深圳市南山区外来工青壮年猝死尸检病理分析

目的在对深圳市南山区青壮年外来工猝死流行病学特征进行调查的基础上,进一步对心源性猝死病例心肌缝隙连接蛋白43（Cx43）检测,观察其在心肌的分布特征,探讨其分布变化与猝死的关联性。方法筛选尸检案例30例,分为冠心病心肌梗死组（5例）、心脏传导系统病变组（5例）、肥厚型心肌病组（6例）及对照组（14例）。采用免疫组织化学染色和图像分析技术对不同组别Cx43阳性单位进行定量检测,分析其阳性表达在各组间的差异,所得数据行统计学分析。结果冠心病心肌梗死组、心脏传导系统病变组和肥厚型心肌病组共占猝死患者的53.3%,这3组患者Cx43染色在心肌闰盘处明显减弱或缺失,胞质内散在或心肌边缘处可见少量着色;对照组未见明显变化。结论冠心病心肌梗死组、心脏传导系统病变组和肥厚型心肌病组占猝死较高比例,且这3组患者Cx43分布发生了变化,推测是猝死性心律失常的结构基础。     

左归丸对化疗性卵巢早衰小鼠卵巢组织中缝隙连接蛋白43表达的影响

目的:研究左归丸对环磷酰胺(CTX)致卵巢早衰(POF)小鼠卵巢组织中缝隙连接蛋白43(Cx43)表达的影响,探讨左归丸治疗化疗性POF的作用机制。方法:将72只小鼠按体质量随机分为空白组、模型组、戊酸雌二醇片组(阳性对照,0.000 13 g/kg)和左归丸低、中、高剂量组(13.65、40.95、122.85 g/kg),每组10只。除空白组外,其余各组小鼠均ip CTX复制POF模型,每天1次,连续20 d;并同时ig相应药物,每天1次,连续30 d。末次给药2h后,分别采用反转录-聚合酶链反应法和Western blot法检测卵巢组织中Cx43 mRNA和蛋白表达;并采用免疫组化法检测卵巢组织中Cx43蛋白的分布。结果:与空白组比较,模型组大鼠卵巢组织中Cx43 mRNA及蛋白表达均明显减弱,卵泡与颗粒细胞中Cx43蛋白的分布明显减少(P<0.01);与模型组比较,戊酸雌二醇片组和左归丸中、高剂量组小鼠卵巢组织中Cx43 mRNA及蛋白表达均明显增强,卵泡与颗粒细胞中Cx43蛋白的分布明显增加(P<0.01)。结论:左归丸可上调CTX致POF小鼠卵巢组织中Cx43 mRNA及蛋白表达,增加Cx43在卵泡及颗粒细胞间的分布,改善缝隙连接功能,这可能是其治疗POF的作用机制之一。     

心房颤动患者右心房L型钙通道变化同心肌细胞结构改变的关系

目的：探讨心房颤动（房颤）的发生、房颤转复后窦性心律的维持与相关细胞结构和超微结构变化的关系.方法：53例心外科手术患者,其中窦性心律者26例（窦律组）,房颤患者27例（房颤组）.术前行超声心动图检查.手术中取右心房组织,石蜡包埋制作病理切片,HE染色后测定细胞直径.电镜检查超微结构变化.提取组织mRNA,逆转录-聚合酶链反应（RT-PCR）方法测定L-钙通道α1亚基的基因转录水平.结果：同窦律组比较,房颤组L-钙通道α1亚基mRNA水平降低,细胞直径增加,心肌出现不同程度的肌溶解现象.细胞直径同心房大小呈正相关,L-钙通道α1亚基mRNA水平与细胞直径呈负相关.结论：L-型钙通道α1亚基表达变化同房颤后细胞结构改变有关.     

奈比洛尔对自发性高血压大鼠心肌连接蛋白43重构的影响

目的探讨奈比洛尔对自发性高血压大鼠(SHR)心肌连接蛋白43(Cx43)重构的影响。方法动物随机分成:SHR组(等量蒸馏水,灌胃),奈比洛尔组(奈比洛尔8 mg·kg^(-1)·d^(-1),灌胃),WKY(Wistar-Kyoto)组(等量蒸馏水,灌胃)。给药8周,期间每周测定尾动脉收缩压。试验结束后,3%戊巴比妥钠(30 mg/kg)麻醉大鼠,分离心脏,利用免疫组织化学方法和蛋白质印迹技术观察心肌Cx43的分布和表达的影响。结果与WKY组相比,SHR收缩压持续上升,奈比洛尔则可显著降低收缩压。免疫组织化学与蛋白质印迹法结果显示,WKY组Cx43阳性表达规则分布于闰盘,SHR组Cx43阳性颗粒排列紊乱位于侧边或胞内,且蛋白表达水平明显降低。奈比洛尔可使Cx43规则分布于闰盘处,Cx43表达升高。结论奈比洛尔在显著降压的同时,可改善SHR心肌Cx43分布和表达。     

