容积敏感性外向整流氯离子通道的研究进展

容积敏感性外向整流氯离子通道对维持细胞容积平衡、影响细胞凋亡、调节细胞的电活动等多种生物功能发挥重要的作用。本文就近年有关容积敏感性外向整流氯离子通道的生理特性和其在凋亡性细胞容积调节、心肌细胞缺血／再灌注损伤及心律失常等病理情况下的作用研究作一综述。     

大麻素受体在海马CA1区锥体神经元的功能表达

目的观察大麻素受体在孤立的海马CA1区锥体神经元的功能表达。方法将出生15~20d的Wistar大鼠取脑,急性分离出单个CA1区锥体神经元,用膜片钳技术记录神经元电活动,观察非选择性大麻素受体激动剂Win55212-2(5μmol/L)对神经元静息电位、动作电位、自发发放频率的影响。根据Win55212-2对膜电位的影响分为超极化组(n=7)和去极化组(n=6)。组织切片活性用MTT染色法检测。结果与给药前比较,超极化组神经元给药中动作电位频率和膜电压显著降低[0Hz vs(4.3±3.2)Hz,P0.05;(-57.0±4.6)mVvs(-54.1±3.8)mV,P0.01];与给药中比较,给药后动作电位频率及膜电压显著升高,差异有统计学意义(P0.01)。与给药前比较,去极化组神经元给药中动作电位频率显著降低,膜电压显著升高(P0.01);与给药中比较,给药后动作电位频率显著升高,膜电压显著降低,差异有统计学意义(P0.05)。结论 CA1区锥体神经元可能存在大麻素受体功能表达且不限于大麻素受体1;激活大麻素受体可能通过不同的机制起到抑制CA1区锥体神经元的作用。     

氯离子通道阻断剂对大鼠离体海马神经元凋亡的影响

目的 通过观察氯离子(Cl-)通道阻断剂对一氧化氮(NO)供体3-吗啉斯德酮胺(SIN-1)诱导的大鼠离体海马神经元凋亡的效应,探讨Cl-通道在缺血性脑损伤中的作用.方法 离体培养12 d的SD大鼠海马神经元,随机分为正常对照组、SIN-1处理组、SIN-1处理后和Cl-通道阻断剂组,对各组神经元分别在相应的时间点进行Hoechst荧光染色观察凋亡细胞数和MTT实验定量检测神经元的存活率.结果 SIN-1能明显诱导神经元凋亡(P<0.05),SITS和DIDS呈剂量依赖性地抑制NO诱导的神经元损伤,提高神经元的存活率,与SIN-1组相比差异显著(P<0.05).结论 Cl-通道阻断剂对NO诱导的大鼠海马神经元凋亡有一定的保护作用.     

海马神经元钙离子通道的研究进展

海马功能的完整不仅依赖于结构的完整,同时也依赖于各种神经递质的参与及其质和量的正常。钙离子水平的异常将导致细胞功能障碍,甚至死亡。海马神经元上的钙通道与海马功能的发挥也有密切关系。L型钙通道参与神经系统的多种重要生理功能,包括长时程增强和抑制、学习和记忆等,而海马是实现这些功能的主要脑区。海马不同亚区表达不同的钙     

二十二碳六烯酸对大鼠心室肌细胞钠通道和瞬时外向钾通道的影响

目的 研究二十二碳六烯酸(DHA)对大鼠心室肌细胞钠通道电流(INa)和瞬时外向钾通道电流(Ito)的动力学影响.探讨DHA抗心律失常的机制.方法 采用膜片钳技术在全细胞模式下,记录20、40、60、80、100和120μmol/L DHA对大鼠心室肌细胞INa和Ito的影响.结果 (1)DHA对INa呈浓度依赖性阻滞,使稳态失活曲线左移、失活后恢复时间延长,对稳态激活曲线无影响.在指令电压-30 mV,上述浓度DHA对INa阻滞分别为(1.51±1.32)%、(21.13±4.62)%、(51.61±5.73)%、(67.62±6.52)%、(73.49±7.59)%和(79.95±7.62)%(P<0.05,n=20),DHA对INa的半效作用浓度为(47.91±1.57)μmol/L.(2)DHA对Ito呈浓度依赖性阻滞,使稳态失活曲线左移、失活后恢复时间延长,对稳态激活曲线无影响.在指令电压+70 mV,上述浓度DHA对Ito阻滞分别为(2.61 ±0.26)%、(21.79±4.85)%、(63.11±6.57)%、(75.52 ±7.26)%、(81.82 ±7.63)%和(84.33±8.25)%(P<0.05,n=20),DHA对Ito的半效作用浓度为(49.11±2.68)μmol/L.结论 DHA对钠通道和瞬时外向钾通道的抑制作用可能是其抗心律失常机制之一.

