超极化激活环核苷酸门控阳离子通道4在大鼠心肌成纤维细胞和心肌细胞中的表达

目的 ：探讨超极化激活环核苷酸门控阳离子通道4(HCN4)在大鼠体内和体外心肌成纤维细胞、心肌细胞中的表达分布及其增龄变化。方法 ：SD大鼠分为新生组（1～3 d）,成年组（17个月）和老年组（22个月）,每组8只,其中4只用于冰冻切片,另4只用于PCR;取新生SD大鼠心,酶消化法分离培养原代心肌成纤维细胞和原代心肌细胞。用免疫荧光、免疫组织化学检测原代心肌成纤维细胞和原代心肌细胞HCN4的表达;免疫印迹、实时荧光定量PCR检测原代心肌成纤维细胞HCN4的表达,用实时荧光定量PCR检测不同月龄大鼠心肌组织HCN4的表达。结果 ：体外细胞培养显示,成纤维细胞和原代心肌细胞中均表达HCN4。组织切片显示,新生、成年和老年大鼠心肌成纤维细胞和部分心肌细胞表达HCN4。P1～P4代成纤维细胞的免疫印迹和实时荧光定量PCR结果显示HCN4随着成纤维细胞代次增加,表达量逐渐降低;不同月龄大鼠左心室的实时荧光定量PCR结果显示随着大鼠月龄增加,HCN4的表达量逐渐降低,其结果与培养的细胞表达一致。结论 ：HCN4在心肌成纤维细胞和部分心肌细胞中表达。随增龄变化,心肌细胞和成纤维细胞HCN4的表达量逐...     

不同发育时段大鼠心肌组织中超极化激活环核苷酸门控阳离子通道4阳性细胞的分布特征

目的 探讨超极化激活环核苷酸门控阳离子通道4(HCN4)在不同发育时期大鼠心肌组织中各类细胞的表达规律,为进一步研究HCN4对心肌的作用提供形态学依据。方法 出生1 d、1月、3月、6月健康SD大鼠各15只。取新鲜心脏,石蜡切片和冷冻切片,利用免疫组织化学和荧光技术观察不同发育时段大鼠心肌组织HCN4阳性细胞的表达分布情况及形态学特征。利用Western blotting 技术观察左右心室HCN4的蛋白含量。 结果 免疫组织化学和荧光染色结果显示,HCN4阳性细胞在出生后1 d、1月、3月、6月的血管平滑肌细胞,心内膜和血管的内皮细胞,心外膜的间皮细胞、成纤维细胞,大量心房肌细胞和少量心室肌细胞、传导组织细胞均有表达,但不同部位的表达细胞数量不均;Western blotting结果显示,心室HCN4蛋白随着月龄的增加表达逐渐降低。 结论 HCN4在大鼠心肌组织中的表达随年龄的增加逐渐下降,但增殖能力较强的细胞持续存在,提示HCN4可能对心肌组织中的多种细胞的生存起调控作用。     

人超极化激活环核苷酸门控阳离子通道4基因重组腺病毒构建心脏生物起搏的实验研究

目的 对心肌细胞标记分子和与起搏活动相关的人超极化激活环核苷酸门控阳离子通道4（hHCN4）的表达进行检测,并进行细胞电生理学研究.方法 将20只成年新西兰大白兔分为对照组（10只）和实验组（10只）,对照组经心外膜注射腺病毒至左心室心尖部,实验组则注射hHCN4重组腺病毒.术后3d内每天描记两组大白兔体表心电图,术后第7天分离颈部双侧迷走神经干并进行双侧或单侧电刺激,以标测电极在心尖部进行起搏（刺激脉宽2～4 ms,刺激电压5～8V,刺激频率20 ～ 30次/s）,并记录同步肢体6导联心电图.免疫荧光检测两组大白兔心脏组织和实验组大白兔肝、肾、肺组织的hHCN4表达.膜片钳记录实验组大白兔基因转染后心尖部心肌单细胞动作电位起搏相关离子流If和LCa.结果 术后3d内心电图都没有记录到两组大白兔的室性心律失常事件.在术后第7天进行颈部迷走神经干电刺激时,实验组大白兔室性逸搏节律的频率明显快于对照组[（60±8）次/min比（42±6）次/min,P＜0.05],其室性逸搏节律起源于注射部位附近.实验组大白兔注射局部的心脏组织较对照组hHCN4蛋白表达明显增强,其肝、肾、肺组织未检测到该蛋白的表达.膜片钳记录到实验组大白兔基因转染后心尖部心肌单细胞存在起搏电流If和LCa.结论 hHCN4导入心肌可以使通道蛋白在局部过度表达,提高室性异位起搏点的频率.     

