东亚钳蝎毒BmK-9-(2)对豚鼠心室肌细胞钠通道的影响

目的 :观察东亚钳蝎毒 Bm K- 9- (2 )对豚鼠心室肌细胞钠通道的作用。方法 :采用全细胞膜片钳方法。结果 :Bm K- 9- (2 )使钠通道峰值电流增加 2 7%。峰值钠电导增加 1 7%。钠通道未失活成分增加 8%。 Bm K- 9- (2 )对心肌细胞钙通道没有明显影响。结论 :Bm K- 9- (2 )可激活钠通道并使钠通道失活过程延长     

东亚钳蝎毒素多肽纯化组份BmK AS-1对大鼠背根神经节神经元河豚毒素不敏感型钠通道电流的作用

目的 研究国产东亚钳蝎（ｂｕｔｈｕｓ ｍａｒｔｅｎｓｉ Ｋａｒｓｃｈ,ＢｍＫ）毒素多肽的纯化单体组分ＢｍＫ ＡＳ－１对大鼠奶神经节（ＤＲＧ）神经元河豚素不敏感（ＴＴＸ－Ｒ）钠电流的作用。方法 采用膜片箝全细胞记录方法,观察ＢｍＫ ＡＳ－１对单个ＤＲＧ细胞ＴＴＸ－Ｒ钠电流幅值的影响。结果 ＢｍＫ ＡＳ－１剂量依赖地抑制ＴＴＸ－Ｒ钠电流,且高剂量（１０μｇ／ｍｌ）ＢｍＫ ＡＳ－１几乎完全抑制ＴＴＸ－Ｒ     

Inhibitory effects of recombinant neurotoxin BmK IM on seizures induced by pentylenetetrazol in Rats

Askeyelementsofsignaltransmission ,voltage gatedsodiumchannelsplayacriticalrolebothinactionpotentialpropagationinexcitabletissuesandinneurotransmitterreleasefromnerveterminals Atthesametime ,thesechannelsarealsothetargetsofneurotoxinsfromvariousscorpionspecies Theeffectsofthesetoxinsonsodiumchannelsinvolvemodificationstochannelgatingandmembranepermeability 1,2Ithasbeendemonstratedthatmostscorpionneurotoxinsarelong chainproteinswith 60 -80aminoacids,cross linkedbyfourdisulfidebridges Generally …     

miR-340对背根神经节神经元兴奋性和钠通道电流的作用

目的：观察miR-340对小鼠背根神经节神经元兴奋性和电压依赖性钠通道电流的作用。方法：采用全细胞膜片钳技术记录神经元放电和钠通道电流。结果：在给予miR-340-3p前DRG神经元的膜静息电位为-49.50±4.76 mV,灌流miR-340-3p后膜的静息电位为-52.13±5.32 mV,差异无统计学意义（P＞0.05）,提示miR-340未影响神经元静息膜电位。在给予miR-340-3p前诱发动作电位数为1.8±0.75个,灌流miR-340-3p后诱发动作电位数为5.6±0.51个,差异具有统计学意义（P＜0.05）,提示miR-340可增高神经元诱发放电频率。电流-电压曲线表明在激活电压从-30 mV至0 mV时,miR-340-3p明显增加钠通道电流密度,差异具有统计学意义（P＜0.05）。激活曲线也表明,miR-340-3p使钠通道激活曲线左移,半激活电压从-22.6 mV增加至-27.4 mV。结论：miR-340对背根神经节神经元的兴奋作用可能是介导坐骨神经损伤神经病理性疼痛的机制之一。     

Whole-cell recordings of voltage-gated Calcium, Potassium and Sodium currents in acutely isolated hippocampal pyramidal neurons

