甘珀酸对癫痫大鼠脑组织缝隙连接蛋白43表达的干预及其意义

目的 探讨癫痫大鼠脑组织缝隙连接蛋白43(connexin43,Cx43)表达及缝隙连接阻断剂甘珀酸(carbenoxolone,CBX)对其表达的影响.方法 健康SD大鼠64只,随机分为正常对照组、致痫组、致痫+生理盐水组(致痫+NS组)和致痫+CBX干预组(致痫+CBX组).采用免疫组化染色方法和实时荧光定量逆转录-聚合酶链反应方法分别测定氯化锂-匹罗卡品癫痫模型大鼠脑组织Cx43免疫阳性细胞数及Cx43mRNA表达水平,并观察腹腔注射CBX对Cx43表达的影响.结果 与正常对照组比较,致痫大鼠脑组织Cx43免疫阳性细胞数及其mRNA表达差异倍数在致痫1 h开始明显增多,并持续至7 d(P＜0.05).大鼠注射CBX后Cx43免疫阳性细胞数及其mRNA表达差异倍数均明显低于同一时间点致痫组和致痫+NS组(P＜0.05),海马区Cx43免疫阳性细胞数及其mRNA表达差异倍数在注射CBX 7 d后降至正常水平,而皮层区Cx43免疫阳性细胞数及其mRNA表达差异倍数在注射CBX 3 d后降至正常水平.结论 星形胶质细胞Cx43参与了癫痫发作和发展过程.CBX减少致痫大鼠脑组织Cx43表达,具有较明确的抗癫痫作用.     

缝隙连接蛋白Cx43在SD大鼠电点燃癫痫模型中的表达

目的探讨缝隙连接蛋白(Cx)在癫痫形成中的作用。方法将32只雄性SD大鼠随机分为4组,分别造模。观察SD大鼠行为,记录脑电图。通过免疫蛋白印迹的方法检测Cx43在大鼠海马区的表达变化。通过药物对癫痫电点燃模型成模过程的干预来观察癫痫形成与缝隙连接蛋白间的关系。结果模型组大鼠出现癫痫发作。蛋白印迹显示Cx43的表达异常增高。药物干预的2组SD大鼠模型痫性发作滞后,发作频率降低,程度减轻,痫性脑电波波幅降低,蛋白印迹显示Cx43的表达增高受到抑制。结论缝隙连接蛋白是癫痫形成的物质基础。抑制Cx43的过度表达,减少异常缝隙连接的形成,癫痫形成也受到抑制,从而预防癫痫产生。     

难治性颞叶癫痫病灶中Cx32与Cx43表达的研究

目的分析难治性癫痫患者脑组织中缝隙连接蛋白32（Cx32）、连接蛋白43（Cx43）表达,探讨缝隙连接在癫痫发生发展中的作用。方法对30例难治性癫痫患者（癫痫灶组）进行皮质电图监测下手术切除痫灶,采用免疫组化染色法（二步法）检测癫痫灶及10例周边脑组织（周边脑组织组）Cx32、Cx43表达,并进行统计学分析。结果癫痫灶组及周边脑组织组Cx43、Cx32均有表达,Cx43在癫痫灶组中表达明显增多（U=4．066,P〈0．001）;而癫痫灶组及周边脑组织组中Cx32的表达比较差别无统计学意义（U=1．866,P〉0．05）。结论缝隙连接在癫痫发生、发展中有重要作用,长期癫痫发作可造成神经元凋亡。     

缝隙连接蛋白43在癫痫持续状态过程中的表达变化

[目的]观察缝隙连接蛋白43(Cx43)在大鼠癫痫持续状态过程中的表达变化及Cx43阻断剂对大鼠痫性发作的作用.[方法]健康雄性大鼠18只随机分为三组,每只6只,分别为Cx43组、Cx43阻断组(干预组)、空白对照组.观察各组大鼠行为学、脑电图(EEG)变化并应用免疫组化方法检测海马Cx43的表达.[结果]致痫后Cx43组大鼠出现典型癫痫持续状态,持续4~6 h,EEG上记录到癫痫波,注药后4 h注药侧海马Cx43表达显著增加(P<0.05);干预组EEG放电次数较Cx43组显著减少(P<0.05);注药后4 h注药侧海马Cx43表达明显低于Cx43组(P<0.05).[结论]缝隙连接蛋白Cx43参与癫痫持续状态过程,Cx43阻断剂可能通过影响神经胶质细胞缝隙连接的形成进而影响癫痫的发生、发展过程.     