缝隙连接蛋白43在脑心综合症中的作用研究

目的检测缝隙连接蛋白43(connexin43,Cx43)在脑心综合症模型大鼠心室肌中的表达及分布,以探讨其与脑缺血后心律失常的关系。方法用线栓法栓塞大鼠右侧大脑中动脉制备脑心综合症模型,检测心电图,电镜下观察心肌细胞的亚显微结构,采用Westernblot和免疫组织化学的方法,检测心室肌中Cx43表达和分布的改变。结果电镜显示脑缺血后,心肌组织闰盘内桥粒和缝隙连接结构及分布改变,与正常对照组相比,Western blot和免疫组织化学结果均显示脑缺血后2h心室肌中Cx43的表达水平明显降低,而缺血后24h心室肌中Cx43的表达增加(P<0·05)。心电图显示,发生心律失常之前10个心动周期的心电QT间期较脑未梗死前延长(P<0·01)。结论大鼠右侧局灶性脑缺血所引起的心律失常可能与心室肌Cx43蛋白表达异常并导致的QT间期延长有关。     

皮肤恶性黑色素瘤患者Cx43表达水平及预后影响因素分析

目的 探讨皮肤恶性黑色素瘤患者缝隙连接蛋白43（Cx43）的表达水平及其预后影响因素。方法 选取2015年1月至2016年12月河北省沧州市中心医院收治的56例皮肤恶性黑色素瘤患者为研究对象，比较患者癌组织和癌旁组织的Cx43表达水平，根据癌组织Cx43的表达水平分为高表达组和低表达组，各28例。比较两组患者的临床病理特征和生存状况，采用多因素Cox回归模型分析患者预后的影响因素。结果 皮肤恶性黑色素瘤患者癌组织Cx43相对表达水平低于癌旁组织，差异有统计学意义（t=2.392，P<0.05）。低表达组原发灶-淋巴结-远处转移（TNM）分期为Ⅲ~Ⅳ期、Clark分级为Ⅴ级的比例高于高表达组，中位生存时间低于高表达组，差异均有统计学意义（χ²=5.976、5.793、16.875，P<0.05）。癌组织Cx43低表达、TNM分期为Ⅲ~Ⅳ期、发病部位为非肢端是皮肤恶性黑色素瘤患者预后的影响因素（HR=2.156、3.156、1.896，P<0.05）。结论 皮肤恶性黑色素瘤患者的癌组织Cx43表达水平明显低于癌旁组织，癌组织Cx43低表达、TNM分期晚、非肢端发病是其预后的影响因素。     

人脑胶质瘤中Cx43mRNA及其蛋白的表达意义

目的 探讨Cx43基因表达与胶质瘤恶性程度的关系。方法采用免疫组化及原位杂交技术检测Cx43基因的表达。结果Cx43mRNA及其蛋白表达随着胶质瘤组织学分级的提高呈下降趋势,Cx43蛋白表达结果与Cx43mRNA的表达结果之间呈显著正相关（P〈0．05）。人脑胶质瘤中Cx43mRNA及蛋白表达与肿瘤分级呈负相关。结论Cx43基因表达与胶质瘤恶性进展密切相关。     