超极化激活环核苷酸门控阳离子通道4、连接蛋白43和平足蛋白在小鼠胚胎心的表达与心传导系的发生

目的 探讨小鼠胚胎心传导系的发生机制。方法 用抗心肌肌球蛋白重链（MHC）、抗超极化激活环核苷酸门控阳离子通道4（HCN4）、抗缝隙连接蛋白43（CX43）和抗平足蛋白（podoplanin）抗体,对40只胚龄9~16d小鼠胚胎心进行连续石蜡切片并免疫组织化学或免疫荧光染色。结果 胚龄9d,较强的HCN4阳性表达集中在MHC阴性的静脉窦壁,随心脏发育,HCN4较强阳性表达逐渐向窦房结转移。胚龄11d开始,CX43阴性表达显示部位特异性。CX43阴性染色经窦房结沿右心房背侧壁和左、右静脉瓣向房室管背侧壁延伸。胚龄13d,左、右静脉瓣与房间隔底部融合后,进一步延续为房室管背侧壁发育中的CX43阴性染色的房室结,继而与室间隔顶部CX43阴性的房室束相连。胚龄9~10d,在MHC阳性心肌、心包腔背侧壁脏壁中胚层心肌前体细胞及静脉窦周间充质均显示podoplanin阳性表达。胚龄11~13d,podoplanin阳性间充质细胞沿心脏外表面扩展形成podoplanin阳性间皮样心外膜。结论 心脏发育早期,主起搏点位于静脉窦壁,起搏电位的产生早于收缩功能的发生。CX43阴性心肌是发育中的心传导系心肌,在胚龄11d即可观察到心传导系早期雏形。podoplanin参与促进心肌前体细胞向心肌细胞的分化。     

大鼠脊髓背角HCN4通道在糖尿病神经痛中作用的研究

目的:观察脊髓背角超极化激活环核苷酸门控通道4(HCN4)在糖尿病神经痛(DNP)发生与发展过程中的作用。方法:选择4～5周龄SD大鼠,随机分为3组:对照组、链脲佐菌素STZ组和ZD7288+STZ组。单次腹腔注射60 mg/kg STZ制备大鼠糖尿病模型,注射STZ 2周后鞘内给予HCN通道拮抗剂ZD7288。采用von Frey丝测定机械性缩足反射阈值(MWT),Western Blot法检测大鼠脊髓背角HCN4通道表达,ELISA法检测背角cAMP含量。结果:大鼠腹腔注射STZ 2周后MWT明显缩短,产生糖尿病神经痛,鞘内给予HCN通道拮抗剂ZD7288可显著延长糖尿病大鼠的MWT; DNP大鼠脊髓背角HCN4通道表达及c AMP含量显著增高,鞘内注射ZD7288可显著降低DNP大鼠脊髓背角HCN4通道表达及c AMP含量的升高。结论:脊髓背角HCN4通道表达增加促进了糖尿病神经痛的发生和发展,cAMP含量增加可能参与了HCN4通道的作用。     

