【作者】 马雪莲；

【导师】 刘传勇； Wolfgang Kunze；

【作者基本信息】 山东大学 ， 生理学， 2009， 博士

【摘要】 内脏痛是许多胃肠道疾病最常见的症状之一,但其发生机制还不清楚。目前认为,内脏异常疼痛的产生主要归结为外周敏感化和中枢敏感化。各种原因引起的内脏组织和初级传入神经元的敏感化可以诱导和易化中枢敏感化,所以了解初级神经元敏感化的机制,探索降低其兴奋性的方法对干预痛觉信号的传导、控制疼痛具有重要意义。内脏感觉主要通过结状神经节与脊髓背根神经节（dorsal root ganglia,DRG）传入到次级感觉神经元,其中一般的内脏感觉主要通过结状神经节传导,而痛觉主要通过DRG神经元传导。按其直径不同,DRG神经元可以分为大型神经元（39-50μm）、中型神经元（33-38μm）和小型神经元（19-27μm）三类。其中,中、小型神经元主要发出细髓的Aδ纤维和无髓的C类纤维,传递伤害性感觉,与痛觉产生密切相关。因此,DRG中的中小神经元是内脏痛觉信号传导与调控的初级靶点。电压依赖性钠通道决定神经细胞动作电位的产生和传导,它的改变对于神经元的兴奋性具有重要的影响。钠通道由一个α亚单位和两个β亚单位组成。目前已经克隆出九种α亚单位,并据此将电压依赖性钠通道分为9种类型,即Nav1.1-Nav1.9。根据其对河豚毒素（tetrodotoxin,TTX）的敏感性又可分为河豚毒素敏感（TTX-sensitive,TTX-S）和河豚毒素不敏感（TTX-resistant,TTX-R）两种类型。其中,Nav1.1、Nav1.2、Nav1.3、Nav1.4、Nav1.6和Nav1.7属于TTX-S钠通道,而Nav1.5,Nav1.8和Nav1.9属于TTX-R钠通道。Nav1.3、Nav1.7、Nav1.8和Nav1.9参与痛觉信号的调控。一些诱发疼痛的病理性因素影响DRG细胞中钠通道的表达和功能,改变DRG细胞的兴奋性。BDNF（brain-derived neurotrophic factor）是一种神经营养因子,广泛存在于中枢与外周神经系统中。以往的研究认为,BDNF的主要作用是调节突触的可塑性和神经递质的释放,参与学习和记忆过程。但近来越来越多研究发现,BDNF也参与对痛觉传导的调控。在脊髓中,BDNF由DRG细胞合成后被运输到中枢端,在脊髓后角释放,与第二级感觉神经元上的Trk B受体结合,调节突触传递和脊髓痛觉的传导,导致中枢神经元兴奋性的改变。BDNF还可以通过自分泌和旁分泌的方式调节DRG神经元的兴奋性。肠道益生菌是与人类共生的、具有治疗或保健作用的细菌,主要包括乳酸杆菌（Lactobacillus reuteri）和双歧杆菌。它们能够调节炎症引起的肠道敏感性的改变,调节肠道内在感觉神经元的兴奋性,对一些肠道疾病包括肠易激综合征（irritable bowel syndrome,IBS）和炎症性肠道疾病等所引起的腹部不适症状具有明显的改善作用。由于重复的结直肠扩张刺激（colorectal distention,CRD）能够诱导内脏高敏感性,本课题研究该模型大鼠DRG神经元兴奋性的改变及其机制,探讨钠通道和BDNF在DRG兴奋性改变中所起的作用,并进一步研究乳酸杆菌对DRG兴奋性改变的调节作用及其相关机制。本课题将揭示DRG神经元兴奋性的改变在内脏高敏感性发生中的作用,并为寻找新的镇痛药物提供依据。[研究目的]1探讨CRD对大鼠远端结肠DRG神经元兴奋性的影响。2探讨CRD后大鼠DRG神经元电压依赖性钠通道表达和功能是否发生改变。3研究CRD对大鼠DRG神经元BDNF表达的影响及外源性BDNF在CRD致大鼠DRG神经元敏感化过程中所起的作用。4探讨乳酸杆菌对CRD后大鼠DRG神经元敏感性的影响及其机制。[研究方法和结果]1结直肠扩张内脏痛模型的建立1.1 DRG神经元的逆行标记健康雄性SD大鼠,腹腔注射麻醉,打开腹腔,找到结肠远端,向结肠壁注射1,1-dioctadecyl-3-3-3-3-tetramethylindocarbocyanine（DiI,5mg/ml）,注射6到10个点（1.0μl/injection）,缝合腹壁。1.2 CRD内脏痛模型的建立注射DiI两周后,大鼠腹腔内注射氯胺酮（75mg/kg）与甲苯噻嗪（10mg/kg）麻醉,利用Barostat system（Distender,G & J Electronic Inc）给予1小时重复的CRD刺激（80 mm Hg,30s on,30 s off）,对照组只给予氯胺酮与甲苯噻嗪腹腔注射麻醉,不给予CRD刺激。2扩张肠段MPO活性检测大鼠处死后,取扩张的远端结肠段,根据试剂盒（南京建成生物工程研究所）说明书检测单位结肠重量中MPO的活性。结果显示,同no CRD组相比,在分别给予CRD后1h、3h、6h、12h、24h,其MPO活性未见明显增加。3 RT-PCR检测CRD后不同时间段DRG中BDNF mRNA含量的表达提取DRG中mRNA,检测发现在CRD后3h、6h、12h、24h,DRG中的BDNF mRNA含量明显增高。4 ELISA检测CRD后不同时间段DRG中BDNF蛋白含量的变化提取DRG中蛋白,检测发现同正常DRG相比,CRD后1h、3h、6h、12h、24h,DRG中BDNF蛋白含量明显增高。5 DRG细胞的培养给予CRD刺激之后,立刻颈椎脱臼牺牲大鼠,取出腰骶段脊柱,取出两边的DRG;置于Krebs溶液中;加入胶原酶Ⅰ（1mg/ml）和胰酶（0.25%）,37℃消化50min,接种于包被了PLL的35mm培养皿中,37℃,5%CO₂,95%O₂培养过夜。6膜片钳记录6.1 CRD细胞自发放电在电流钳模式下,I=0,记录细胞自发放电的情况。结果发现,no CRD组,自发放电的细胞占7.3%;CRD组自发放电细胞数量有所增加占9.4%,两者未见显著性差异。6.2 CRD刺激之后动作电位的产生和各项指标的检测记录刺激动作电位产生所需要的基强度和2倍、3倍基强度刺激产生动作电位的数量。结果显示,在给予1h重复CRD刺激后,DRG神经元产生动作电位基强度降低,2倍、3倍基强度刺激诱发DRG神经元产生动作电位的数量增多,说明CRD后DRG神经元的兴奋性明显增高。6.3 CRD对大鼠DRG神经元钠电流的影响电极电阻控制在2-4 MΩ,所有的钠电流在细胞成为全细胞之后5分钟进行记录,串联电阻被补偿85-90%。分别记录no CRD组和CRD组大鼠DRG细胞钠通道的激活、失活及复活电流的变化。结果发现,在CRD后,大鼠DRG细胞上TTX-S和slow TTX-R钠通道的激活和复活过程没有发生明显的改变,而其失活曲线向去极化方向移动,表明其失活电压依赖性发生改变。