Abstract：

Objective To investigate the effects of docosahexaenoic acid(DHA)on sodium channel current(INa)and transient outward potassium channel current(Ito)in rat ventricular myocytes and to evaluate potential anti-arrhythmic mechanisms of DHA.Methods INa and Ito of individual ventricular myocytes were recorded by patch-clamp technique in whole-cell configuration at room temperature.Effects of DHA at various concentrations(0,20,40,60,80,100 and 120 μmol/L)on INa and Ito were observed.Results (1) INa was blocked in a concentration-dependent manner by DHA,stably inactivated curves were shifted to the left,and recover time from inactivation was prolonged while stably activated curves were not affected by DHA.At-30 mV,INa was blocked to(1.51 ±1.32)%,(21.13±4.62)%,(51.61 ±5.73)%,(67.62 ±6.52)%,(73.49±7.59)%and(79.95±7.62)%in the presence of above DHA concentrations(all P<0.05,n=20),and half-effect concentration(EC50)of DHA on INa was(47.91±1.57)μmol/L(2) Ito were also blocked in a concentration-dependent manner by DHA,stably inactivated curves were shifted to the left,and recover time from inactivation was prolonged with increasing concentrations of DHA,and stably activated curves were not affected by DHA.At+70 mV,Ito was blocked to(2.61 ±0.26)%,(21.79±4.85)%,(63.11 ±6.57)%,(75.52 ±7.26)%,(81.82 ±7.63)%and(84.33±8.25)%,respectively,in the presence of above DHA concentrations(all P<0.05,n=20),and the EC50 of DHA on Ito was(49.11±2.68)μmol/1.Conclusion The blocking effects of DHA on APD and Ito may serve as one of the anti-arrhythmia mechanisms of DHA.     

多因素对海马延迟整流钾电流调控的研究进展

延迟整流钾电流是钾离子电流家族中的重要一员,具有缓慢激活和缓慢失活的特征,是动作电位复极化的主要作用电流,广泛分布于海马神经元。延迟整流钾电流在维持海马神经元膜静息电位、膜复极化速率以及调节神经元放电、动作电位时程、神经递质释放中起重要作用。近年发现,延迟整流钾电流受多种因素调控,如磷酸化、缺氧、神经递质和蛋白质等。很多人类神经疾病,如阿尔茨海默病和癫痫等都与海马神经元延迟整流钾电流有关。     

兔心室肌细胞外向整流性氯电流的研究

目的探讨兔心室肌细胞上外向整流性氯电流的特性。方法应用全细胞膜片钳技术记录兔心室肌单个细胞的电流。分别替代钾离子、氯离子,螯合钙离子,以及加入不同电流通道阻滞剂,观察不同条件下电流的变化。结果特定条件下,阻断兔心室肌细胞钾和钙电流后,移除胞外钙离子后加入EGTA可以引出一个明显的外向整流性的外向电流,被钙离子抑制;主要载流离子为氯离子,替代钾离子对电流影响不大;可以被氯离子阻滞剂氟灭酸阻滞;利用EGTA螯合钙离子以后,电流幅度与EGTA浓度表现出一定的浓度依赖性。结论此电流可能是钙离子抑制的氯电流。它是快激活、慢失活并且呈电压依赖性。     

脑缺血对阿尔茨海默病模型大鼠认知功能及海马病理的影响

目的 观察脑缺血对阿尔茨海默病(AD)大鼠认知功能及海马病理的影响,探讨脑缺血在AD病程进展中的作用.方法 大鼠海马注射凝聚态Aβ1-40成功建立AD模型后,再于海马注射内皮素-1建立脑缺血条件,检测脑缺血后AD大鼠认知功能、海马内Aβ的沉积、海马神经元的丢失及异常磷酸化tau表达的变化.结果 脑缺血后AD大鼠认知功能明显下降,海马内Aβ的沉积增加,海马神经元丢失增加,异常磷酸化tau的表达增加(P＜0.05或P＜0.01).结论 脑缺血促进了海马内Aβ的沉积、神经元的丢失和异常磷酸化tau的表达,最终导致了AD大鼠认知功能障碍的加重,提示脑缺血可加剧AD的病程进展,防治脑缺血可能减缓AD的进展.     