缝隙连接蛋白-43在糖尿病膀胱豚鼠模型中的表达及意义

目的 探讨缝隙连接蛋白-43（Cx43）在豚鼠糖尿病膀胱（DCP）中的表达情况及其意义。方法 50只豚鼠 随机分为DCP组（n=30）、枸橼酸钠组（n=10）、空白对照组（n=10）,DCP组豚鼠单次腹腔注射链脲佐菌素建立糖尿病 模型,利用尿动力学检测筛选出糖尿病膀胱豚鼠;枸橼酸钠组豚鼠单次腹腔注射枸橼酸钠溶液;空白对照组豚鼠单 次腹腔注射生理盐水;通过免疫组织化学染色法观察Cx43在3组膀胱逼尿肌中的定位及分布,采用Western blot技术 检测3组膀胱逼尿肌组织内Cx43蛋白的表达。结果 免疫组织化学染色结果显示,DCP组（筛选出DCP豚鼠共10只 纳入研究）Cx43特异性染色强度较其他2组明显降低,枸橼酸钠组和空白对照组阳性染色强度对比无明显差异。 Western blot检测发现,DCP组膀胱组织Cx43蛋白表达（0.52±0.02）低于空白对照组（0.68±0.02）和枸橼酸钠组（0.70± 0.03）（P＜0.01）,空白对照组与枸橼酸钠组膀胱组织Cx43蛋白表达差异无统计学意义（P＞0.05）。结论 逼尿肌组 织Cx43的表达降低在糖尿病膀胱的形成过程中起重要作用,可能成为研究糖尿病膀胱发病机制的新出发点。     

缝隙连接在缺血后处理保护大鼠脑缺血再灌注损伤中的作用及可能机制

目的：探讨缝隙连接在缺血后处理保护大鼠脑缺血再灌注损伤中的作用及其可能的机制。方法：60只雄性SD大鼠随机分为假手术（sham）组、缺血再灌注（ischmia／reperfusion,I／R）组、缺血后处理（ischemicpost—conditioning,IP0）组、甘珀酸（carbenox010ne,CBX）干预缺血再灌注（I／R＋CBX）组和甘珀酸干预缺血后处理（IPO＋CBX）组。采用线栓法建立大鼠大脑中动脉栓塞模型;Longgs法进行神经功能评分;TTC染色法和HE染色法分别检测大鼠脑梗死体积和脑组织形态学变化;WesternBlot法检测脑组织中缝隙连接蛋白43（Cx43）、蛋白激酶C（PKC）蛋白的表达。结果：与sham组相比,I／R组神经功能评分显著增高,脑梗死体积增大,细胞排列紊乱、胞核固缩等组织形态学改变显著;IP0可使I／R组损伤减轻;CBX可以进一步增强IPO对I／R损伤的保护作用。WesternBlot结果显示,I／R组较sham组Cx43表达增多,PKC表达降低;IPO组较I／R组Cx43表达降低（P〈0．01）,PKC表达增高（P〈O．01）,同时,IPO＋CBX组较IPO组也有类似的改变。结论：IPO可通过抑制缝隙连接而减轻I／R损伤,其机制可能与影响PKC蛋白的表达有关。     

MicroRNA在心房颤动的心房重构过程中的应用研究

目的研究和分析microRNA（miRNA）在心房颤动的心房重构过程中的作用。方法选取于2012年9月至2013年11月在该院接受治疗的心房颤动患者198例作为研究对象,依据患者实际病况分成心房颤动组（104例）和窦性心律（窦律）组（94例）,均行外科开胸术治疗,术中取样两组患者右心耳组织进行检测和分析,记录两组患者心房组织中miRNA的表达量和Cx43mRNA表达量,并采取苏木精-伊红染色观察患者心房组织形态的变化情况。结果心房颤动患者心房组织形态学发生明显变化,主要表现为心房细胞异常增大,肌纤维异常增粗且断裂,同时表现出大小不均、排列紊乱、细胞核增大、间质纤维化明显等特点。心房颤动组miRNA在心房组织中的表达量[（0.614 9±0.175 7）×10^3]明显低于窦律组[（2.298 6±0.390 6）×10^3],差异有统计学意义（t=39.743,P〈0.01）;但两组Cx43mRNA表达量比较,差异无统计学意义（t=1.224,P〉0.05）;心房颤动组Cx43蛋白表达量[（0.576 2±0.201 0）×10^3]显著低于窦律组[（1.145 0±0.373 9）×10^3],差异有统计学意义（t=13.507,P〈0.01）。结论心房颤动患者心房重构明显;同时该类患者心房组织中miRNA的表达量明显下降,致使其相关的靶基因Cx43蛋白的表达量也随之下调,二者呈非负相关,说明miRNA并不能直接通过对Cx43的表达调控而参与到心房重构中。     

Involvement of ERK1/2 in Cx43 depression induced by macrophage migration inhibitory factor in atrial myocytes

