大鼠海马神经干细胞外向电压门控性钾通道的电生理特性

目的 研究大鼠海马源神经干细胞(NSC)外向电压门控性钾通道(Kv)的电生理特性.方法 利用无血清培养、单细胞克隆技术,体外培养SD大鼠海马组织源NSC.以免疫细胞化学方法对NSC及其分化后的子代神经细胞进行鉴定.应用膜片钳技术全细胞模式记录不同外向Kv通道的电生理特性.结果 体外培养SD大鼠海马组织源NSC表达巢蛋白(nestin),在体外可诱导分化产生分别表达Tuj1和GFAP的细胞.全细胞膜片钳可记录到三种外向Kv通道:一种缓慢激活的、对四乙基胺(TEA)敏感的延迟整流钾电流(IDR);一种快速激活和失活、可被4-氨基吡啶(4-AP)阻断的外向瞬时钾电流(IA),其在+20 mV和+50 mV的电流密度分别为11 pA/pF±3 pA/pF和29 pA/pF ±7 pA/pF(标本数=11);一种可被iberiotoxin(IbIX)抑制的大电导钙激活钾通道(BKCa),钳制电位+80mV时,加入100 nM IbTX前后的电流密度分别为56 pA/pF ±4 pA/pF和45 pA/pF±4 pA/pF(标本数=6.P＜0.05).结论 体外培养的未分化状态下SD大鼠海马源NSC至少含有IDR、IA和BKCa三种外向Kv通道.     

MiRP1对异源转染的CHO细胞上HCN1和HCN2通道电生理特性的调节

目的 评价Mink相关肽1(MiRP1)对异源转染的CHO细胞上的HCN1、HCN2通道电生理特性的影响.方法 将MiRP1质粒DNA分别和HCN1、HCN2质粒DNA共转染CHO-K1细胞,用全细胞膜片钳记录细胞膜上的HCN通道电流.结果 MiRP1显著增加HCN2 [(37.8±4.8) pA/pF(n=11) vs (25.9±1.7) pA/pF(n=10); -140 mV, P<0 05]的电流密度,但是对HCN1的电流大小没有影响[(41.3±10.9) pA/pF(n=13) vs (34.9±7.8) pA/pF(n=11); P>0 05];MiRP1可加快HCN1[时间常数:(180.9±8.6) ms vs (306.1±45.6) ms; -80 mV, P<0 05]和HCN2 [时间常数:(391.8±33.6) ms vs (763.5±106.1) ms; -80 mV, P<0 05]激活动力学;MiRP1对HCN1及HCN2通道激活的电压依赖性没有影响.结论 MiRP1可加快HCN1和HCN2通道激活,且增加HCN2的电流表达.     

马桑内酯对大鼠海马神经元钠电流的影响

目的 研究马桑内酯(coriaria lactone,CL)对大鼠海马神经元钠离子通道电流的影响,探讨该影响在CL致痫中的意义.方法 利用全细胞膜片钳技术,在急性分离的大鼠海马神经元上记录钠电流,观察CL对电流幅度的影响.结果 经0.1 mg/mL、0.2 mg/mL的CL作用后,海马神经元钠电流有不同程度的增加[CL 0.1 mg/mL组的最大峰值电流密度为(-90.11±14.02) pA/pF,增幅为17.32%±8.52%;CL 0.2 mg/mL组的最大峰值电流密度为(-111.52±6.65) pA/pF,增幅为37.98%±4.91%];经与对照组相比,CL 0.2 mg/mL组(P＜0.05)的变化较CL 0.1 mg/mL组(P＞0.05)明显.结论 CL使海马神经元电压依赖性钠电流的幅度增大,从而改变细胞的兴奋性,引发异常放电.     

大鼠海马锥体神经元延迟整流钾电流的发育变化

目的 研究大鼠海马锥体细胞延迟整流钾电流(Ik)在出生后不同发育阶段的变化.方法 采用膜片钳全细胞记录模式比较3个不同年龄组,即出生后7～10天(P7-10组)、25～30天(P25-30组)和56～60天(P56-60组)Wistar大鼠急性分离的海马锥体神经元Ik的变化及通道动力学和药理学特征.结果 随着动物的发育,Ik的电流密度上调,由p-10的(32±13)pA/pF逐渐增加到P25-30的(54±20)pA/pF和P56-60的(70±18)pA/pF.随年龄增大,Ik的激活曲线左移,其半数最大激活电位(V1/2)由-12.2mV逐渐增加到-17.8 mv,而斜率因子无明显变化.四乙铵(TEA)可剂量依赖性地抑制Ik,其中P7-10组的Ik对低浓度的TEA(2和5mmol/L)较P25-30及P56-60组更敏感.结论 在大鼠海马锥体神经元的发育过程中Ik逐渐增加,并伴有通道激活动力学和药理学特性的改变.上述变化可能与海马锥体神经元的成熟以及认知功能的日益完善有关.     