GABA与脑缺血再灌流后海马迟发性神经元损害

GABA 是海马主要抑制性神经递质。实验性脑缺血再灌流后海马 GABA 发生急剧升高,又显著降低于正常的双相改变;再灌流后海马 CA1区持续兴奋性增高、返回抑制减弱,齿状回和 CA3区却是兴奋性减低、返回抑制增强;若事先给一定剂量 GABA 能模拟剂能完全或部分保护 CA1区不受缺血损害;因而推测 GABA 改变、海马内原性抑制降低致兴奋—抑制失衡,可能是脑缺血再灌流后海马迟发性神经元损害的因素之一。     

葛根素对去卵巢大鼠认知功能及海马神经元超微结构的影响

目的 观察不同浓度葛根素对去卵巢大鼠认知功能及海马CA1区、CA3区神经元电镜下形态的影响.方法 SD大鼠72只,随机分为葛根素高、中、低剂量组、阳性对照组(倍美力组)、模型组、假手术组,每组12只,除假手术组外,均摘除大鼠卵巢造成去势模型.灌胃30 d后,用Morris水迷宫检测大鼠认知功能的变化,用透射电镜观察海马CA1区、CA3区神经元形态变化.结果 在训练第6天,Morris水迷宫试验测试大鼠第一次经过平台的时间(潜伏期)与模型组比较,假手术组、阳性对照组和葛根素高剂量组有统计学意义(P＜0.05);大鼠在120 s内经过平台的次数与模型组比较,假手术组、阳性对照组和葛根素高剂量组有统计学意义(P＜0.01);与低剂量组比较,葛根素高剂量组有统计学意义(P＜0.05).透射电镜结果表明,葛根素在不同浓度(30～ 120 mg/kg)范围内,随着浓度的增大,抑制海马神经元细胞凋亡的作用越强,并有一定的剂量依赖关系.结论 葛根素可以促进去卵巢大鼠学习、记忆功能的恢复,减轻因雌激素缺乏而引起的大鼠海马神经元凋亡作用.     

Effect of Trophic Factors on Delayed Neuronal Death Induced by in Vitro Ischemia in Cultivated Hippocampal and Cortical Neurons

The effect of trophic factors on neuronal survival after 30 min oxygen and glucose deprivation (in vitro ischemia) was studied in primary hippocampal and cortical neuronal cultures of rat.In vitro ischemia was produced at 37°C by placing cultures in glucose-free medium, the oxygen content of which was removed by gassing with pure argon. Afterin vitro ischemia neurons were allowed to recover either in serum-free minimal, essential medium (MEM) or in MEM containing 5% native horse serum, 100 ng/ml basic fibroblast growth factor (bFGF) or 10 ng/ml transforming growth factor-ß1 (TGF-ß1), respectively. Cultures that recovered in serum-free medium suffered a progressive type of neuronal injury: survival of either cortical or hippocampal neurons declined from about 60% after 1 h to 50% after 3 h, 40% after 6 h and less than 20% after 24 h. Addition of serum proteins to the incubation medium did not influence early survival (up to 3–6 h) but significantly improved survival after 24 h (more than 40% in both hippocampal and cortical cultures). Addition of TGF-ß 1 and bFGF had only minor effects. These data show that serum reduces delayed ischemic cell death by a mechanism which is different from that of TGF-ß1 or bFGF protection.     

Neuroprotection by melatonin against ischemic neuronal injury associated with modulation of DNA damage and repair in the rat following a transient cerebral ischemia

In the present study, double fluorescence staining combined with confocal laser scanning microscopy analysis were used to examine the effects of melatonin on ischemia-induced neuronal DNA strand breaks and its possible mechanisms in a transient middle cerebral artery (MCA) occlusion model. Results showed that melatonin dose-dependently reduced infarct areas and decreased both DNA double and single strand breaks (DSB and SSB) and enhanced cell viability in the peri-ischemic brain regions. Furthermore, Bcl-2 induction in the ischemic brain was further enhanced by melatonin treatment. Double staining analysis indicated that the cells costained for Bcl-2 and TdT-mediated-deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL), a DSB marker, displayed a relative regular morphology compared with the cells only stained with TUNEL. Transient ischemia induced an expression of excision repair cross-complementing factor 6 (ERCC6) mRNA, a gene essential for the preferential repair of nuclear excision repair, in the injured neurons. Double labeling showed that ERCC6 only co-localized with proliferating cell nuclear antigen (PCNA), a member of the nuclear excision repair complex, but not with TUNEL. Melatonin further and statistical significantly up-regulated ERCC6 mRNA expression in the peri-ischemic region of rat brains. The results suggest that neuroprotection by melatonin against ischemic injury may be related to modulation of apoptosis and DNA repair capacity.     

