【作者】 蔺芳菊；

【导师】 杨晓苏；

【作者基本信息】 中南大学 ， 神经病学， 2009， 硕士

【摘要】 研究背景及目的失眠是最为常见的一种睡眠障碍,是多种躯体、精神和行为疾病所具有的常见临床表现,但其确切的发生机制至今仍不明了。对睡眠发生的相关解剖结构研究发现,觉醒与睡眠主要与脑干的网状结构有关,其有睡眠诱导功能的脑干网状抑制系统(ascendingreticular inhibitory system,ARIS)。神经系统内阳离子-Cl~-共转运体(cation-chloride cotransporters,CCCs)是一个非常重要的影响和维持Cl~-浓度平衡的家族。目前报道的七种阳离子-Cl~-共转运体中,神经系统内仅表达KCC1、KCC2、KCC3和NKCC1四种,其中,K~+-Cl~-共转运体2(K~+-Cl~-cotransporter 2,KCC2)负责将神经元内Cl~-向外转运,而Na~+,K~+-2Cl~-共转运体1(Na~+,K~+-2Cl~-cotransporter1,NKCC1)则负责将胞外的Cl~-向胞内转运,二者构成一对平衡体,从而维持神经元内Cl~-浓度的稳定和平衡。阳离子-Cl~-共转运体的功能异常,可引起中枢神经系统的Cl~-的浓度异常,进而导致临床上多种神经系统疾病,如癫痫和持续性疼痛等,提示阳离子-Cl~-共转运体可能通过影响GABA能神经元,在神经兴奋/抑制的平衡中扮演了重要角色,而失眠正是一种神经兴奋/抑制功能失衡导致的临床疾病。作为经典的治疗失眠的药物,地西泮(安定)等属苯二氮卓受体激动剂,它们可通过促进GABA与GABA_A受体的结合而使Cl~-通道开放的频率增加,更多的Cl~-内流,引起突触膜超极化,产生快抑制性突触后电位(inhibitory postsynaptic potential,IPSP),而发挥神经抑制功能。而这个过程中,阳离子-Cl~-共转运体KCC2和NKCC1会发生什么同步变化呢?目前国内外尚未见相关报道。本研究拟采用腹腔注射PCPA制成大鼠失眠模型,并同步设立生理盐水对照组和地西泮干预组,分别检测各组大鼠脑干组织中KCC2mRNA和NKCC1 mRNA的表达变化,并检测细胞内氯离子浓度,以探讨其在失眠发病机制中的作用。方法1.动物分组及处理:采用清洁级成年雄性Sprague-Dawley(SD)大鼠为实验对象,随机分为3组,模型组:按照用量PCPA 300mg/Kg,用略碱性生理盐水(pH:7-8)溶解PCPA配制成混悬液,予以腹腔注射1ml/100g,每日1次,8:30-9:00 AM,连续2天,制作实验性失眠大鼠模型;对照组:与模型组同步对照,以生理盐水代替PCPA腹腔注射;干预组:自PCPA造模之后,按照地西泮片0.92mg/Kg的剂量,用蒸馏水溶解地西泮片配制成9.2%水溶液,予以灌胃,每日一次,9:00-9:30 AM,连续5天。2.将各组受试动物于治疗后第6天断头处死,取大鼠脑干组织,液氮内保存。3.RT-PCR法半定量检测大鼠脑干KCC2和NKCC1 mRNA的表达。4.用氯离子荧光探针MQAE,结合激光共聚焦显微镜检测脑干组织细胞内Cl~-浓度及变化。结果1.与对照组相比,模型组KCC2mRNA表达明显降低(P＜0.01),干预组较模型组表达增加(P＜0.01),但仍低于对照组(P＜0.01)。2.与对照组相比,模型组NKCC1mRNA表达稍增加,但无显著性差异(P＞0.05),干预组较对照组和模型组均降低(P＜0.01)。3.与对照组相比,模型组细胞内氯离子浓度升高(P＜0.01),干预组较模型组降低(P＜0.01),但仍较对照组高,差异无显著性(P＞0.05)。结论1、脑干KCC2 mRNA表达下调及细胞内Cl~-浓度的升高可能与失眠的发病有关。2、地西泮可能通过上调KCC2 mRNA和下调NKCC1 mRNA的表达,以及降低细胞内Cl~-浓度而发挥镇静催眠的作用。更多还原