5-HT受体亚型在原代培养大鼠脊髓背角神经元的表达

为探讨脊髓背角内 5 -HT发挥作用的受体类型及其胞内信号转导机制 ,本研究首先利用免疫荧光技术对胎鼠脊髓背角神经元的产率进行了观察 ,再利用反转录 PCR方法观察了 5 -HT受体 14种亚型 m RNAs在原代培养神经元中的表达。原代培养的背角神经元生长状态良好 ,存活时间达到 14～ 2 1d。对培养的背角细胞用神经元特异性标志物—神经细胞核蛋白 (Neu N)进行免疫荧光检测的结果表明 ,神经元的产率超过 90 % ;用 PCR方法在培养的背角神经元中检测到了 5 -HT1 A、5 -HT1 B、5 -HT1 D、5 -HT1 F、5 -HT2 A、5 -HT2 C、5 -HT3、5 -HT4 、5 -HT5A、5 -HT5B、5 -HT6 和 5 -HT7等受体亚型 m RNAs的表达。但上述各受体亚型的表达水平存在差异 ,未检测到 5 -HT1 E和 5 -HT2 B受体亚型 m RNAs的表达。结果表明 ,本研究建立的实验方法可满意地获得原代培养脊髓背角神经元 ;这些神经元不但表达多种受体亚型 ,而且表达类型与以往在成年大鼠脊髓背角观察到的表达状况基本一致。上述结果为进一步开展 5 -HT作用机制的体外研究奠定了基础。     

CGRP样阳性终末在脊神经结扎模型大鼠脊髓背角浅层内的表达变化

目的:观察降钙素基因相关肽(calcitonin gene-related peptide,CGRP)样阳性终末在L5脊神经结扎模型大鼠脊髓背角浅层内的表达变化。方法:建立大鼠L5脊神经结扎模型(L5 spinal nerve ligation model,SNL),建模后分别在1、3、5、7 d和14 d不同时间点通过von Frey丝检测大鼠后爪的机械性痛敏,然后在大鼠分别存活7 d和14 d时灌注取材,采用免疫组织化学染色结合平均光密度(optical density,OD)分析的方法以及免疫电镜标记技术检测脊髓背角浅层内CGRP样阳性物质的表达变化。结果:(1)行为学结果显示:模型组大鼠的机械性痛敏的阈值明显降低,与正常组比较有显著性差异(p<0.05),提示建模成功;(2)免疫组织化学染色显示:正常大鼠脊髓背角浅层内存在大量密集分布的CGRP样阳性终末,主要分布于脊髓背角的I层和II层。SNL模型大鼠在结扎7 d和14 d后脊髓背角浅层内CGRP样阳性产物的表达明显增多,其平均OD值分别为38.30±3.11和35.70±2.36,与正常对照组(25.10±2.30)比较,具有显著性差异(P<0.05);(3)电镜结果显示:CGRP样免疫活性物质只分布于轴突终末内,且主要与树突结构形成非对称性突触。结论:CGRP样免疫阳性物质在SNL模型大鼠脊髓背角浅层内的表达增加,提示CGRP样阳性终末在伤害性信息的传递中具有重要作用。     

钙激活钾通道参与重症失血性休克大鼠肠系膜细动脉平滑肌细胞膜的超极化

目的血压降低、血管反应性低下是导致创伤、脓毒性等休克病人死亡的重要原因之一.我室曾提出了致血管反应性降低的细胞膜超极化理论,并报道了ATP敏感钾通道在血管反应性低下中的作用认为随着休克的发展,组织细胞缺血、缺氧加重,ATP耗竭,细胞内乳酸堆积,从而激活了ATP敏感钾通道,使细胞膜超极化,血管舒张.除ATP敏感钾通道外,在微动脉平滑肌细胞中还有两种钾通道,即内向整流钾通道(Kir)和钙激活钾通道(KCa).其中钙激活钾通道(KCa)在血管平滑肌细胞上尤其丰富,而且在血管平滑肌细胞的肌源性调节中起着重要的作用.本文目的在于进一步阐明钙激活钾通道在休克大鼠肠系膜细动脉平滑肌细胞膜超极化中的作用,从而为临床治疗提供理论基础.     

