核苷酸和P2Y2受体与伤害性感受的研究进展

P2受体为以ATP、UTP及其类似物激活的受体，分为促离子型P2X受体和促代谢型P2Y受体两大类，其各自又分为许多亚型。许多证据显示，P2Y，特别是P2Y2受体，在痛觉信息调节和痛觉过敏中起着重要作用。ATP和UTP作为P2Y2受体的天然激动剂，是伤害性感受和痛觉过敏信息传递中的重要介质。P2Y2受体参与疼痛的调节可能与辣椒素受体有关。膜片钳分析小鼠背根神经节神经元证明了是P2Y2而非P2Y1受体在ATP诱发的TRPV1介导的热痛过敏反应中起作用。原位杂交组织化学研究结果也显示大鼠腰段脊髓背根神经节中TRPV1mRNA与P2Y2mRNA共同表达。这些证据证明了促代谢型P2Y，受体介导背根神经节神经元中核苷酸对VR1的增敏效应及参与伤害性感受的调节。     

大鼠嘌呤P2Y4受体在神经系统中表达的实验研究

目的 研究P2Y4受体在成鼠脊髓，脊神经节及坐骨神经的表达及分布情况，为研究ATP／UTP对周围神经再生的作用机制提供理论基础。方法 取成鼠脊髓、脊神经节及坐骨神经(SD大鼠，雌雄不拘)，采用原位杂交和逆转录一聚合酶链式反应(RT-PCR)方法，观察嘌呤P2Y4受体的分布与表达，并对RT-PCR的结果进行测序。结果 原位杂交与RT-PCR结果显示，脊髓灰质有P2Y4R的表达，主要分布在中央管上皮细胞和前角，运动神经元呈强阳性，白质中为阴性。坐骨神经也可见到P2Y4受体的表达，主要分布在雪旺细胞及其内的血管内皮细胞。脊神经节则没有P2Y4受体的表达。结论 大鼠的P2Y4受体可能主要分布在输出纤维，在感觉神经原纤维几乎无表达。     

慢性神经病理性痛大鼠背根神经节P2Y2受体mRNA的表达

目的评价背根神经节（DRG）P2Y2受体mRNA的表达在大鼠慢性神经病理性痛中的作用。方法SPF级大鼠24只,体重150～200g,雌雄不拘,随机分为2组：假手术组（S组）和慢性神经痛组（N组）,每组12只。N组结扎左侧坐骨神经建立大鼠慢性压迫性损伤（CCI）模型,S组只暴露,不结扎左侧坐骨神经。2组分别于CCI前1d、CCI后1、4、7、10、14 d进行行为学观察,测定大鼠双后肢热痛阈和机械痛阈,并分别于CCI后7、14 d各取6只大鼠断颈处死,取双侧L（4-6）背根神经节,RT-PCR法测定P2Y2受体mRNA的表达。结果两组CCI后4 d热痛阈、机械痛阈明显降低（P〈0.05）,持续至CCI后14 d;N组左侧DRG P2Y2受体mRNA表达较右侧明显下调,CCI后14 d较CCI后7 d下调（P〈0.05）;与S组右侧比较,N组DRG P2Y2受体mRNA表达差异无统计学意义（P〉0.05）。结论背根神经节P2Y2受体mRNA的表达下调参与了大鼠慢性神经病理性痛的形成。     

P2X3受体介导急性痛反应大鼠脊髓背角星形胶质细胞P物质受体和谷氨酸受体表达的变化

P2X3受体在背根感觉神经节特异表达,参与疼痛的信号传递.大鼠足底注射ATP等P2X3受体激动剂可诱发急性伤害性反应[1].研究表明,疼痛的发生和维持不仅与神经元有关,脊髓胶质细胞在某些因素诱导下,也参与了疼痛产生的过程[2],而其是否参与了由P2X3受体介导的急性疼痛信号传导,目前尚不清楚.本研究拟评价P2X3受体介导急性痛反应大鼠脊髓背角星形胶质细胞内P物质受体NK-1和谷氨酸受体NMDA1表达的变化,以探讨脊髓背角星形胶质细胞在急性痛信号传导中的作用.     