Connexin 43 (Cx43) plays an important role in the pathogenesis of atrial fibrillation (AF). The present study sought to investigate the effect of macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, on Cx43 expression and activity and determine the intracellular signalling pathways. Cx43 protein and mRNA levels were assayed using immunofluorescence, real‐time polymerase chain reaction (PCR), and western blot. We found that increased MIF and extracellular regulated protein kinases (ERK) expression was accompanied by a significant reduction in Cx43 protein expression in atrial tissues from patients with AF compared with those with sinus rhythm. In cultured atrium‐derived myocytes (HL‐1 cells), mouse recombinant‐MIF (rMIF, 20 or 40 nmol/L, 24 hours) down‐regulated gene and protein expression of Cx43 in a concentration‐dependent manner. U0126, a specific inhibitor of mitogen‐activated protein kinase kinase (MAPKK) could reverse the decrease in expression of Cx43 protein induced by rMIF. Further studies revealed that rMIF (40 nmol/L, 15, 30, and 45 minutes) was able to stimulate phospho‐Erk1/2 (Thr202/Tyr204) production in a time‐dependent manner. These results suggest that MIF is involved in the pathogenesis of AF, probably by down‐regulating the protein and gene expression of Cx43 via ERK1/2 kinase activation. Our findings represent a potential pathogenic mechanism in AF.     

A new role for extracellular Ca2+ in gap-junction remodeling: studies in humans and rats

We wanted to elucidate whether extracellular calcium may regulate the expression of the cardiac gap-junction proteins connexin 40 and connexin43. In the free wall of the left atria of 126 cardiac surgery patients with either sinus rhythm (SR) or chronic atrial fibrillation (AF), we determined the expression of the cardiac gap-junction proteins Cx43 and Cx40 by Western blot and immunohistology. For deeper investigation, we incubated cultured neonatal rat cardiomyocytes at 2 or 4 mM Ca⁺⁺ for 24 h and determined intercellular coupling, Cx40, Cx43 protein and mRNA expression, protein trafficking and sensitivity to verapamil (10–100 nM), cyclosporin A (1 μM),and BMS605401 (100 nM), a specific inhibitor of Ca²⁺-sensing receptor (CaSR). We found in patients that both Cx are up-regulated in AF in the left atrium (by 100–200%). Interestingly, Cx40 was mainly up-regulated, if total serum calcium was ≥2.2 mM, while Cx43 was independent from extracellular [Ca⁺⁺]. In cultured cells, 4 mM Ca⁺⁺-exposure lead to up-regulation of Cx40, but not Cx43. We found enhanced Cx40 in the plasma membrane and reduced Cx40 in the Golgi apparatus. The membrane Cx40 up-regulation resulted in enhanced gap-junction intercellular coupling with a shift in the Boltzmann fit of voltage-dependent inactivation indicating a higher contribution of Cx40 as revealed by dual whole cell voltage clamp experiments. BMS605401 could prevent all Ca²⁺-induced changes. Moreover, cyclosporin A completely abolished the Ca²⁺-induced changes, while verapamil was ineffective. We conclude that extracellular calcium (24 h exposure) seems to up-regulate Cx40 but not Cx43.     

右美托咪定对快速心房起搏后兔心房电生理学特性及Cx43 Cx40表达的影响

目的 探讨右美托咪定对快速心房起搏后兔心房电生理学特性及Cx43、Cx40表达的影响。方法 选择成年雄兔24只,随机分为对照组（C组）、快速心房起搏组（RAP组）与快速心房起搏+右美托咪定灌流组（RAP+DEX组）,每组8只。制备Langendorff离体心脏灌注模型,通过快速心房起搏构建房颤模型。分别检测3组心房90%单相动作电位复极时程（MAPD₉₀）、心房有效不应期（ERP）、ERP与MAPD₉₀比值（ERP/MAPD₉₀）、房颤诱发率及持续时间,取心房组织,采用Western-bolt法和免疫荧光法检测Cx43、Cx40的蛋白含量和分布。结果 T₁~T₃时,3组MAPD₉₀比较,差异有统计学意义（P<0.05）。RAP组MAPD₉₀随时间的推移有逐渐下降趋势（P<0.05）,不同的处理方式和时间对MAPD₉₀有交互作用（P<0.05）。T₃时,RAP组ERP、ERP/MAPD₉₀、Cx43和Cx40蛋白含量均低于C组和RAP+DEX组,房颤诱发率高于C组和RAP+DEX组,差异有统计学意义（P<0.05）。3组房颤持续时间比较,差异无统计学意义（P>0.05）。电镜下,RAP组Cx43和Cx40分布不规律且侧面分布增多,而C组和RAP+DEX组Cx43和Cx40分布较规律且主要集中在两端。结论 右美托咪定可抑制房颤时的心房电重构,降低房颤的易感性,其机制可能与其抑制Cx43、Cx40的表达下调和再分布有关。     