6.4 BDNF对正常及CRD大鼠DRG神经元兴奋性的影响研究发现,no CRD组在加入5ng/ml BDNF 5min后,DRG动作电位阈值及动作电位的产生频率都未见变化,而幅值、去极化的速率明显下降;在CRD组中神经元加入同样剂量的BDNF后,DRG神经元动作电位的阈值明显升高,给予3倍基强度刺激诱发DRG神经元产生动作电位的数量减少。说明BDNF下调CRD后DRG神经元的敏感性。6.5乳酸杆菌对正常及CRD大鼠DRG神经元敏感性的影响。用乳酸杆菌（5×10⁹CFU/ml）连续灌胃对正常大鼠DRG神经元的电生理特性没有影响,但明显升高CRD后DRG神经元动作电位产生的基强度,降低动作电位的去极化速率及产生频率,说明乳酸杆菌下调CRD后DRG神经元的兴奋性。[结论]1远端结肠受到80mmHg的CRD刺激1h后,可以引起相应节段DRG细胞兴奋性明显增加。2 CRD后DRG神经元电压依赖性钠通道失活动力学发生改变,这可能是CRD导致DRG细胞高敏感性的发生的原因之一。3内源性BDNF可能下调CRD后DRG神经元的兴奋性。4乳酸杆菌抑制CRD引起的DRG神经元的敏感化,对内脏痛具有潜在的治疗作用。更多还原

【Abstract】 Visceral pain is an important phenomenon of some gastrointestinal（GI） diseases, but the molecular and cellular mechanisms are still not clear.The hypersensitivity of the nociceptive receptor,the primary sensory neuron and the central nervous system （CNS） are the main causes of visceral hyperalgesia.The GI tract has the potential to provide subconscious and conscious awareness of injury.The physiological or non-noxious stimuli are transmitted through vagal afferents with their cell bodies in nodose ganglion.But the pain is transmitted through the spinal afferents with their cell bodies in the dorsal root ganglia（DRG）.DRG neurons are classified into three main groups according to their sizes: large-sized（39-50μm diameter）,medium-sized（33-38μm diameter） and small-sized （19-27μm diameter） neuron.The spinal afferent Aδand C fibers,which transmit pain input to the dorsal corner in the spinal cord,are originated from medium-sized and small-sized neurons.So,the two kinds DRG neurons are the crucial "gateway" of visceral pain transmission.Voltage-gated sodium（Nav） channels play fundamental roles in the generation and conduction of neuronal action potentials.They are comprised ofα-subunit forming the pore,and auxiliaryβ-subunits.To date,nine kinds ofα-subunits have been cloned, and Nav were correspondingly classified into nine types,Nav1.1-Nav1.9.According to their sensitivity to tetrodotoxin（TTX）,sodium channels have been classified to TTX-sensitive channels（TTX-S） and TTX-resistant channels（TTX-R）.Nav1.1, Nav1.2,Nav1.3,Nav1.4,Nav1.6 and Nav1.7 are TTX-S channels and Nav1.5,Nav1.8 and Nav1.9 are TTX-R channels.Nav1.3,Nav1.7,Nav1.8 and Nav1.9 are thought to be involved in the modulation of the pain sensation by modulating the neurons excitability.But the detailed mechanisms are not clear.BDNF（brain-derived neurotrophic factor） is a kind of neurotrophic factor and is widely distributed in the peripheral and central nervous system.It is synthesized in the DRG,transported to the central terminals of the primary afferents,released into the spinal dorsal horn,and binds to the trkB receptors on second-order sensory neurons.It might play significant roles in synaptic plasticity,signal transmission,learning and memory.Nowadays,more and more studies found that BDNF also functions as a mediator of