大鼠坐骨神经ATP受体mRNA表达的实验研究

目的：证实大鼠坐骨神经上ATP受体P2y的分布 。方法：梨状肌下缘以远5mm处切取长5mm的坐骨神经，同时切取主动脉3mm作阳性对照；组织总mPNA提取，RT-PCR cDNA第一链合成，反应所得产物于2%琼脂糖凝胶电泳，1цg/ml溴化乙啶染色（EB），观察，照相。结果：在大鼠坐骨神经上有3种ATP受体亚型表达（P2y2、P2y4和P2y6mRNA表达）。结论：大鼠坐骨神经上有3种ATP受体亚型分布，细胞我ATP可能通过此三种受体或其中之一完成轴突诱向作用，P2y受体与神经损伤修复有关。     

坐骨神经损伤及神经生长因子对背根神经节中TrkA及其mRNA表达的调控

目的 研究周围神经损伤、神经再生及外源性神经生长因子对脊神经背根神经节中TrkA及TrkA mRNA表达的影响。方法 将24只SD大鼠坐骨神经从神经中段切断后，外膜缝合修复神经。实验组动物的胫后肌内每日注射NGF200U，术后3、7、14、28d，采用免疫组织化学及原位杂交方法，检查大鼠坐骨神经损伤、神经再生及外源性NGF靶肌肉注射后脊神经背根神经节中TrkA及TrkA mRNA表达。结果 正常背根神经节中有大量的神经元表达TrkA及TrkA mRNA；坐骨神经损伤后，背根神经节中TrkA的表达下降，术后7d组最低，28d接近正常；NGF靶肌肉注射后，背根神经节中TrkA的表达明显增高。结论 神经损伤后背根神经节中TrkA的表达降低，靶肌肉注射外源性NGF背根神经节中TrkA的表达增加，表明靶肌肉注射可作为NGF的有效给药途径.     

急性吗啡耐受或炎性痛大鼠脊髓背角和背根神经节嘌呤受体P2X4表达的变化

目的 评价急性吗啡耐受或炎性痛大鼠脊髓背角和背根神经节嘌呤受体P2Y4 (P2X4R)表达的变化.方法 雄性SD大鼠30只,采用随机数字表法,将大鼠随机分为3组:生理盐水组(NS组,n=5)、急性吗啡耐受组(M组,n=5)和炎性痛组(F组,n=20).F组建立福尔马林炎性痛模型,M组和NS组鞘内注射吗啡或生理盐水,10 μg(10 μl)/次,2 h/次,连续6次,最后1次给药后2h处死取脊髓背角和背根神经节.NS组和M组分别于每次给药后(T1-6)测定热刺激缩足反射潜伏期(PWL),F组于致炎后4、7、10和14d时随机取5只大鼠测定PWL后处死取脊髓背角和背根神经节,采用免疫组化法检测P2X4R表达.结果 与基础值比较,F组致炎后4～14 d PWL明显降低,M组T1-5时PWL升高,T6时PWL差异无统计学意义(P＞0.05).与NS组比较,M组脊髓背角和背根神经节P2X4R表达上调,F组在致炎后第4天脊髓背角和背根神经节P2X4R表达上调,于第7天时P2X4R表达至峰值,第10天和第14天时P2X4R表达逐渐下调(P＜0.01).结论 急性吗啡耐受或炎性痛大鼠脊髓背角和背根神经节P2X4R表达上调,该变化可能与急性吗啡耐受和炎性痛的形成有关.     

大鼠睾丸痛模型中NMDA受体的表达研究

目的:NMDA受体的NR1和NR2B亚基对于痛觉的产生和维持起着关键作用,本文旨在探究NMDA受体在大鼠睾丸痛模型中的表达及可能机制。方法:参照Yoshioka等的方法建立大鼠睾丸痛模型,分实验组和对照组,其中对照组大鼠睾丸注射0.2 ml生理盐水,实验组大鼠睾丸注射0.2 ml 2%醋酸溶液,后进行行为学检测。建模成功后4 h采用Western印迹、RT-qPCR、免疫荧光检测NMDA受体在背根神经节和脊髓背角中的表达。结果:建模后,实验组大鼠逃避反应潜伏期明显降低,于4 h达到最低值[(4.15±0.84) s],显著低于对照组[(12.32±1.05) s,P0.05]。建模后4 h,RT-qPCR, Western印迹、免疫荧光结果显示:与对照组相比,实验组大鼠背根神经节中NR2B mRNA和蛋白表达明显升高(P0.05),脊髓背角中NR2B的表达无明显变化;实验组和对照组大鼠的背根神经节和脊髓背角的NR1表达也无明显差异。结论:NMDA受体在大鼠睾丸痛发病过程中具有重要作用,疼痛前期便可通过上调NMDA受体NR2B亚基表达介导外周痛敏形成,介导睾丸疼痛的发生。     