Objective:To record Calcium, Potassium and Sodium currents in acutely isolated hippocampal pyramidal neurons. Methods:Hip-pocampal CA3 neurons were freshly isolated by 1 mg protease/3 ml SES and mechanical trituration with polished pipettes of progressively smaller tip diameters. Patch clamp technique in whole-cell mode was employed to record voltage-gated channel currents. Results:The procedure dissociated hippocampal neurons, preserving apical dendrites and several basal dendrites, without impairing the electrical characteristics of the neurons. Whole-cell patch clamp configuration was successfully used to record voltage-gated Ca2+ currents, delayed rectifier K+ current and voltage-gated Na+ currents. Conclusion:Protease combined with mechanical trituration may be used for the dissociation of neurons from rat hippocampus. Voltage-gated channels currents could be recorded using a patch clamp technique.     

加巴喷丁对大鼠背根神经节神经元高电压激活钙电流的影响

目的 探讨加巴喷丁对正常大鼠与神经病理性痛大鼠不同损伤部位的背根神经节神经元高电压激活(HVA)钙电流的影响及其原因.方法 雄性SD大鼠,周龄4～6周,采用左侧L5脊神经结扎(SNL)术制备大鼠神经病理性痛模型.于结扎后第14天时采用酶消化法急性分离结扎侧L4背根神经节神经元(SNL-L4组)、L5背根神经节神经元(SNL-L5组),另取正常大鼠L4,5背根神经节神经元作为对照组.采用全细胞膜片钳技术记录加巴喷丁对3组神经元HVA钙电流的作用.结果 在10、100、300 μmol/L浓度下,加巴喷丁对SNL-L5组峰值钙电流的抑制率[(18.5±1.7)%、(32.0±2.6)%、(32.7±2.8)%]高于SNL-L4组[(16.0±1.9)%、(26.9±2.0)%、(27.4±2.3)%]和对照组(均P<0.05).加巴喷丁作用后,SNL-L5组钙通道稳态失活曲线向超级化方向移动幅度较其他两组明显.与其他两组比较,SNL-L5组加巴喷丁敏感性钙电流中N型钙电流比例升高(均P<0.05).结论 加巴喷丁对神经病理性痛大鼠L5背根神经节神经元HVA钙电流的抑制作用增强,其可能与神经损伤后钙通道激活特性的改变以及N型钙电流比例的升高有关.

Abstract：

Objective To compare the effects of gabapentin on high-voltage-activated calcium (HVA) current in dorsal root ganglion (DRG) neurons in normal and nerve-injured rats and understand the reasons of their differences. Methods Pathogen-free male SD rats(weight 180-220 mg)aged 4-6 weeks were used. The animals were anesthetized with intraperitoneal pentobarbital sodium 50 mg/kg. L5 spinal nerve was ligated between DRG and sciatic nerve and cut distal to the ligature. The animals were decapitated at Day 14 post-operation. L5 (SNL-L5 group) and L4 DRGs (SNL-L4 group) were respectively isolated and the ganglionic neurons enzymatically dissociated. The control group of rats was not operated. The lumbar DRG neurons of normal rats were treated similarly. The HVA-Ca2+ current was recorded by the technique of whole cell patch clamp. Results Compared with the SNL-L4 group [(16.0±1.9)%, (26.9±2.0)%, (27.4±2.3)%] and the control group, gabapentin inhibited the peak calcium current highlier at 10, 100 and 300 μmol/L in the SNL-L5 group [(18.5±1.7)%, (32.0±2.6)%, (32.7±2.8)%] (P<0.05). The steady-state inactivation curves shifted to more hyperpolarized potentials in the SNL-L5 group. The N-type relative contribution to the gabapentin-sensitive HVA-Ca2+ current was markedly elevated in the SNL-L5 group compared with that in other two groups (P<0.05). Conclusion Gabapentin enhances the inhibition of HVA-Ca2+ current in injured DRG neurons following spinal nerve ligation in rats. The alteration in the activation of electrophysiological properties and the increase of N-type relative contribution to the total HVA-Ca2+ current may be involved in the mechanism.     