大麻素抑制坐骨神经损伤导致的神经痛及脊髓背角HCN4通道表达

目的:观察CB1受体在坐骨神经缩窄性损伤(CCI)所致的神经痛中的作用及对CCI大鼠脊髓背角HCN4通道表达的影响。方法:7~8周龄SD大鼠分为4组:(1)sham组(假手术组);(2)CCI组;(3)CP55940+CCI组;(4)AM251+CP55940+CCI组。采用von Frey电子测痛仪测定各组大鼠损伤侧机械缩足阈值(MWT);免疫印迹法检测损伤侧L_4~L_6脊髓背角HCN4的表达。结果:CCI术后1~14 d大鼠MWT明显降低,呈现稳定的机械痛敏;鞘内给予大麻素受体激动剂CP55940(0.05 mg/kg)可显著升高CCI大鼠MWT(P0.05);预先给予CB1受体拮抗剂AM251(0.05 mg/kg)可明显阻断CP55940的镇痛效果(P0.05)。免疫印迹检测结果显示,CCI大鼠损伤侧L_4~L_6脊髓背角HCN4通道表达明显增加(P0.05);鞘内给予CP55940可显著降低CCI大鼠的HCN4表达(P0.05),CP55940抑制HCN4表达的效应可被AM251阻断(P0.05)。结论:脊髓CB1受体激活对外周神经损伤导致的神经痛具有良好的镇痛作用,其镇痛效应可能与抑制神经痛大鼠脊髓背角HCN4通道表达有关。     

ClC-3在神经病理性痛大鼠脊髓背角及背根神经节的表达及其参与痛行为调控的研究

目的:观察电压门控性氯通道(voltage-gated chloride channel,ClC)3型在腓总神经结扎神经病理性痛模型大鼠脊髓背角(spinal dorsal horn,SDH)和背根神经节(dorsal root ganglion,DRG)内的表达变化及阻断氯离子通道后痛行为的改变。方法:应用免疫组织化学染色法、蛋白印迹法以及痛行为检测观察ClC-3在神经病理性痛大鼠SDH和DRG的变化和作用。结果:在正常大鼠,ClC-3主要位于DRG神经元胞膜;在SDH,ClC-3阳性纤维主要位于Ⅰ层。在腓总神经结扎大鼠,1周内结扎侧背角Ⅰ层及DRG的ClC-3表达增加,2～4周表达逐渐减少,在DRG也观察到相同的现象。给予氯离子通道阻断剂后,腓总神经结扎大鼠的痛阈下降。结论:ClC-3在神经病理性痛早期表达上调,随病程发展逐渐下降;阻断ClC-3可使大鼠痛阈下降。     

Diabetes alters protein expression of hyperpolarization-activated cyclic nucleotide-gated channel subunits in rat nodose ganglion cells

Vagal afferent neurons, serving as the primary afferent limb of the parasympathetic reflex, could be involved in diabetic autonomic neuropathy. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in the vagal afferent neurons and play an important role in determining cell membrane excitation. In the present study, the protein expression and the electrophysiological characteristics of HCN channels were investigated in nodose ganglion (NG) afferent neurons (A-fiber and C-fiber neurons) from sham and streptozotocin (STZ)-induced diabetic rats. In the sham NG, HCN1, HCN3, and HCN4 were expressed in the A-fiber neurons; and HCN2, HCN3, and HCN4 were expressed in the C-fiber neurons. Compared to the sham NG neurons, diabetes induced the expression of HCN2 in the A-fiber neurons besides overexpression of HCN1 and HCN3; and enhanced the expression of HCN2 and HCN3 in C-fiber neurons. In addition, whole-cell patch-clamp data revealed diabetes also increased HCN currents in A-fiber and C-fiber neurons. However, we found that diabetes did not alter the total nodose afferent neuron number and the ratio of A-fiber/C-fiber neurons. These results indicate that diabetes induces the overexpression of HCN channels and the electrophysiological changes of HCN currents in the A- and C-fiber nodose neurons, which might contribute to the diabetes-induced alteration of cell excitability in the vagal afferent neurons.     