p27kip1和Skp2在大鼠坐骨神经切断后脊髓中的表达

目的 观察坐骨神经切断后大鼠脊髓中p27^kip1和Skp2的表达变化。方法 将成年SD大鼠随机分为正常对照组和实验组。对实验组实行坐骨神经切断术。用Western blot和免疫组织化学技术检测脊髓中p27^kip1和Skp2的表达变化。结果 Western blot结果表明坐骨神经切断后脊髓中p27^kip1表达持续下降，Skp2表达上调。免疫组织化学结合免疫荧光双标技术显示，在正常组和实验组，p27^kip1和Skp2在脊髓神经元和胶质细胞均有表达。在腹角神经元，损伤后p27^kip1 免疫染色减弱，Skp2增强。坐骨神经损伤前后脊髓腹角p27^kip1和Skp2阳性细胞数目改变呈负相关（r=-0．6892，P〈0．01）。结论 坐骨神经损伤可影响脊髓中p27^kip1和Skp2的表达水平及亚细胞定位，两者改变呈负相关。     

背根神经节P2X3受体在大鼠切口痛形成中的作用

目的 评价背根神经节P2X3受体在大鼠切口痛形成中的作用.方法 健康雄性SD大鼠24只,体重200～220 g,随机分为3组(n=8):对照组(C组)、切口痛组(IP组)和P2X3受体拮抗剂组(A组).IP组和A组制备大鼠左后趾部切口痛模型.模型建立后30 min,A组左后爪底皮下注射P2X3受体特异性拮抗剂TNP-ATP 200 nmol,C组和IP组注射等容量生理盐水.采用累积疼痛评分法,评定注药后1 h内大鼠疼痛行为学变化.注药后2 h时处死大鼠,取背根神经节,测定P2 X3受体表达和细胞内Ca2+浓度.结果 与C组比较,IP组和A组累积疼痛评分和Ca2+浓度升高,P2X3受体表达上调(P＜0.05);与IP组比较,A组累积疼痛评分和Ca2+浓度降低,P2X3受体表达下调(P＜0.05).结论 背根神经节P2X3受体参与了大鼠切口痛的形成,其机制可能与升高细胞内Ca2+浓度有关.     

银杏酮酯对大鼠坐骨神经损伤后神经生长因子基因表达的影响

目的 观察银杏酮酯（EGb50）对大鼠坐骨神经损伤后神经生长因子（NGF）mRNA表达的影响。方法 取SD大鼠78只，其中72只切断右侧坐骨神经并缝合，随机分成损伤对照组与实验组，另6只为正常对照组。实验组给予EGb50200mg／kg／d溶于1．0ml生理盐水中灌胃，损伤对照组每天给予生理盐水1．0ml灌胃，正常对照组不作任何处理。分别于术后1、3、7、14、21及28d取吻合口远段的神经、相应节段的脊神经节及脊髓，采用逆转录-多聚酶链反应技术检测所取组织中NGFmRNA的表达水平。结果 实验组坐骨神经中NGFmRNA的表达在术后7、14和21d明显高于对照组（P〈0．05）。术后7d和14d，实验组脊神经节及脊髓中NGFmRNA的表达高于对照组（P〈0．05）。结论 大鼠坐骨神经损伤后用银杏酮酯治疗，在早期可促使坐骨神经及相应节段脊神经节和脊髓组织中的NGFmRNA的表达增加。     