连接蛋白43在颞叶局灶性脑皮层发育不良中的表达

目的探讨连接蛋白43(Cx43)在局灶性脑皮层发育不良(focal cortical dysplasia,FCD)致痫机制中的作用。方法选择手术治疗的颞叶FCD 43例作为观察组,同期正常人脑标本10例作为对照组,采用免疫组织化学方法比较两组Cx43表达情况。结果免疫组织化学检测结果显示观察组平均每个视野Cx43免疫反应阳性细胞为(19.5±3.3)个,对照组平均每个视野Cx43免疫反应阳性细胞为(7.6±3.7)个,观察组明显高于对照组,差异有统计学意义(P0.05);单纯型FCD患者平均每个视野Cx43免疫反应阳性细胞为(14.5±4.3)个,结合型FCD患者平均每个视野Cx43免疫反应阳性细胞为(22.6±3.8)个,结合型FCD患者Cx43表达较单纯型FCD患者明显增高,差异有统计学意义(P0.05)。结论 Cx43参与FCD致痫机制,结合型FCD和单纯型FCD致痫机制可能存在差异。     

心室纤颤时心肌缝隙连接蛋白Cx43的变化

目的 观察心室纤颤(室颤)发生后缝隙连接蛋白Cx43的表达以及缝隙连接改造剂ZP123对Cx43表达的影响.方法 按照随机数字表法将30只家猪分为假手术组、模型组和ZP123干预组,每组10只.以80 V电压持续刺激动物5 s诱发室颤;致颤前15 min ZP123组给予ZP123 1μg/kg静脉推注+ZP12310μg·kg-1·h-1微量泵泵入;模型组泵入生理盐水50 ml;假手术组动物不致颤也不补液.室颤持续8 min后开胸取左心室游离壁心肌,用免疫荧光结合激光共聚焦显微镜技术检测Cx43的分布及水平,用蛋白质免疫印迹法(Western blotting)定量检测Cx43蛋白表达.结果 假手术组Cx43荧光信号强,分布均匀;模型组Cx43荧光信号弱,呈不均一分布;ZP123干预组Cx43荧光信号增强,不均一分布减轻.与假手术组比较,模型组心室肌组织Cx43荧光信号面积百分比、积分吸光度(A)值及蛋白表达均明显下降[面积百分比:(0.64±0.36)%比(1.27±0.19)%,积分A值:15 201±2 613比30 634±4 975,Cx43蛋白表达:0.72±0.08比0.97±0.07,均P＜0.05];与模型组比较,ZP123干预组Cx43表达[面积百分比(0.96±0.16)%,积分A值22 100±4 404,Cx43蛋白表达0.82±0.04]均明显升高(均P＜0.05).结论 室颤发生时心肌组织Cx43表达减少;应用ZP123可减少或逆转Cx43的降解.     

Caspase-3在戊四氮急性致痫大鼠脑组织中的表达

目的 研究caspase-3在戊四氮(pentylenetetrazole，PTZ)急性致痫大鼠脑组织中的表达情况。方法 免疫组织化学SP法显示海马及皮层caspase-3阳性细胞，CMIAS图像分析系统半定量分析caspase-3蛋白的含量。结果 癫痫发作后海马及皮层caspase-3蛋白的表达水平均增加，且海马组织较皮层组织增加更为明显。结论 Caspas-3参与了癫痫后脑组织神经元的凋亡过程；在癫痫发作所致的神经元损伤中，海马较皮层可能更为敏感。     

食管鳞癌间隙连接蛋白Cx32和Cx43的检测及意义

摘要：目的：检测间隙连接蛋白（connexin,Cx）32和43在食管鳞癌的表达,探讨其在食管鳞癌发生、发展中的作用及临床意义。 方法：用免疫组织化学技术（Elivision法）检测78例食管鳞癌组织和22例癌旁食管鳞状上皮组织中Cx32和Cx43的表达情况。 结果：癌旁食管鳞状上皮中Cx32和Cx43主要表达于细胞膜,阳性表达率均显著高于癌组织。Cx32和Cx43在食管鳞癌细胞膜表达与组织的分化程度、浸润深度、淋巴结转移及临床分期均有一定相关性,而与性别、年龄和病变部位无关。 结论：Cx32和Cx43的表达随着食管鳞癌病理分级的增高而降低,其异常表达可能是食管鳞癌发生、发展的重要因素之一。     