Nociceptive neurones in the superficial dorsal horn of cat lumbar spinal cord and their primary afferent inputs

Summary The morphology, background activity and responses to stimulation of primary afferent inputs of small neurones in the superficial dorsal horn which could only be excited from the skin by noxious stimulation were investigated by intracellular recording and ionophoresis of HRP. Neurones which gave similar responses to afferent stimulation were morphologically heterogeneous with respect to dendritic tree geometry and axonal projection, but were located around the lamina I/II border. Cutaneous excitatory receptive fields responding to noxious stimulation were generally small; most neurones had more extensive inhibitory fields responding to innocuous mechanical stimulation, in many cases overlapping the excitatory fields. Generally, stimulation of the excitatory field resulted in depolarization of the neurone and increased action potential firing, and stimulation of the inhibitory field resulted in hyperpolarization. Electrical stimulation of peripheral nerves revealed the existence of converging excitatory inputs carried by different fibre groups, and all neurones received an inhibitory input activated at low threshold. Excitatory responses were short-lived and occurred consistently in response to repeated stimulation. Central delay measurements gave evidence of a number of A monosynaptic inputs but only one A monosynaptic input; inhibitory inputs along A fibres were polysynaptic. The constant latency and regularity of the C response suggested monosynaptic connections. Low intensity stimulation of inhibitory inputs elicited a short-lived i.p.s.p. which increased in amplitude with increasing stimulus strength until it disappeared into a more prolonged hyperpolarization. This was associated with inhibition of background action potentials, and increased in duration with increasing stimulus strength up to C levels, indicating an A and C component. It is suggested that the level of excitability of these neurones depends on the relative amounts of concurrent noxious and innocuous stimulation, and that the resultant output, which is conveyed mainly to other neurones within the spinal cord, could modulate reflex action at the spinal level as well as affecting components of ascending sensory pathways.Supported by grant no. 11853/1.5 from the Wellcome Trust     

酪氨酸家族激酶在大鼠脊髓背角LTP中的作用及其机制

目的: 探讨酪氨酸家族激酶(SFKs)在大鼠脊髓背角C纤维诱发电位的长时程增强(LTP)中的作用及其机制。 方法: 用在体电生理方法检测SFKs抑制剂对高频刺激坐骨神经诱导的脊髓背角LTP的影响;用Western blotting方法检测高频刺激诱导脊髓LTP的不同时点大鼠脊髓背角磷酸化SFKs的表达情况;用免疫荧光双染方法检测高频刺激诱导脊髓LTP后大鼠脊髓背角磷酸化SFKs表达的细胞学定位。 结果: SFKs的抑制剂(PP2或SU6656)可以完全阻断LTP的诱导,甚至将LTP逆转为长时程抑制(LTD);高频电刺激大鼠坐骨神经诱导脊髓LTP后15 min开始,刺激同侧的脊髓背角中磷酸化SFKs表达明显增加,并且这些高表达的磷酸化SFKs仅仅存在于脊髓小胶质细胞中。 结论: 在脊髓背角,小胶质细胞中的SFKs 是诱导LTP的必要条件,抑制SFKs及其下游分子可能有助于治疗病理性疼痛。     

哮喘小鼠C_7～T_5节段脊髓后角BDNF及其受体TrkB的表达研究

目的探讨在哮喘发病中,脑源性神经营养因子(BDNF)及其高亲和力受体酪氨酸激酶B(TrkB)在哮喘小鼠C7～T5节段脊髓后角内的表达变化。方法BALB/c小鼠20只,按随机数字表法均分为正常对照组和哮喘组,利用AniRes2005肺功能仪测小鼠气道阻力、免疫荧光方法和Western blot方法检测各组小鼠C7～T5节段脊髓后角内BDNF及其高亲和力受体TrkB的表达变化。结果哮喘组小鼠吸气阻力和呼气阻力明显高于正常组(P<0.01),哮喘模型建立成功。免疫荧光结果显示哮喘组C7～T5节段脊髓后角内BDNF及TrkB阳性产物的平均光密度值(MOD)显著高于正常对照组(P<0.01),Western blot方法检测也得到了相同的结果。结论哮喘小鼠C7～T5节段脊髓后角内BDNF及TrkB的表达升高。     