pain.Furthermore,BDNF itself could modulate the hypersensitivity of the DRG cells in a paracrine or autocrine manner.Probiotics,including Lactobacillus reuteri（L.reuteri） and Bifidobacterium,are health-promoting commensal bacteria that exert therapeutic or preventative roles to some gastrointestinal diseases.Beneficial effects of probiotics have been described in traveler’s diarrhea,irritable bowel syndrome,and inflammatory bowel disease.In addition,probiotics can modulate postinflammatory gut hypersensitivity and alter neuropeptide expression in neurons.Based on the above backgrounds,we hypothesized that the change of the DRG neuron sensitivity was involved in the visceral hyperalgesia in some GI disease,and this hypersensitity of the DRG neurons might caused by the changes of the expression and electrical property of the sodium channels on the soma of the DRG neuron. BDNF and probiotics might modulate the sensation of pain by modulating the sensitivity of DRG neurons.In order to test this hypothesis,we use repeated noxious colorectal distention（CRD） to make the rat model of visceral hyperalgesia and monitor the sensitivity of the DRG neurons by whole cell recording. [Objective]1 To explore the effects of noxious CRD on the excitability of rat DRG neurons.2 To explore the effect of CRD on the expression and function of voltage-gated sodium channels.3 To explore the expression of BDNF in DRG following CRD and the effect of exogenous BDNF on the excitability of DRG neuron.4 To explore if L.reuteri could modulate the hypersensitivity of DRG neurons after CRD.[Methods and results]1 The noxious CRD model1.1 Male adult Sprague-Dawley rats were anesthetized with ketamine（75mg/kg, i.p.） and xylazine（10mg/kg,i.p.）.A midline laparotomy was performed,and the descending colon was carefully exposed.1, 1-dioctadecyl-3-3-3-3-tetramethylindocarbocyanine（DiI） was injected into six to ten sites in the colon wall.Then,the abdomen was closed.1.2 Forteen days later,the rats were anesthetized again as above and treated with repeated distention for 1h（80 mm Hg,30s on,30 s off） by Barostat system.The vehicle control group was anesthetized but not treated with CRD.2 Measure of myeloperoxidase（MPO） activityThe rats were sacrificed and the MPO activity of the distal colon was measured. The MPO activities did not change following CRD.3 The BDNF mRNA levels in DRG（PCR）The total mRNA were extracted from DRG and the amount of mRNA in DRG was measured by RT-PCR.The BDNF mRNA in DRG were significantly increased from 3 h to 24 h following CRD.4 The BDNF protein levels in DRG（ELISA）The total protein were extracted from DRG and the amount of BDNF protein was mesured by ELISA.The expression of BDNF protein in DRG were significantly increased from 1h to 24h following CRD. 5 Cell dissociation and cultureAfter CRD,the rat was