产前应激加剧慢性子代应激诱导的子鼠学习记忆能力损伤

目的探讨产前应激是否进一步加剧慢性应激所致的6月龄雄性APPswe/PS1dE9子鼠学习记忆损伤及其可能机制。方法以 APPswe/PS1dE9双转基因小鼠为研究对象,实验分为产前慢性应激-子代慢性应激组( TT组)、产前慢性应激-子代正常处理组( TC组)、产前正常处理-子代慢性应激组( CT组)和产前正常处理-子代正常处理组( CC组)。应用Morris水迷宫检测子代小鼠的空间学习、记忆能力,通过HE染色观察小鼠海马CA3区神经元的病理组织形态,采用Congo red染色检查小鼠脑组织的淀粉样斑块,采用 Western blot 法检测小鼠海马组织淀粉样前体蛋白(APP)、淀粉样前体蛋白β位点分裂酶1(BACE1)和β-淀粉样蛋白1-42( Aβ42)的表达量,运用ELISA法检测血清皮质酮含量。结果与CC组相比, CT组小鼠的逃避潜伏期
　　和游泳距离延长(P<0．05),平台象限游泳时间和穿越平台次数减少(P<0．05);海马CA3区损伤的神经元数目明显增加(P<0．05),排列疏松紊乱,脱失现象明显,核固缩、浓染;脑组织淀粉样斑块数目增多;海马组织APP、BACE1和Aβ42的表达量升高( P <0．05);血清皮质酮浓度升高(P<0．05)。与CT组相比, TT组小鼠的逃避潜伏期和游泳距离进一步延长(P<0．05),平台象限游泳时间和穿越平台次数进一步减少( P <0．05);脑组织淀粉样斑块和海马CA3区损伤的神经元数目进一步增加(P<0．05);海马组织APP、BACE1和Aβ42的表达量和血清皮质酮浓度进一步升高(P<0．05)。结论产前应激进一步加剧慢性应激所致的子鼠学习记忆损伤,其机制可能与其升高小鼠血清皮质酮水平,促进APP和BACE1表达,进而增加Aβ42的生成,最终引起海马CA3区神经元损伤有关。     

可溶性Aβ25-35对大鼠海马CA3区神经元延迟整流钾电流的抑制作用

目的研究可溶性β-淀粉样蛋白(Aβ25-35)对大鼠海马CA3区锥体神经元延迟整流钾电流(IK)的影响。方法采用酶消化法急性分离新生大鼠海马CA3区神经元,全细胞膜片钳技术观察加入可溶性Aβ25-35后IK的变化。结果可溶性Aβ25-35对IK的作用具有时间依赖性,IK随Aβ25-35作用时间的延长而减小,加药5～7min后作用趋于稳定。可溶性Aβ25-35对IK有明显抑制作用,加入5μmol/LAβ25-35前后IK峰值分别为(6.987±1.152)和(2.540±0.349)nA(n=8,P<0.01);该抑制作用没有明显的浓度依赖性,1、2.5和5μmol/L3个浓度的Aβ25-35使IK峰值减小率都在60%左右。5μmol/LAβ25-35可显著影响IK激活过程,作用前后的半数激活电压分别为(4.114±0.730)和(-5.463±0.950)mV(n=15,P<0.05),但不改变激活曲线的斜率因子。结论可溶性Aβ25-35对海马CA3区神经元IK的抑制作用可能是其产生神经毒性的作用机制之一。     

慢性低氧对大鼠膈肌细胞L型钙通道电流的影响

目的　研究慢性低氧对急性分离的大鼠膈肌细胞L型钙通道电流的影响。方法　采用标准的全细胞膜片钳 (wholecellpatchclamp)技术 ,记录并比较正常对照组与慢性低氧组大鼠单个膈肌细胞的膜电容 ,L型钙电流的峰值和电流 电压关系曲线。结果　正常对照组与慢性低氧组大鼠单个膈肌细胞的膜电容分别为 (15 0 0± 12 6 )pF和(174 0± 13 3)pF ,低氧组显著大于正常对照组 (P <0 0 5 ,n =6 ) ;L型钙电流的峰值分别为 (- 1 0 5± 0 19)nA和(- 0 97± 0 16 )nA ,两组之间无显著差异。当膜电位处于 - 2 0～ +40mV时 ,慢性低氧组大鼠单个膈肌细胞的L型钙电流密度 [(- 5 6± 0 8)pA/pF]较正常对照组 [(- 7 0± 1 4 )pA/pF]显著下降 (P <0 0 5 ,n =6 )。结论　慢性低氧能使大鼠单个膈肌细胞的膜电容增加 ,L型钙电流的幅度不变 ,因此L型钙电流密度下降。L型钙电流密度下降可能是慢性缺氧时膈肌发生疲劳的重要机制之一     