Possible presence of the ATP-sensitive K channel in isolated spinal dorsal horn neurons of the rat

The ATP-sensitive K⁺ channel (K_ATP channel) is a K⁺ channel inhibited by cytoplasmic ATP. It was originally found in cardiac cells and recently in neuronal cells. Here, we present evidence indicating that the K_ATP channel also exists in spinal dorsal horn neurons: membrane currents were recorded by whole-cell voltage-clamp in spinal dorsal horn neurons isolated from young rats. The outward current was augmented by K_ATP channel activators nicorandil and minoxidil and reduced by the blocker glibenclamide. This glibenclamide-induced change in the current was augmented when the intracellular ATP was lowered and the reversal potential was shifted according to the external K⁺ concentration.     

Thiopental inhibits glycine receptor function in acutely dissociated rat spinal dorsal horn neurons

Whole-cell patch-clamp was used to assess the modulatory effect of thiopental (Thio) on glycine (Gly) receptor in mechanically dissociated rat spinal dorsal horn neurons. It was found that Thio inhibited the amplitude, accelerated the desensitization and prolonged the deactivation of Gly-induced currents (I_Gly) in a concentration-dependent manner. In addition, a rebound current occurred after washout of the co-application of Gly and Thio in most neurons tested. Moreover, the inhibitory effect of Thio was not the result of cross-inhibition between Gly and GABA_A receptors. Furthermore, taurine-induced currents, a low-affinity agonist for Gly receptors, were also markedly inhibited by Thio in a similar way to I_Gly. These results indicate that Thio suppresses Gly receptor function and suggest that Thio anesthetic actions might not be mediated by Gly receptors. We speculate that the weak muscle relaxation and the limited analgesic effects observed during Thio anesthesia may attribute to its inhibitory effects on Gly receptors.     

前列腺酸性磷酸酶在慢性痛大鼠脊髓背角和背根神经节的表达变化

目的:观察前列腺酸性磷酸酶(prostatic acid phosphatase,PAP)在多种慢性痛大鼠脊髓背角(spinaldorsal horn,SDH)和背根神经节(dorsal root ganglion,DRG)内的表达变化。方法:应用免疫组织化学染色法以及免疫荧光多重染色技术在多种慢性痛模型大鼠观察PAP的表达变化。结果:在正常大鼠,PAP阳性反应产物主要位于DRG的中、小型的非肽能神经元,PAP阳性神经元约占DRG神经元总数的64±4.3%;在脊髓背角,PAP阳性纤维和终末主要位于Ⅱ层。在神经病理性痛模型大鼠,术侧脊髓背角Ⅱ层的PAP阳性初级传入终末较对侧减少甚至消失,DRG内PAP阳性神经元较对侧明显减少。在慢性炎性痛模型大鼠,双侧脊髓背角和DRG内PAP的表达未见明显改变。结论:PAP特异地定位于DRG神经元以及脊髓背角Ⅱ层,可能与神经病理性痛信号的传递和加工密切相关。     

小胶质细胞SFKs在ATP诱导的脊髓背角LTP中的作用

目的: 研究Src家族激酶(SFKs) 在5'-三磷酸腺苷(ATP)诱导的脊髓背角长时程增强(LTP)中的作用。方法: 雄性SD大鼠(250-280 g)。在体电生理记录脊髓腰膨大部背角浅层神经元C-纤维诱发电位,Western blotting和免疫组织化学观察脊髓背角SFKs的磷酸化水平和表达部位。结果: ATP应用后30 min和60 min,磷酸化的Src-家族激酶(p-SFKs)的水平明显升高;p-SFKs只表达于小胶质细胞中,星型胶质细胞和神经元中没有表达。脊髓表面应用SFKs抑制剂阻断ATP诱导的LTP。结论: 小胶质细胞SFKs可能在ATP诱导的脊髓背角C-纤维诱发电位LTP中发挥重要作用。