GABAA受体γ2亚单位在海人酸颞叶癫痫大鼠海马内的表达

目的:探讨癫痫发作后GABAA受体γ2亚单位在海马各区的动态表达以及氯硝西泮干预对其表达的影响。方法:健康成年雄性SD大鼠40只,随机分为对照组5只,致痫组15只,干预组15只,干预对照组5只。大鼠海马CA3区注射海人酸建立颞叶癫痫模型,干预组大鼠在致痫前予以氯硝西泮灌胃。于致痫后6 h、12 h和1 d采用免疫组化法检测各组大鼠海马CA1及CA3区γ-氨基丁酸A受体γ2亚单位(GABAARγ2)的动态表达水平。结果:致痫组在海人酸给药后6 h、12 h及1 d,海马CA3区GABAARγ2蛋白表达均显著低于对照组(P0.01);CA1区GABAARγ2蛋白表达也下降,注射后1 d显著低于对照组(P0.01)。干预组在海人酸注射后1 d CA1和CA3区GABAARγ2蛋白表达低于对照组(P0.05);海人酸注射后6 h、12 h及1 d,CA3区GABAARγ2蛋白表达均高于同时间点致痫组(P0.05),CA1区于海人酸注射后1 d,GABAARγ2蛋白表达显著高于同时间点致痫组(P0.01)。结论:海人酸诱导的颞叶癫痫模型中,海马GABAARγ2蛋白表达减少,氯硝西泮可缓解颞叶癫痫导致的GABAARγ2蛋白表达减少。     

腺苷受体激动剂对致痫大鼠海马神经细胞bcl-2, Bax基因表达的影响

背景癫痫发作后大脑海马神经元有明显受损,而癫痫后神经细胞损害有坏死和凋亡两种形式,在癫痫神经损害中起着重要作用.腺苷作为内源性神经保护递质,可以抑制兴奋性氨基酸的释放、氧自由基的产生以及一氧化氮的作用,同时还有改善脑血流以及抗惊厥作用.但有关腺苷与癫痫后细胞凋亡之间的关系尚不完全清楚.目的观察腺苷受体激动剂2-CAdo对癫痫大鼠海马神经细胞bcl-2,Bax基因表达的影响,进一步探讨腺苷抗惊厥及脑保护的作用机制.设计以实验动物为研究对象,完全随机对照实验研究.单位一所油田总医院的儿科和普外科,一所大学医院儿内科.材料实验于2002-10/2003-03在哈尔滨医科大学实验动物学部及病理教研室完成.体质量200～250 g健康成年Wistar大鼠104只,雌雄各半.动物随机分为正常组8只,致痫组32只,致痫+2-CAdo组32只,致痫+生理盐水组32只.干预按1.5 mg/kg腹腔注射马桑内酯(由哈尔滨医科大学药理学部提供)建立动物癫痫模型,全部大鼠于注射后5 min出现抽搐,持续时间一两分钟.致痫+2-CAdo组于马桑内酯注射前1 h及抽搐后1 h经尾静脉注射2-CAdo(由ICN公司提供)剂量为0.6 mg/kg,致痫+生理盐水组于马桑内酯注射前1 h及抽搐后1 h经尾静脉注射等量的生理盐水.主要观察指标海马CA1区bcl-2,Bax基因表达阳性细胞数.结果癫痫发作后24 h海马CA1区神经细胞bcl-2表达量增多,48 h明显下降,72 h仅有少量表达,7 d时其表达量再度升高.而Bax表达则在癫痫发作24 h开始增多,48 h显著增多,72 h表达达高峰,7 d表达量最少.致痫+2-CAdo组各相应时间点bcl-2表达量较致痫组、致痫+生理盐水组明显增高(P＜0 05),Bax表达量较致痫组、致痫+生理盐水组明显减少(P＜0.05),有统计学意义.结论2-CAdo能够减少癫痫发作后海马神经细胞的凋亡,对神经细胞有一定的保护作用.     