海马神经干细胞体外诱导分化过程中延迟整流钾通道的电生理特性

目的 研究大鼠海马源神经干细胞(NSC)分化前及其体外诱导分化的神经元样细胞在不同发育阶段延迟整流钾电流(IDR)的电生理特性.方法 利用无血清培养、单细胞克隆技术,体外培养SD大鼠海马组织源NSC.应用膜片钳技术全细胞模式记录IDR的电生理特性.结果 IDR的电流密度在NSC分化前和体外分化(DIV)0～6 d分别为45 pA/pF±4 pA/pF和56 pA/pF±10 pA/pF(+50 mV,标本数=9),而在DIV＞6 d的发育过程中保持稳定;IDR的半数最大激活膜电位(V1/2)在NSC分化前和DIV 0～6 d分别为9 mV±3 mV和12 mV±3 mV(标本数=9,P＜0.05),激活曲线右移,斜率参数K值无明显改变,但IDR激活特性在DIV＞6 d的发育过程中无明显改变;IDR的失活特性NSC分化前后及不同发育阶段的神经元样细胞中均无改变.结论 NSC分化/发育过程中,IDR的特性改变均发生在DIV 0～6 d,提示IDR通道在神经发育过程中发挥作用,且发育初始阶段对于细胞功能的成熟尤为重要.     

KCNE2对Kv4.3通道功能的调节作用

目的研究KCNE2对人类心肌细胞瞬间外向钾电流的主要α亚基-Kv4.3功能的调节。方法通过基因转染技术将Kv4.3或Kv4.3与KCNE2cDNA转入COS-7细胞株,采用膜片钳全细胞记录方式记录通道电流。结果KCNE2对Kv4.3功能有明显调控作用:减小Kv4.3通道电流密度;Kv4.3单独表达组通道电流密度为375.13±112.87pA/pF(n=11),KCNE2与Kv4.3共表达组电流密度为152.96±33.71pA/pF(n=16);减慢Kv4.3通道激活和衰减,在 60mV电压刺激下通道激活达峰值的时间由4.82±0.32ms(n=11)延长至20.41±2.13ms(n=16),P<0.05;通道电压依赖性失活发生正向移位,半数失活电压由-53.62±1.24mV(n=8)移至-46.58±1.6mV(n=10);通道从失活中恢复的速度加快,恢复时间常数由193.43±17.98ms缩短137.71±18.29ms,(n=7,P<0.05)。结论KCNE2可能作为人类心肌细胞膜Kv4.3钾离子通道一个重要的辅助亚基-β亚基参与Ito通道功能的调节。     

加巴喷丁对急性分离大鼠三叉神经节神经元高电压激活钙电流的影响

目的:评价加巴喷丁对急性分离大鼠三叉神经节神经元高电压激活钙电流的影响.方法:清洁级SD雄性大鼠,150～200 g,酶消化急性分离双侧三叉神经节神经元,采用全细胞膜片钳技术记录三叉神经节神经元高电压激活钙电流.记录加巴喷丁10、30、100 μmol/L (G1-3组,n=10)作用下的钙电流,计算电流抑制率;并绘制浓度依赖曲线和100 μmol/L加巴喷丁作用下钙电流-电压曲线、钙通道激活曲线和稳态失活曲线.计算半数激活电压和半数失活电压.结果:加巴喷丁(10、30、100 μmol/L)均能抑制三叉神经节神经元钙电流(P<0.05),且抑制率随浓度增加而增大(P<0.05).加入100 μmol/L加巴喷丁后,钙电流峰值降低,激活曲线和稳态失活曲线分别向超极化方向移动,半数激活电压和半数失活电压均降低(P<0.05),且稳态失活曲线移动程度大于激活曲线.结论:加巴喷丁能够抑制三叉神经节神经元高电压激活钙电流,可能与其主要改变钙通道失活动力学特征有关.该作用可能为加巴喷丁治疗三叉神经痛作用机制之一.     