蛋白激酶A和蛋白激酶C对大鼠背根神经节神经元P2X3受体介导的内向电流的调节作用

目的 探讨蛋白激酶A(PKA)和蛋白激酶C(PKC)对分离培养的背根神经节(DRG)神经元ATP受体P2X3功能的调节作用.方法 分离培养大鼠DRG神经元,给予外源性ATP诱导出瞬时型内向电流,通过全细胞膜片钳记录的方法观察P2X3和P2X2/3受体特异性阻断剂TNP-ATP对这一电流的影响,在此基础上观察PKA和PKC激动剂对ATP诱导的瞬时型内向电流的调节作用.结果 在分离培养的DRG神经元上,ATP诱导的瞬时型电流可以被TNP-ATP抑制,其抑制效应呈剂量依赖性,半数有效剂量(IC50)为(21.7±7.6)nmol/L.PKA激动剂forskolin(1 μmol/L)和PKC激动剂PMA(1 μmol/L)均可以快速、可逆地抑制ATP诱导的瞬时型电流.结论 在大鼠分离培养的DRG神经元,PKA和PKC可能通过磷酸化调节抑制P2X3受体的功能,从而抑制ATP诱导的瞬时型内向电流,提示蛋白激酶对P2X3受体的调节作用可能参与痛觉的形成.     

糖尿病大鼠背根神经节神经元TTX-R钠离子通道电流变化及意义

目的:通过研究大鼠糖尿病神经病理性疼痛(DNP)中背根神经节(DRG)神经元TTX-R钠离子通道特征的变化,以探讨此疾病引起痛觉过敏可能的发生机制。方法:8只SD大鼠腹腔注射链尿佐菌素诱导制作糖尿病神经病理性疼痛模型(DNP组),取L5⁶DRG,贴壁消化法行神经元培养,电生理全细胞膜片钳记录离子通道电流;8只同月龄大鼠为正常对照。结果:大鼠糖尿病神经病理性疼痛小直径DRG神经元TTX-R INa电流密度较对照组增高。与对照组比较激活曲线向超极化移动3.9 mV(P<0.05),DNP组具有重复放电的神经元的比率增加。结论:大鼠糖尿病神经病理性疼痛小直径DRG神经元TTX-R钠通道电流的变化是痛觉过敏形成的原因之一。     

神经病理性痛大鼠背根神经节P2X3受体表达及电生理学特性的改变

目的 观察坐骨神经缩窄性损伤(CCI)致神经病理性痛后大鼠背根节神经元P2X3受体的表达和电生理学特性的变化.方法 应用免疫组化技术观察CCI后不同时间相应节段(L4、L5、L6)脊髓及背根神经节P2X3受体表达的变化;全细胞膜片钳技术观察CCI后不同时间L4、L5、L6节段背根节神经元ATP反应电流的变化.结果 CCI后7、14 d,损伤同侧L4、L5、L6节段背根节P2X3免疫阳性神经元数量明显增加,而且染色加深,相应节段的脊髓背角浅层P2X3免疫反应性亦明显增加.CCI后7、14 d,损伤同侧相应节段背根节产生ATP快反应电流的细胞数量明显增加,电流幅度亦增强.应用非选择性P2X受体拮抗剂Suramin能明显抑制ATP反应电流.结论 坐骨神经缩窄性损伤致神经痛后同侧对应节段的背根节神经元P2X3受体表达明显增加,ATP快反应电流明显增强.     