缺血期补充硝酸甘油对离体大鼠心脏心肌缺血再灌注损伤的作用

目的：观察离体大鼠心脏缺血期补充一氧化氮（ＮＯ）供体对缺血再灌注损伤的影响。　　方法：将２６只离体大鼠心脏缺血３０分,再灌注６０分。分为两组,用药组（１５只）及对照组（１１ 只）。用药组于缺血期给予４．４×１０－ ３ ｍｍ ｏｌ／Ｌ硝酸甘油（ｎｉｔｒｏｇｌｙｃｅｒｉｎ,ＮＴＧ,一种ＮＯ供体）,碳酸氢盐缓冲液灌注。对照组仅给予碳酸氢盐缓冲液灌注。全部心脏均测定ＮＯ释放量、肌酸激酶漏出量及（或）心脏功能。　　结果：用药组大鼠心脏,使用ＮＴＧ表现为两种效应。其中部分大鼠心脏（非心室颤动组,ｎ＝ ７）,ＮＴＧ增加肌酸激酶漏出量,减弱再灌注期心脏功能的恢复,伴随缺血期ＮＯ释放量的增加。对另一部分大鼠心脏（心室颤动组,ｎ＝ ８）,ＮＴＧ减少肌酸激酶的释放,但心脏于再灌注期持续心室颤动,缺血期ＮＯ释放量无明显增加。　　结论：缺血期给予同一剂量ＮＴＧ对心肌缺血再灌注损伤产生双重效应,既可增加心肌损伤,又可减轻心肌损伤。     

Effect of Prenatal Ethanol Exposure on the Developmental Profile of the NMDA Receptor Subunits in Rat Forebrain and Hippocampus

The effects of prenatal ethanol exposure on the NMDARl protein expression (postnatal days 1 and 7) and on the developmental profile of the NMDAR2A and NMDAR2B subunits in rat forebrain and hippocampus were investigated. Forebrain and hippocampal membrane proteins were isolated from pups of various ages (postnatal days 1 to 21) from prenatally ethanol exposed, pairfed and ad libitum control groups. A semiquantitative immunoblot procedure was used with antibodies raised against the NMDAR1, NMDAR2A, and the NMDAR2B subunits to assess the NMDA subunit protein expression. In the samples, NMDAR1 protein expression was unaffected by prenatal ethanol exposure at postnatal day 1 or 7 in both the forebrain and hippocampus. NMDAR2A protein expression levels rose rapidly. In both forebrain and hippocampus during the time frame of study. Renatal ethanol exposure caused a significant reduction in protein expression levels of the NMDAR2A in forebrain through postnatal day 14. NMDAR2B protein expression levels were high throughout the study in both forebrain and hippocampus. Prenatal ethanol exposure significantly reduced protein expression of the NMDAR2B in the forebrain (through postnatal day 14) and hippocampus (up to day 7). The results suggest that there may be a link between the depressed expression of the NMDAR2 subunits and the neurodevelopmental disorders associated with fetal ethanol exposure.     

益肾降浊汤对脑缺血再灌注大鼠海马EAA含量的影响

目的 :观察益肾降浊汤对脑缺血再灌注大鼠海马组织EAA含量的影响。方法 :用氨基酸自动分析仪测定游离氨基酸浓度。结果 :第 1天模型大鼠海马Glu、Asp含量分别是 ( 8.2 6± 2 .13 ) μmol/g、( 2 .10± 0 .93 ) μmol/g ,第 7天含量分别是 ( 7.2 7± 2 .2 0 ) μmol/g、( 1.87± 2 .0 1) μmol/g ,第 15天含量分别是 ( 9.3 6± 2 .2 4) μmol/g、( 1.42± 1.2 1) μmol/g ,均较正常对照组显著降低 (P <0 .0 5～ 0 .0 1)。而中药组大鼠第 1天脑海马Glu、Asp含量分别是 ( 9.90± 3 .2 0 )μmol/g、( 2 .95± 1.73 ) μmol/g ,第 7天含量分别是 ( 10 .49± 2 .3 4)μmol/g、( 3 .0 3± 1.0 9) μmol/g ,第15天含量分别是 ( 11.0 5± 2 .5 4)μmol/g、( 3 .0 5± 1.41) μmol/g ,均高于同期模型组 (P <0 .0 5 )。结论 :益肾降浊汤可提高缺血再灌注后脑组织细胞内的EAA含量 ,调节突出间隙EAA浓度 ,对缺血神经元有保护作用。     