听源性惊厥大鼠脑星形胶质细胞Xx43表达增加对神经元保护的机制

Epilepsyisoneofthecommonandfrequentdiseases,anditharmshumanhealthseriously.Theratwithaudiogenicseizureistheanimalmodelofchronicexperimentalepilepsydeterminedbyheredity.Theseizureiscontrolledandsimilarwithsomeprimaryepilepsyofhuman,soithasbeenregardedasidealanimalmodeltodiscussprimaryepilepsyofhumanandanti-epilepsydrug犤1犦.Withdevelopmentofresearchaboutpathogenicmechanismofepilepsyandfurthercognitionofastrocytefunctionundernormalandpathologicalconditions,astrocytehasbeenthoughtthatitplayarolei…     

Slow ventricular conduction in mice heterozygous for a connexin43 null mutation.

To characterize the role of the gap junction protein connexin43 (Cx43) in ventricular conduction, we studied hearts of mice with targeted deletion of the Cx43 gene. Mice homozygous for the Cx43 null mutation (Cx43 -/-) die shortly after birth. Attempts to record electrical activity in neonatal Cx43 -/- hearts (n = 5) were unsuccessful. Ventricular epicardial conduction of paced beats, however, was 30% slower in heterozygous (Cx43 -/+) neonatal hearts (0.14+/-0.04 m/s, n = 27) than in wild-type (Cx43 +/+) hearts (0.20+/-0.07 m/s, n = 32; P < 0.001). This phenotype was even more severe in adult mice; ventricular epicardial conduction was 44% slower in 6-9 mo-old Cx43 -/+ hearts (0.18+/-0.03 m/s, n = 5) than in wild-type hearts (0.32+/-0.07 m/s, n = 7, P < 0.001). Electrocardiograms revealed significant prolongation of the QRS complex in adult Cx43 -/+ mice (13.4+/-1.8 ms, n = 13) compared with Cx43 +/+ mice (11.5+/-1.4 ms, n = 12, P < 0.01). Whole-cell recordings of action potential parameters in cultured disaggregated neonatal ventricular myocytes from Cx43 -/+ and +/+ hearts showed no differences. Thus, reduction in the abundance of a major cardiac gap junction protein through targeted deletion of a Cx43 allele directly leads to slowed ventricular conduction.     

室性心动过速时连接蛋白43含量与分布的变化

目的　探讨室性心动过速 (室速 )时心肌细胞连接蛋白 4 3(Cx 4 3)含量和分布的变化。方法　实验用日本大耳白兔 2 0只 ,随机分为对照组、30min室速组、6 0min室速组、12 0min室速组 4组。分别通过心室刺激复制室性心动过速动物模型 ,应用激光共聚焦显微镜技术和荧光免疫组织化学方法对其发生心律失常心肌连接蛋白 4 3的含量和分布进行定量分析。结果　 30min室速组连接蛋白 4 3像素密度较对照组减少 18.4 % (P <0 .0 5 ) ,6 0min室速组减少 38.0 % (P <0 .0 1) ,12 0min室速组减少 5 4 .8% (P <0 .0 1)。对照组各层间心肌细胞连接蛋白 4 3含量比较无显著差异 ,但心律失常后 ,中间层心肌细胞连接蛋白 4 3含量较其他层心肌细胞含量下降更明显 (P <0 .0 5或P <0 .0 1)。结论　室性心动过速时连接蛋白 4 3迅速降解 ,其分布也发生明显的改变 ,各层心肌细胞连接蛋白 4 3的降解程度也明显不均一。     

骨髓间质干细胞移植上调心肌梗死区Cx43表达并改善其分布

目的探讨骨髓间质干细胞（MSCs）移植能否上调心肌梗死区缝隙连接蛋白43（connexin43，Cx43）的表达，改善缝隙连接（gapjunction，GJ）的分布。方法4JD只日本大白兔随机分为正常对照组（N组）、假手术组（SH组）、心肌梗死组（MI组）和MSCs组。开胸结扎冠状动脉左前降支制造心肌梗死模型，1h后将5-溴脱氧尿核苷（BrdU）标记的MSCs（MSCs组）或培养基（MI组）移植入心肌梗死区。术后6周取心脏标本，免疫组化法鉴定植入细胞、Cx43蛋白表达与GJ分布。结果免疫组化显示，MSCs组心肌梗死区可见BrdU阳性细胞；与正常心肌比较，MI组心肌梗死中心区及边缘区Cx43显著减少（P〈0．01），GJ分布紊乱；与MI组比较，MSCs组心肌梗死边缘区Cx43表达增an（P〈0．05），GJ分布紊乱改善；在梗死中心区未见明显变化。结论MI可以导致梗死中心区和边缘区Cx43蛋白水平表达下降，GJ分布混乱；同种异体MSCs移植能改善GJ分布的紊乱，上调边缘区Cx43蛋白的表达。MSCs移植有可能减少心肌梗死后心律失常的产生。     