针刺对备用背根节神经元NP-1表达的影响

目的探讨针刺对备用背根节（dorsal root ganglion,DRG）NP-1表达的影响。方法将25只成年雄猫分为5组,即假手术组、单侧备用背根手术7d组、单侧备用背根手术并针刺7d组、单侧备用背根手术14d组和单侧备用背根手术并针刺14d组。通过免疫组化ABC法,观察各组备用DRG中NP-1的免疫反应神经元数目。结果在假手术组,NP-1阳性神经元主要定位于部分中小神经元及少数大神经元胞浆中;部分背根切断7d后NP-1阳性中小神经元数较假手术组明显减少（P〈0．05）,NP-1阳性大神经元数与假手术组者差异无统计学意义（P〉0．05）;而术后14d,NP-1阳性中小神经元数较7d组明显增多（P〈0．05）,且回升至假手术组水平,NP-1阳性大神经元数与手术7d组差异无统计学意义（P〉0．05）;针刺7d组较手术7d组NP-1阳性中小神经元数增多（P〈0．05）,但尚未回升至假手术组水平,NP—1阳性大神经元数减少（P〈0．05）;针刺14d组各类阳性神经元数目与手术14d组及针刺7d组差异均无统计学意义（P〉0．05）。结论针刺可上调NP-1在备用DRG中小神经元的表达。     

脊神经结扎慢性痛大鼠背根神经节细胞超极化激活电流(Ih)的变化

目的:研究超极化激活电流( hyperpolarization-activated current, Ih )在慢性神经源性痛中的作用.方法:以单侧L5和L6脊神经结扎制备神经源性痛大鼠模型,运用全细胞电压钳技术记录和分析背根神经节(dorsal root ganglion, DRG)神经元Ih的表达和激活特征.结果:脊神经结扎后,相应节段小直径DRG神经元表达Ih的细胞比率升高,但大、中直径神经元Ih没有改变,另外大、中、小直径神经元Ih的活性增强.结论:神经损伤导致DRG神经元Ih的表达和电流活性发生改变,提示此种电流参与了神经源性痛"外周敏化"的发生.     

胰岛素样生长因子 1对背根神经节神经元谷氨酸损伤的保护作用

目的探测胰岛素样生长因子 1（IGF 1）对背根神经节（DRG）神经元谷氨酸（Glu）损伤的保护作用。方法Wistar胎鼠DRG神经元分散培养48?h后,用Glu（200?μmol/L）造成神经元损伤,并同时给予IGF 1（10?nmol/L）,观察添加IGF 1和未添加IGF 1孵育的Glu损伤的神经元的活细胞生长状况,并用流式细胞仪检测神经元的凋亡率,用共聚焦激光扫描显微镜（CLSM）检测细胞内钙离子荧光强度。结果用IGF 1孵育的神经元生长状况良好,凋亡率、细胞内钙离子荧光强度均低于无IGF 1孵育的标本。结论IGF 1可通过减低钙离子内流,抑制细胞凋亡,从而对Glu损伤的DRG神经元产生保护作用。     

加巴喷丁对SNL模型大鼠损伤DRG神经元细胞内[Ca2+]i的影响

目的:探讨加巴喷丁对脊神经结扎(SNL)模型大鼠损伤背根神经节神经元胞内游离钙浓度的影响?方法:SD大鼠,雄性35只,4~6周龄,采用左侧L5 SNL术制备大鼠神经病理性疼痛模型?于结扎后15 d采用酶消化法急性分离损伤同侧L5背根神经节神经元(SNL组)和假手术组L5背根神经节神经元(Sham组)?采用流式细胞仪检测10?100和300 μmol/L浓度加巴喷丁对损伤侧L5背根神经节神经元基础及高K+激发胞内游离钙浓度的影响?结果:加巴喷丁剂量依赖性地抑制高钾激发的胞内游离钙浓度的增加(P < 0.05或P < 0.01),但对静息钙无作用?结论:加巴喷丁对神经病理性疼痛大鼠损伤背根神经节神经元高钾激发的钙内流的抑制作用,可能与其抗伤害作用的外周机制相关?     