老年SD大鼠前列腺上皮细胞膜钾离子通道变化及意义

目的探讨雄性SD大鼠前列腺上皮细胞膜钾离子通道电流的变化和对钾通道阻断剂的反应。方法分别取3个月龄成年和12个月龄的老年SD大鼠的背外侧叶前列腺组织,剪成1—2mm^3大小,经消化、培养、免疫组鉴别,将形态正常、贴壁良好的腺上皮细胞用全细胞膜片钳模式记录钾通道电流。结果成年和老年SD大鼠的前列腺上皮细胞膜＋80mV钾电流密度分别为（10．84±1．54）pA／pF vs（18．48±1．7）pA／pF（n=20,P〈0．01）;钙激活型钾通道抑制剂（KCa通道）四乙胺（TEA）对老年大鼠峰电流阻断从19．1±2．9到7．2±3．2,KCa电流被抑制约63％;成年大鼠为9．5±1．8到5．4±3．1,KCa通道电流被抑制约44％。电压依赖型钾通道抑制剂4-AP（四氨基吡啶）对大鼠前列腺上皮细胞膜钾电流有显著阻断效应。结论老年大鼠的前列腺上皮细胞膜钾通道电流比成年大鼠显著增强,同时老年大鼠KCa通道电流对TEA更敏感。由此结果可推论老年大鼠的前列腺上皮细胞分泌功能是降低的。     

Na+/Ca2+ exchanger current and K+ current remodeling in midmyocardial cells of hypertrophic left ventricle]

OBJECTIVE: To investigate the characteristics of Na+/Ca2+ exchanger current (INa+ /Ca2+) and K+ current remodeling in midmyocardial cells of hypertrophic left ventricle for understanding the ionic basis of arrhythmia of the hypertrophic heart. METHODS: Twenty New Zealand rabbits were divided equally into normal control group and operation group, and in the latter, left ventricular hypertrophy was induced in the rabbits by partial ligation of the abdominal aorta. Action potentials, INa+/Ca2+, slowly activating delayed rectifier K+ current (IKs) and rapidly activating delayed rectifier K+ current (IKr) were recorded in the two groups by using whole-cell patch-clamp technique. RESULTS: At the basic cycle length of 2 s, 90% action potential duration (APD90) in control and operation groups was 522.0+/-19.5 ms (n=6) and 664.7+/-32.7 ms (n=7) respectively; at the testing potential of +40 mV, outward INa+/Ca2+ density in the two groups was 0.94+/-0.11 pA/pF (n=9) and 1.30+/-0.11 pA/pF (n=8) respectively; the testing potential of -100 mV elicited inward INa+/Ca2+ density of 0.40+/-0.05 pA/pF (n=9) and 0.56+/-0.02 pA/pF (n=8) respectively. The testing potential of +50 mV induced IKs tail current density of 0.26+/-0.03 pA/pF (n=8) and 0.17+/-0.01 pA/pF (n=9), and IKr tail current density of 0.34+/-0.02 pA/pF (n=8) and 0.23+/-0.02 pA/pF (n=9) respectively. Statistically significant differences were identified between the control and operation groups in all the above indices measured. CONCLUSION: The characteristics of electrical remodeling changes in midmyocardial cells of hypertrophic left ventricle, exhibited by prolonged action potential, up-regulated INa+/Ca2+ and down-regulated IKs and IKr.     

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.     

肾上腺髓质素对脓毒性休克大鼠心肌细胞外向钾电流的影响

目的 研究肾上腺髓质素(ADM)对脓毒性休克大鼠心肌细胞外向钾电流的影响.方法 成年大鼠腹腔注射脂多糖(LPS,10 mg/kg)4 h后,分离心室肌细胞.用全细胞膜片钳的方法 记录瞬时外向钾电流(I_(to)),稳态钾电流(I_(ss))和ATP敏感钾电流(I_(K,ATP)).结果 LPS处理对I_(to)和I_(ss)无影响.休克心肌细胞I_(K,ATP)密度[(2.66 ± 0.56)pA/pF(n=12)]较正常心肌细胞值[(0.27 ± 0.08)pA/pF(n=14)]明显增加(P＜0.01).ADM受体拮抗剂ADM-(22-52)可使增加的I_(K,ATP)得到明显的恢复[(0.69 ± 0.21)pA/pF(n=11),P＜0.01 vs LPS 组].而ADM-(22-52)与100 μmol/L氨基胍联合处理几乎可完全取消I_(K,ATP)的增加.结论 脓毒性休克大鼠心肌细胞I_(K,ATP)被激活.ADM 与NO共同参与了脓毒性休克大鼠心肌细胞I_(K,ATP)的激活.     