Adenoviral gene transfer in the peripheral nervous system

Background Viral vectors have gained widespread use as vehicles for somatic gene transfer, and the targeted expression of foreign proteins by these vectors offers advantages over the systemic administration of the drugs in some therapeutic situations. Selective virus-mediated gene transfer to the peripheral nervous system (PNS), however, remains to be established. There are no data showing efficiency of protein transduction in the PNS, which consists of a variety of cell types, many of which are postmitotic. Methods We prepared the first-generation replication-deficient recombinant adenovirus vectors engineered to express LacZ. Eight-week-old Wister rats were used in this study. Adenovirus vector (5 μl) containing the LacZ gene (5 × 10⁸ pfu) was injected into rat sciatic nerves or the dorsal root ganglia at the level of L5. The sciatic nerves, the dorsal root ganglia, and the spinal cords were obtained 7, 14, 21, and 28 days after injection. Expression of LacZ was assessed by X-gal histochemistry and β-gal immunohistochemistry. Results Following injection of the adenovirus carrying the LacZ gene into the sciatic nerve, LacZ expression was seen mainly in the Schwann cells and the small neurons in the dorsal root ganglion. In contrast, expression was observed in the primary nerve terminals of the spinal dorsal horn and the small to large dorsal root ganglion neurons and the Schwann cells after injection of the vectors into the L5 dorsal root ganglion. There were no side effects in rats with injection in the dorsal root ganglia or the sciatic nerve. Conclusions The present study shows efficient protein transduction by adenovirus vectors in the PNS. It is noted that injection of the virus into the dorsal root ganglia leads to extensive expression of LacZ in the spinal cord, the dorsal root ganglia, and the sciatic nerves.     

Expression of vascular endothelial growth factor and its fetal liver kinase-1 receptor in spinal cord and dorsal root ganglia after neurotomy of sciatic nerve in rats

egenerationofinjured peripheralnervesiscloselyrelatedtosomegrowthfactorssecretedbycentralneurons .Vascularendothelialgrowthfactor (VEGF)isapotentangiogenicfactor ,which possessesspecificmitogenicactivityforvascularendothelialcells ,stimulatestheformationofnascentbloodvesselsandenhancesvascularpermeability .1RecentevidencesindicatethatVEGFcanalsoactdirectlyonneuronstoinduceneurotrophiceffects.Forexample ,VEGF promotestheproliferationofculturedcerebralcorticalneurons ,stimulatestheaxonaloutg…     

大鼠坐骨神经切断后VEGF及其受体flk-1在脊髓与脊神经节中的表达

目的观察大鼠坐骨神经切断后脊髓与神经节组织中血管内皮生长因子(VEGF)及其胎肝激酶-1(flk-1)受体的表达变化规律。方法取成年雄性Wistar大鼠45只，对照组5只，实验组切断双侧坐骨神经后，在术后8h、24h、72h、5d、7d、10d、14d、21d取L4～L6段脊髓与相应脊神经节组织，采用免疫组织化学方法和图像分析技术，对VEGF及其flk-1受体的表达进行检测与分析。结果大鼠正常脊髓与神经节组织中均存在VEGF及flk-1的表达，坐骨神经切断后其表达可反应性地增强，持续一段时间后迅速回落至正常水平。并且flk-1在胶质细胞及白质中的神经纤维也有所表达。结论本实验从中枢神经元的角度揭示VEGF促周围神经再生机制，为今后进一步应用VEGF治疗外周神经损伤奠定基础。     

Reduction of experimental neuroma formation with ricin.

Twenty rat sciatic nerves were bilaterally transected, one as control, and one intraneurally injected with ricin. At 11 weeks, all controls demonstrated large neuromas. Ten injected nerves had no neuroma or a significantly smaller one, while the other 10 had neuroma formation similar to controls. No effect on distant dorsal root ganglia or spinal cord was seen. Thirteen additional rats underwent nerve injection with I125-labeled ricin. At one week, most radioactivity was localized to sciatic nerve, surrounding muscle, and thyroid, with trace amounts in dorsal root ganglia and spinal cord. Intraneural ricin can inhibit neuroma formation in transected nerve, but results in unpredictable uptake of ricin by nerve and excess spillage into surrounding tissues.     