Characteristics of atrial substrates for atrial tachyarrhythmias induced in aged and hypercholesterolemic rabbits

Background: Old age and dyslipidemia increase the occurrence of atrial tachyarrhythmias (ATR). This study investigated the effect of age and hypercholesterolemia on the atrial substrates for ATR. Methods: Five 3‐year‐old rabbits fed standard chow were categorized into an old‐age group, five 3‐month‐old rabbits fed high cholesterol chow were used as a hypercholesterolemia group, and five 3‐month‐old rabbits fed standard chow were controls. Effective refractory period, atrial vulnerability to ATR, expressions of connexin40 (Cx40) and connexin43 (Cx43), phosphorylated c‐Jun N‐terminal Kinase (P‐JNK), and degree of fibrosis in the right (RA) and left (LA) atria were compared. Results: Old‐age and hypercholesterolemia rabbits were more vulnerable to ATR than the controls (18,628 ± 13,981 ms and 30,157 ± 39,548 ms vs 639 ± 325 ms, P < 0.05). Old‐age rabbits had significantly decreased Cx40 expression in both atria (3.9‐fold decrease in RA, P < 0.01 and 4.8‐fold in LA, P < 0.01) and significantly decreased Cx43 in RA (14‐fold, P < 0.01). Hypercholesterolemia rabbits had significantly decreased Cx40 expression in both atrial (18‐fold decrease in RA, P < 0.01 and 17‐fold in LA, P < 0.01) and significantly increased Cx43 expression in LA (five‐fold increase, P < 0.01). Hypercholesterolemia, but not old‐age rabbits, had greater expression of P‐JNK in both atria (1.8‐fold in RA and 2.3‐fold in LA, P < 0.01). There were no significant group differences in ERP or degree of atrial fibrosis in both atria. Conclusions: ATR is more easily induced in the atria of old‐age and hypercholesterolemia rabbits than younger rabbits with normal cholesterol levels. The age and hypercholesterolemia induced changes in gap junctions expression may have partially contributed to the higher atrial vulnerability to ATR. (PACE 2012;1–9)     

The involvement of gap junctions in the delayed phase of the protection induced by cardiac pacing in dogs

The present study has examined the role of GJ (gap junctions) in the delayed anti-arrhythmic effect of cardiac pacing, with particular reference to the time-course changes in Cx43 (connexin43) expression both after pacing (4×5?min, at a rate of 240 beats/min) and 24?h later, when the dogs were subjected to a 25?min occlusion and reperfusion of the LAD (left anterior descending coronary artery). Compared with the SP (sham-paced) controls (n=20), in dogs paced 24?h previously (n=16) there were reductions in arrhythmia severity [e.g. number of VPB (ventricular premature beats) during occlusion 294±78 compared with 63±25; survival from the combined ischaemia/reperfusion insult 20% compared with 78%], and in other ischaemic changes [epicardial ST-segment, TAT (total activation time) and tissue impedance]. Pacing also prevented the ischaemia-induced structural impairment of the intercalated discs, and preserved GJ permeability and Cx43 phosphorylation, without modifying Cx43 protein content. Following cardiac pacing the membrane and total Cx43 protein contents were unchanged up to 6?h, but were significantly reduced 12?h later (preceded by a down-regulation of Cx43 mRNA at 6?h), and returned to normal by 24?h. Interestingly, dogs that were subjected to ischaemia 12?h after cardiac pacing showed increased arrhythmia generation. We conclude that cardiac pacing results in time-dependent changes in Cx43 expression, which may alter GJ function and influence arrhythmia generation during a subsequent ischaemia/reperfusion insult. This effect is manifested in protection 24?h after pacing, but of potential clinical interest is the finding that there is a time interval after pacing during which an ischaemic event may generate severe ventricular arrhythmias.