4-1BB体外刺激的T淋巴细胞抑制小鼠黑色素瘤的作用

目的 探讨4—1BB体外刺激下小鼠T淋巴细胞的增殖情况及对小鼠黑色素瘤的抑瘤效果．方法：在IL-2＋CD3＋CD28基础上,增加4—1BB为刺激条件,体外培养经诱导的小鼠腹股沟淋巴结T细胞,MTT法检测其体外增殖率,并建立小鼠黑色素瘤实验模型,观察所诱导T淋巴细胞的抑瘤效果．结果：增加4—1BB后,T淋巴细胞的增殖率明显增加[（1．15±0．08）％协（0．57±0．06）％,P〈0．01],将所诱导的T淋巴细胞注入小鼠模型,其肺转移灶数目明显减少[（93±6）vs（151±22）,P〈0．01],生存率提高,与对照组比较差异有统计学意义（P〈0．01）．结论：4—1BB在体外可促进T淋巴细胞的增殖,并具有较强的抗黑色素瘤作用．     

PROPERTIES OF VOLTAGE-GATED SODIUM CHANNELS IN DEVELOPING AUDITORY NEURONS OF THE MOUSE IN VITRO

Objective. To investigate the properties of voltage-gated sodium (Na+) channels in developing auditory neurons during early postnatal stages in the mammalian central nervous system.Methods. Using the whole-cell voltage-clamp technique, we have studied changes in the electrophysi-ological properties of Na+ channels in the principal neurons of the medial nucleus of the trapezoid body (MNTB).Results. We found that MNTB neurons already express functional Na+ channels at postnatal day 1 (P1), and that channel density begins to increase at P5 when the neurons receive synaptic innervation and reach its maximum (~3 fold) at P11 when functional hearing onsets. These changes were paralleled by an age-dependent acceleration in both inactivation and recovery from inactivation. In contrast, there was very little alteration in the voltage-dependence of inactivation.Conclusion. These profound changes in the properties of voltage-gated Na+ channels may increase the excitability of MNTB neurons and enhance their phase-l     

Effects of Chinese herbs on multiple ion channels in isolated ventricular myocytes

Background Shensong Yangxin (SSYX) is one of the compound recipe of Chinese materia medica. This study was conducted to investigate the effects of SSYX on sodium current (I(Na)), L-type calcium current (I(Ca,L)), transient outward potassium current (I(to)), delayed rectifier current (I(K)), and inward rectifier potassium currents (I(K1)) in isolated ventricular myocytes. Methods Whole cell patch-clamp technique was used to study ion channel currents in enzymatically isolated guinea pig or rat ventricular myocytes. Results SSYX decreased peak I(Na) by (44.84±7.65)% from 27.21±5.35 to 14.88±2.75 pA/pF (n=5, P&lt;0.05). The medicine significantly inhibited the I(Ca,L). At concentrations of 0.25, 0.50, and 1.00 g/100 ml, the peak I(Ca,L) was reduced by (19.22±1.10)%, (44.82±6.50)% and (50.69±5.64)%, respectively (n=5, all P&lt;0.05). SSYX lifted the I-V curve of both I(Na) and I(Ca,L) without changing the threshold, peak and reversal potentials. At the concentration of 0.5%, the drug blocked the transient component of I(to) by 50.60% at membrane voltage of 60 mV and negatively shifted the inactive curve and delayed the recovery from channel inactivation. The tail current density of I(K) was decreased by (30.77±1.11)% (n=5, P&lt;0.05) at membrane voltage of 50 mV after exposure to the medicine and the time-dependent activity of I(K) was also inhibited. Similar to the effect on I(K), the SSYX inhibited I(K1) by 33.10% at the test potential of –100 mV with little effect on reversal potential and the rectification property. Conclusions The experiments revealed that SSYX could block multiple ion channels such as I(Na) I(Ca,L), I(k), I(to) and I(K1), which may change the action potential duration and contribute to some of its antiarrhythmic effects.     

