NAc nerve cells[Ca2+]i and CaM activity;Immunohistochemical staining detection CaMK Ⅱ and after CREB positive expression,and CaMKⅡand after CREB phosphorylation;Real-time fluorescent quantitative PCR detection VTA and NAc neurons CaMK II alpha subunit mRNA expression and CaMK Ⅱ alpha subunit phosphorylation level;Western Blot detection VTA and NAc neurons CaMK Ⅱ alpha protein expression and CaMK Ⅱ alpha phosphorylation level.SPSS18.0 software package was used to statistical analysis,measurement data to plus or minus S said,mean comparison between groups using single factor analysis of variance,covariance together with LSD method when compared between two groups,the variance is used when Tamhane’S T2 test,P<0.05,the difference was statistically significant.Result:1.The body quality change:after acupuncture intervention,compared with control group,model group rats significantly lower body quality,statistically significant difference(P<0.01).Compared with model group,body acupuncture group and the curative group rats quality specific significant difference(P<0.05).Compared with acupuncture group,the curative group of rats body quality there was no significant difference(P>0.05).2.Morris water maze test results:in the directional navigation experiments,the control group,model group,acupuncture group,the curative group rats with the increase of training test times,each rat escape latent period shorten.At the same time point compared with control group,model group rats escape significantly prolong the incubation period(P<0.01).At the same time point compared with model group,acupuncture group and the curative group significantly shorten the escape latency of rats(P<0.01).At the same time point compared with acupuncture group,rat escape latent period shorten electric acupuncture group(P<0.05).In the search space experiments,compared with control group,model group rats significantly extend across platform(P<0.05).Compared with model group,acupuncture group and the curative group rats crossing platform number increased significantly(P<0.05,P<0.01).Compared with acupuncture group,the curative group rats crossing platform number increased significantly(P<0.05).3.HE staining results:normal group:the brain tissue staining is clear,neat,nerve cells in the form of cytoplasm nucleus boundaries clear,no tissue edema,scattered in a small amount of inflammatory cells,blood vessels without the congestion.Model group:brain tissue structure is loose,owe neat,nerve cells form part of the nucleus pycnosis neurons and glial cells increased obviously,increased number of inflammatory cells,capillary swelling.Acupuncture group and the curative group:brain tissue pathologic conditions have obvious change,main show is loose significantly reduce brain tissue,reduced Numbers of nucleus pycnosis neurons,glial cells and inflammatory cells number also reduce,capillary swelling reduce.4.Blood listing amine neurotransmitter content:compared with control group,model group rats serum DA,5-HT,NE content increased significantly,statistically significant difference(P<0.01).Acupuncture group and the curative group of morphine withdrawal rats serum DA,5-HT,NE content is significantly reduced,compared with model group,the difference statistically significant(P<0.01).Compared with acupuncture group,serum DA electric acupuncture group,5-HT,NE content reduced,statistically significant difference(P<0.05).5.Flow cytometry results:acupuncture group and the curative group rat VTA and NAc nerve cells[Ca2+]i and CaM activity than model group significantly reduced(P<0.05,P<0.01).Inhibition compared with acupuncture group,electric acupuncture intervention of chronic morphine dependence naloxone in nerve cells caused by acute urged withdrawal[Ca2+]i and CaM activity dramatically increases more significantly(P<0.05).6.Immunohistochemical results:compared with model group,acupuncture and cupping intervention can significantly reduce morphine withdrawal rats VTA and NAc neurons CaMKⅡ positive cells expression(P<0.05,P<0.01),decrease after CREB positive cells expression(P<0.05,P<0.01),inhibit after CREB phosphorylation(P<0.05,P<0.01),and electric acupuncture grpup is more significant than acupuncture group(P<0.05).-7.A real-time fluorescent quantitative PCR results:acupuncture and cupping intervention can significantly lower CaMK Ⅱ alpha mRNA expression,inhibition of CaMKⅡ alpha subunit phosphorylation,with significant difference compared with model group(P<0.05,P<0.01),and the curative group is more significant than acupuncture group(P<0.05).8.Western Blot results:acupuncture and cupping intervention can significantly lower CaMK Ⅱ alpha mRNA,protein expression,inhibition of CaMKⅡ alpha subunit phosphorylation,with significant difference compared with model group(P<0.05,P<0.01),and electric acupuncture group is more significant than acupuncture group(P<0.05).Conclusion:1.Electric acupuncture can reduce acute chronic morphine dependence naloxone urged withdrawal rats lost body quality,could shorten the morphine withdrawal rats platform to escape the incubation period,increase the cross platform,to improve the spatial learning and memory ability.2.Electric acupuncture can improve pathomorphism changes of morphine withdrawal rats neurons.3.Electric acupuncture can reduce morphine withdrawal rats blood listing amine neurotransmitter DA,5-HT,NE content.4.Electric acupuncture can inhibit chronic morphine dependence naloxone in acute urged withdrawal caused by intracellular[Ca2+]i and CaM activity rise sharply,cut the VTA and NAc neurons CaMK Ⅱ positive expression,cut the VTA and NAc neurons CaMK Ⅱ alpha subunit mRNA expression and protein expression,inhibition of CaMK Ⅱ alpha subunit phosphorylation,cut after CREB nuclear transcription factor protein expression,and suppress after CREB phosphorylation.5.Will inhibit morphine withdrawal reaction,the ability of learning and memory in the rat model of morphine withdrawal by Ca2+-CaM-CaMK Ⅱ-after CREB signaling pathways play its biological effect.更多还原