sacrificed immediately and the spinal colum was completely removed,transferred to a beaker containing ice-cold sterile Krebs solution. DRG were exposed by dissection from both sides and collected from L1-S5 spinal levels.After incubated 50min in collagenase typeⅠ（1mg/ml） and trypsin（0.25%） at 37℃,the dissociated neurons were plated onto 35-mm-diameter Petri dishes previously coated with PLL.The neurons were then incubated overnight at 37℃with 5%CO₂ and 95%O₂.6 Electrophysiological recording6.1 Spontaneous discharge in DRG neuronsThe spontaneous discharge of the DRG DiI labled neurons was recorded in current clamp mode,I=0.The frequency of the spontaneous discharge in no CRD group were 7.3%（6/82）,and that in the CRD group it was 9.4%（6/64）.There were no significant difference betweent the two groups.6.2 Passive membrane and active membrane parameters in DRG neurons.With the amplifier in current clamp mode,action potential and passive membrane characteristics were measured.After CRD,the rheobase of the DRG neurons was significantly reduced and the number of APs discharged at 2×and 3×rheobase were increased compared with that in no CRD group.6.3 The effects of CRD on sodium current in DRG.Only DiI-labled DRG neurons were selected for this study.The patch pipettes were pulled with resistance 2-4 MΩ.After establishment of a whole cell recording,series resistance were compensated to 85-90%.The results showed that the activation and recovery currents of TTX-S and slow TTX-R channels in the CRD group have no difference from that in no CRD group.But theV_1/2 of inaction current shifted to the right.This implies that CRD change the inactivation kinemics of the sodium channel.6.4 The effects of exogenous BDNF on the excitability of DRG neurons in CRD and no CRD groupsExogenous BDNF reduced the AP_dV/dt and the amplitude of AP of the DRG neuron in no DRG group.But in CRD group,exogenous BDNF increased the rheobase, decreased AP_dV/dt and the number of APs discharged at 3×rheobase.So,it it clear that exogenous BDNF decreased the excitability of the DRG neurons in CRD group.6.5 The effect of L.reuteri ingestion on the excitability of DRG neurons in CRD and no CRD groupsIn the DRG neuron of the CRD group,9 days of L.reuteri ingestion increase the rheobase,decreased the AP_dV/dt and decreased the number of APs discharged at 2×rheobase intensity stimulation..[Conclusion]1 Repeated 80mmHg CRD stimulation in distal colon can cause increased excitability of DRG neurons.2 CRD might increase the excitability of the DRG neuron by changing inactivation kinemics of sodium channels.3 Endogenous BDNF might downregulate the hypersensitivity of DRG neurons following CRD.4 Ingestion of L.reuteri downregulated the sensitivity of the DRG neurons following CRD.It may exert therapeutic or preventative role to visceral pain.更多还原