【Abstract】 BackgroundsInsomnia is one of the most common sleep disorder,and it also is a very common clinical manifestation of many physical,mental and behavioral diseases;however,its mechanism is not clear enough so far.As for anatomic structure of sleep,it is widely accepted that ascending reticular inhibitory system(ARIS) of brainstem is closely related to wakefulness-sleep since ARIS performs its sleep-inducing function.GABA_AR in brain is a kind of ionotropic receptor which consists of recognition site of GABA,benzodiazepine and chloride ion channel. Classical sedative and hypnotic drugs like diazepam excite GABA_AR, open Cl~- channel to make Cl~- influx,then synaptic membrane hyperpolarize to form quick postsynaptic inhibitory potential.Cation-Chloride Cotransporters(CCCs) is just a key family which plays very important role in maintaining the balance of concentration between intracellular chloride ion and extracellular chloride ion.Among seven types of CCCs,only KCC1,KCC2,KCC3 and NKCC1 are expressed in central nerve system.KCC2 is in charge of neuronal Cl~-extrusion while NKCC1 is responsible for neuronal Cl~- intrusion in order to form a pair of substances to maintain balance of concentration of Cl~-.A lot of researches have proved that dysfunction of CCCs induce many nerve system diseases related to abnormality of concentration of Cl~-,like epilepsy and lasting pain.All of these give us a hint that CCCs may interrupt GABA function.And insomnia is such a kind of disease induced by neuron excitation/inhibition dysfunction.As for classical drug for insomnia,diazepam,a kind of benzodiazepine receptor,is able to promote GABA to connect to GABA_A receptor,increase the frequency of chloride ion channel opening,and make more chloride ion influx so that synapse membrane hyperpolarize and form inhititory postsynaptic potential.In this process,what kind of change will happen to KCC2 and NKCC1 simultaneously? There is no report pertinent to it so far in the world.ObjectivesTo make PCPA-induced experimental insomnia model,and design normal saline contral group and diazepam interference group at the same time,in order to investigate the possible roles of KCC2 and NKCC1 in mechanism of insomnia.Methods1.Grouping and building model:18 male adult Sprague-Dawley (SD) rattus were divided into three randomized groups.Model group:PCPA was injected intraperitoneally by 300mg/kg/d 8:30-9:00 AM for 2 days;Control group:NS was injected intraperitoneally instead of PCPA at the same time;Interference group:diazepam was administrated intragastricly by 0.92mg/kg/d for 5 days at 9:00-9:30 AM after making model.2.The expressions of KCC2mRNA and NKCC1mRNA in the brainstem were detectec by RT-PCR.3.The concentration of intracellular Cl~- in brainstem was detected by fluorescence probe MQAE with the help of laser confocal microscopy.Results1.Comparing with control group,the expression of mRNA of KCC2 in model group was down-regulated(p＜0.01);however,it is up-regulated in interference group comparing with model group(p＜0.01),and still down-regulated comparing with control group(p＜0.01).2.Comparing with control group,the expression of mRNA of NKCC1 in model group was a little up-regulated,but there was no statistical difference;however,it is down-regulated in interference group comparing with model group(p＜0.01) and control group(p＜0.01).3.Comparing with control group,the concentration of intracellular Cl~- in model group is up-regulated(p＜0.01);it is down-regulated in interference group comparing with model group(p＜0.01),and higher than control group without statistical diference(p＞0.05).Conclusions1、Insomnia may have something to do with the down-regulation of KCC2 mRNA and rise of intracellular concentration of Cl~-.2、The mechanism of sedative-hypnotic diazepam may have something to do with the up-regulation of KCC2 mRNA, down-regulation of NKCC1 mRNA,and decrease the intracellular concentration of Cl~-.更多还原