大鼠脑缺血后可逆性蛋白磷酸化水平变化的实验研究

目的：研究大鼠脑缺血后可逆性蛋白磷酸化水平的变化。方法：采用线栓法制作大鼠永久性大脑中动脉闭塞模型,免疫荧光染色法检测神经颗粒素（Ng）、磷酸化Ng（p-Ng）和钙调神经磷酸酶（CaN）在脑缺血不同时间的变化情况。结果：脑缺血3h p-Ng开始增加,1d后达高峰（P〈0．05）,随着缺血时间的延长,P-Ng水平逐渐下降且不再恢复;缺血6hNg总蛋白无明显变化,缺血1d后呈下降趋势;脑缺血6hCaN开始发挥去磷酸化作用,3d时达到高峰（P〈0．05）,以后随着缺血时间的延长,CaN的去磷酸化作用逐渐丧失。结论：急性脑缺血后神经元内的可逆性蛋白磷酸化水平发生失衡,蛋白磷酸化／去磷酸化作用参与了缺血性神经元损伤过程。     

Effects of Dietary Restriction and Metal Supplementation on the Accumulation of Iron-Laden Glial Inclusions in the Aging Rat Hippocampus

The mechanisms responsible for the pathological deposition of iron and other redox-active metals in the aging and degenerating mammalian CNS remain poorly understood. We previously demonstrated that normal aging and pharmacological (oxidative) stressors promote the transformation of astroglial mitochondria to iron-laden, diaminobenzidine (DAB)-positive cytoplasmic inclusions in sub-cortical regions of the rat brain. In the current study, we demonstrate that (1) numbers of DAB-positive glial granules in the rat dorsal hippocampus, an area implicated in learning and memory, progressively increase between 3, 12 and 22 months of age; (2) dietary restriction (40%), a manipulation that attenuates many mammalian aging processes, has no effect on the age-related accumulation of these gliosomes in the rat hippocampus; and (3) the latter can be accelerated by dietary supplementation of iron and copper. Our data support the view that dietary exposure to iron and/or copper in adult life can impact the sequestration of redox-active metals in aging hippocampal astroglia.     

Influence of Hydrostatic Pressure and Cationic Type on the Diffusion Behavior of Chloride in Concrete

The durability of the concrete in underground and marine engineering is affected by the underground and ocean environment. Chloride diffusion coefficient under hydrostatic pressure is a key parameter of concrete durability design under corresponding conditions. Therefore, this paper studies the diffusion behavior of chloride in different diffusion source solutions by experiment and simulation. Based on the experimental results, this paper proposes a new chloride diffusion model under the coupling effect of diffusion and convection. The interaction of ions and compounds in the diffusion source solutions, concrete pore fluid, and concrete material are considered in the new chloride diffusion model. The experimental results show that chloride diffusion rate is significantly affected by hydrostatic pressure, which increases with the increase of hydrostatic pressure. The chloride diffusion coefficient shows a certain difference in difference diffusion source solutions. The chloride diffusion coefficient in divalent cationic diffusion source solutions is the largest, the chloride diffusion coefficient in the divalent and monovalent cationic compound ones is in the middle, and the chloride diffusion coefficient in the monovalent cationic ones is the smallest. There is a linear relationship between the chloride diffusion coefficient and the hydrostatic pressure whether in single or combined cationic diffusion source solutions.     

Role of ischemia in postinfarction heart failure: Hypothetical considerations based on use of verapamil in the DAVIT II study

Summary Based on a retrospective analysis of the DAVIT II database, we attempted topropose subgroups of post-infarction patients with heart failure, who might benefit or not benefit from antiischemic medical intervention. In DAVIT II, patients were randomized in a double-blind fashion to either verapamil 360 mg/day or placebo in the second week after the infarct and followed up to 18 months. The endpoint was the first major event; that is, death or reinfarction. In patients with heart failure but no ischemia, the event rate was 17.6% in the verapamil-treated patients and 16.5% in the placebo group (hazard verapamil: 1.01; 95% CL: 0.57, 1.79). In patients with both heart failure and residual ischemia, the event rate was 11.3% and 17.8% in the verapamil and placebo groups, respectively (hazard verapamil: 0.60; 95% CL: 0.24, 1.52). Although the latter reduction in major events was not statistically significant, we propose, in accordance with other studies, that in postinfarction patients with both heart failure and residual myocardial ischemia, the harmful negative inotropic effects of antiischemic drugs might be outweighed by their antiischemic effects, by means of which the prognosis might be improved. Patients without ischemia but with heart failure may, however, be at a disadvantage from such treatment.