Electrophysiological properties of delayed rectifier outward K+ currents in the differentiation and development of hippocampal neural stem cells

OBJECTIVE: To study the developmental electrophysiological properties of delayed rectifier outward K+ currents (IDR) in undifferentiated NSC and NSC-derived neurons at various time points of adult SD rat hippocampus in vitro. METHODS: Neural stem cells were isolated from the hippocampus of adult SD rats with serum-free incubation and single-cell cloning technique. Electrophysiological recordings of IDR were performed using whole-cell patch clamp. RESULTS: The current density of IDR increased in NSC-derived neurons DIV 0-6 d whereas remained constant in DIV > 6 d. The current density of IDR at +50 mV was 45 pA/pF +/- 4 pA/pF and 56 pA/pF +/- 10 pA/pF in undifferentiated NSCs and NSC-derived neurons DIV 0-6 d respectively (n = 9). The activation process of IDR was also altered in DIV 0-6 d whereas remained constant in DIV > 6 d. The positive shift in steady-state activation curve of IDR revealed an increase of V1/2, however the slope factors K remained unchanged. V1/2 was 9 mV +/- 3 mV and 12 mV +/- 3 mV in undifferentiated NSCs and NSC-derived neurons DIV 0-6 d respectively (n = 9, P < 0.05). The inactivation properties of IDR were not altered before and after differentiation. CONCLUSION: Electrophysiological characteristics of IDR were all altered in DIV 0-6 d, suggesting the essential role of IDR in neurogenesis and early stage of differentiation/development process is very important for the functional mature of neuron.     

Evidence for expression of a nifedipine resistant L-type calcium current in human atrial cardiomyocytes

In the heart, two types of calcium currents were described, the L- and T-type. In addition to these two types, a dihydropyridine-resistant Ca2+ component has been described to be up-regulated in rat ventricular cardiomyocytes during their differentiation- dedifferentiation process. The aim of our study is to examine if such calcium current component is present in human cardiomyocytes. The patch clamp technique was used to record Ca2+ current in atrial cells. In the presence of 2 microM nifedipine, residual current was activated (-2.7 +/- 0.7 pA/pF, n = 6) in the same voltage range as the L-type, nifedipine-sensitive Ca2+ current (-2.1 +/- 0.4 pA/pF, n = 6), but its steady-state inactivation was negatively shifted by 10 mV. This nifedipine-resistant Ca2+ current was completely blocked by 500 microM cadmium chloride and significantly enhanced by 1 microM isoproterenol (-7.5 +/- 0.5 pA/pF, n = 6; p <0.01). These results give evidence that a nifedipine-resistant Ca2+ current, similar to the one which has been shown to be developmentally expressed in rat ventricular cardiomyocytes, is observed in human atrial cells. Its molecular identity, its expression level as well as its role in pathophysiologic conditions remain to be studied.     

β2-nAChR促进小鼠海马CA1和CA3锥体神经元GABAA受体的功能成熟

目的探讨β2-烟碱型乙酰胆碱受体（β2-nAChR）在海马CA1和CA3锥体神经元的A型γ-氨基丁酸受体（GABAA-R）发育中 的作用。方法应用β2-nAChR基因敲除小鼠（β2-KO组）制备急性分离的海马CA1和CA3锥体神经元,应用穿孔膜片钳记录技 术记录GABAA-R选择性激动剂蝇蕈醇在CA1和CA3锥体神经元诱导的GABA电流,测试其平衡电位（EMus）和动力学指标,并 与野生型小鼠（WT组）进行比较。结果β2-KO组小鼠（n=4）CA1锥体神经元（n=7）的EMus为-31.7±3.5 mV,与WT组相比向去极 化偏移（P<0.05）;CA3锥体神经元（n=4）的EMus为-16.1±4.6 mV,同样较WT组偏向去极化方向（P<0.01）;与WT组小鼠不同,β2- KO组小鼠CA3和CA1神经元的EMus差异有统计学意义（P<0.05）。β2-KO组小鼠CA1和CA3神经元上都显示GABAA-R的失 敏显著减慢,衰减时间分别为2.2±0.2 s、3.2±0.1 s（WT组为1.6±0.1 s、2.3±0.1 s,P<0.05或P<0.01）。结论含β2的nAChR可能参 与促进小鼠海马CA1和CA3锥体细胞中GABAA-R的功能成熟。