脑源性神经营养因子在猫备用背根节的表达变化

为探讨脑源性神经营养因子（ＢＤＮＦ）在备用背根节（ＤＲＧ）的表达变化情况,对２０只成年雄性家猫（其中１５只行单侧Ｌ６备用根手术后分别存活３、６、１２天）Ｌ６ＤＲＧ行ＢＤＮＦ免疫组化染色。结果：①正常猫Ｌ６ＤＲＧ神经元以胞体直径３２μｍ和４４μｍ为分界划分出大中小三类;②正常Ｌ６ＤＲＧ内有５０．８７％的神经元呈ＢＤＮＦ阳性反应,备用根手术侧其ＢＤＮＦ阳性神经元数量增多,反应增强,以术后６天变化最显著     

PTD-BDNF对海马神经元钠、钾、钙离子通道的影响

目的研究PTD-BDNF对大鼠海马神经元细胞钠、钾、钙离子通道的影响。方法全细胞膜片钳技术记录海马神经元细胞电压依赖性钠、钾、钙通道电流,观察并分析PTD-BDNF对离子通道电流的影响。结果与对照组比较,PTD-BDNF、BDNF使海马神经元钠通道电流强度峰值分别增加39.35%和41.99%（n=4,P〈0.01）;使钠通道电流-电压关系曲线下移（n=4,P〈0.01）;使海马神经元钙通道电流强度峰值分别增加39.20%和38.72%（n=4,P〈0.01）;使钙通道电流-电压关系曲线下移（n=4,P〈0.01）;使海马神经元钾通道电流IA、IK在20mv刺激处分别比对照组降低29.03%、28.06%和29.76%、33.38%;使钾电流-电压关系曲线下移（n=4,P〈0.01）。PTD-BDNF与BDNF两者之间比较差异均无统计学意义（P〉0.05）。结论PTD-BDNF显示了与脑源性神经营养因子相似的电压依赖性离子通道的调节作用,说明PTD-BDNF与脑源性神经营养因子在调节神经元静息膜电位、神经兴奋性方面有相同的分子基础。     

部分去背根猫Sema3A及其受体NP-1在脊髓和背根节的表达

目的研究部分去背根对神经生长抑制因子Sema3A在脊髓及其受体NP-1在备用背根节内表达的影响。方法建立猫的单侧备用背根模型,分别在术后7d、14d取L3、L5、L6脊髓节段及手术侧L6背根节进行免疫组织化学ABC法染色,同时设置正常对照组。脊髓背角内检测Sema3A免疫阳性产物的平均光密度值(OD值),背根节内计数NP-1阳性中小神经元数。结果在L3脊髓节段,Sema3A的表达术后7d(0.25±0.14)、14d(0.27±0.09)较正常对照组(0.37±0.87)减弱(P<0.05);在L5脊髓节段,Sema3A的表达术后7d(0.26±0.11)较正常组减弱(P<0.05),术后14d(0.33±0.09)时有所恢复;L6脊髓节段各组Sema3A的表达无明显变化。背根节内,术后7dNP-1阳性中小神经元数(30.85±10.26)较正常组(45.06±12.47)减少(P<0.05),14d时阳性中小神经元数(40.73±12.25)较7d时有所增加(P<0.05)。结论脊髓部分去背根后,Sema3A及其受体NP-1表达的时空变化可能与脊髓损伤后可塑性有关。     

受损背根节神经元持续性钠流的检测

目的:检测损伤大鼠背根节(DRG)神经元持续性钠流(persistent sodium current, Inap)并研究其与阈下膜电位振荡的关系. 方法: 术后3～6 d慢性痛大鼠模型背根节急性分离,消化离散成单细胞,对中小型DRG神经元运用全细胞膜片钳技术记录. 结果: 电压钳下,钳制电位在-80 mV,给予斜波去极化到-30 mV时,细胞产生一持续时间150～200 ms,幅值89～200 pA的持续性内向钠流,且对100 nmol/L TTX敏感.电流钳下,产生阈下膜电位振荡的细胞,给予100 nmol/L TTX后,振荡消失.结论:在受损DRG神经元上检测到的持续性钠流可能是产生阈下膜电位振荡的基础之一.