神经生长因子保护脊髓背根神经节的实验研究

坐骨神经损伤后，在相应节段脊髓神经元可出现变性。为了观察外源性神经生长因子（ＮＧＦ）的保护作用而设计了本实验。方法是切断ＳＤ大鼠单侧坐骨神经，损伤局部给予外源性ＮＧＦ。借助酶组织化学及图像分析检测坐骨神经切断及应用ＮＧＦ后所引起的脊髓内抗氟化物酸性磷酸酶（ＦＲＡＰ）的活性变化，以探讨ＮＧＦ对脊髓背根神经节的保护作用。结果发现：坐骨神经切断可导致脊髓内ＦＲＡＰ含量降低，从而提示坐骨神经损伤可损害背根神经节；应用ＮＧＦ可显著减少这一酶含量的降低，从而首次从体内酶学水平证实ＮＧＦ对脊髓背根神经节有明显的保护作用。     

银杏叶提取物对大鼠坐骨神经切断后HSP 70在脊髓与脊神经节中表达的影响

目的 研究周围神经损伤后脊髓热休克蛋白(heat shock proteirns,HSPs)的表达变化,及银杏叶提取物EGb 761对其影响,探讨银杏叶提取物对周围神经损伤的保护机制.方法 取成年雄性SD大鼠144只,随机分成三组:对照组、坐骨神经切断组、坐骨神经切断+银杏叶提取物干预组[术后每天用EGb 761(100 mg·kg-1*d-1,溶于2 ml SAL)灌胃,直到取材],每组48只.术后6 h、12 h、1 d.2 d,4 d、7 d、14 d、28 d取L4～6节段脊髓节段,采用免疫组织化学方法检测HSP 70在脊髓前角及脊神经节中的表达.结果 正常大鼠脊髓与神经节中均有少量HSP 70表达,在坐骨神经切断后表达迅速增加,持续一段时间后又回到正常水平,用EGb 761干预后,HSP 70表达早期较未干预组表达低,后期表达增高,且表达持续时间长.结论 银杏叶提取物EGb 761可以增加神经损伤后HSP 70表达,可能是银杏叶提取物保护神经,促进神经再生作用的机制.     

P2X receptors in sensory neurones

P2X receptors are a family of ligand-gated ion channels responsive to ATP. Seven subtypes have been identified which form homo-multimeric or hetero-multimeric pores. P2X3 receptors are selectively expressed predominantly on small-diameter nociceptive sensory neurones in the dorsal root, trigeminal and nodose ganglia, particularly the non- peptidergic subpopulations labelled with the lectin IB4. P2X2/3 labelling is also present in inner lamina II of the spinal cord and in sensory nerve projections to skin and viscera, but few receptors are present in skeletal muscle. P2X3 receptors are down-regulated after peripheral nerve injury and their expression can be regulated by glial cell-derived neurotrophic factor. P2X receptor activation of sensory neurones has been demonstrated in in vivo pain models, including the rat hindpaw and knee-joint preparations, as well as in inflammatory models. P2X4 and/or P2X6 receptors in the CNS also seem to be involved in pain pathways. Non-nociceptive P2 receptors on sensory nerves are present in muscle and on sensory endings in the heart and lung that initiate reflex activity involving vagal afferent and efferent nerve fibres. The sources of ATP involved in nociception and non-nociceptive sensory nerve stimulation are discussed as well as a novel hypothesis about purinergic mechanosensory transduction.      

Alterations in retrograde axonal transport in streptozocin-induced diabetic rats

Retrograde axonal transport in the sciatic nerve of rats with streptozocin-induced diabetes was studied by the [3H]N-succinimidyl propionate [( 3H]NSP) method. The accumulation of retrogradely transported labeled proteins in the dorsal root ganglia and the ventral horn of spinal cord 1 day after [3H]NSP injection was not statistically significantly different from controls in rats diabetic for 1 or 14 days at the time of [3H]NSP injection. However, accumulation of labeled proteins in the dorsal root ganglia 7 days after [3H]NSP injection was reduced by 35% and transport to the ventral horn of spinal cord 7 days after [3H]NSP injection was reduced by 70% at the same time points. Partial control of the diabetes with insulin resulted in a partial reversal of these deficits. The early occurrence of defects in retrograde transport suggests that such defects may play a role in the pathogenesis of the neuropathy.