The antiarrhythmic peptide rotigaptide (ZP123) increases gap junction intercellular communication in cardiac myocytes and HeLa cells expressing connexin 43

We investigated the effects of rotigaptide (ZP123), a stable hexapeptide with antiarrhythmic properties, on gap junction mediated intercellular communication in contracting rat neonatal cardiac myocytes, HL-1 cells derived from cardiac atrium and in HeLa cells transfected with cDNA encoding Cx43-GFP, Cx32-GFP, Cx26-GFP, wild-type Cx43 or wild-type Cx26.Intercellular communication was monitored before and after treatment with rotigaptide following microinjection of small fluorescent dyes (MW<1 kDa). The communication-modifying effect of rotigaptide was confined to cells expressing Cx43 since the peptide had no effect on dye transfer in HeLa cells expressing Cx32-GFP, Cx26-GFP or wild-type Cx26. In contrast, HeLa cells expressing Cx43-GFP exposed to 50 nM rotigaptide for 5 h showed a 40% increase in gap junction mediated communication.Rotigaptide (50 nM) increased intercellular dye transfer in myocytes and atrial HL-1 cells, where Cx43 is the dominant connexin. However, it caused no change in cell beating rates of cardiac myocytes.Western blot analysis showed that rotigaptide did not modify the overall level of Cx43 expression and changes in the phosphorylation status of the protein were not observed.We conclude that the effects of rotigaptide were confined to cells expressing Cx43.     

蝙蝠葛碱对家兔急性房颤连接蛋白40重构的影响及其机制

目的　观察快速心房起搏所致急性心房颤动 (房颤 )对家兔心房肌组织Ca2 + 含量和连接蛋白 4 0 (Connexin 4 0 ,Cx4 0 )重构的影响以及钙通道阻滞剂蝙蝠葛碱 (DAU)的干预作用 ,并对其作用机制进行探讨。方法　 32只家兔随机分为 3组 :对照组 (n =8)、房颤组 (n =12 )、DAU组 (n =12 )。经颈内静脉将电极置入右心房 ,对照组不予心房起搏 ,另外两组以 6 0 0beat·min-1行快速心房起搏以诱发房颤 ,并且DAU组于快速起搏前 30min按 5mg·kg-1静脉给予DAU ,其余两组则给予等量的生理盐水。用生化方法检测右心耳组织Ca2 + 含量 ,并分别用Westernblot和免疫荧光标记激光共聚焦显微镜检测Cx4 0的含量和分布。结果　房颤组心房组织Ca2 + 含量高于对照组 ,Cx4 0含量低于对照组 (P均 <0 0 1) ,房颤组Cx4 0分布不均一 ;蝙蝠葛碱组Ca2 + 、Cx4 0含量分别低于和高于房颤组 (P均 <0 0 5 ) ,Cx4 0分布不均一的程度较房颤组减轻。结论　快速心房起搏诱发急性房颤可引起家兔心房肌Ca2 + 含量升高、Cx4 0含量降低和分布不均一 ,蝙蝠葛碱能有效抑制Ca2 + 含量升高、减轻Cx4 0重构 ,钙超载可能参与房颤Cx4 0重构。     

Chronic regulation of the expression of gap junction proteins connexin40, connexin43, and connexin45 in neonatal rat cardiomyocytes

Gap junction channels form the basis of intercellular communication in the heart. In the working myocardium, the connexin43 (Cx43) is most abundantly found, whereas connexin40 (Cx40) is expressed in the atria and in the conduction system [together with low levels of connexin45 (Cx45)]. However, little is known about the differential regulation of the connexins by pathophysiologically stimuli such as tumor necrosis factor alpha (TNFalpha). Inasmuch as TNFalpha may play a contributory role in the concert of factors involved in the pathophysiology of heart failure and because this cardiac disease often leads to ventricular reentrant arrhythmia, the goal of our study was to find out whether TNFalpha may influence the expression of the cardiac connexins connexin43, connexin40, and connexin45. Neonatal rat cardiomyocytes were exposed to TNFalpha (10, 40, 100, 400, and 1000 pg/ml) for 24 h with or without additional treatment with the mitogenic-activated protein kinase (MAP-kinase) inhibitors SB203580 [4-(4-fluorophenyl)-2-(4-methyl-sulfinylphenyl)-5-(4-pyridyl)-1H-imidazole; 10(-5) M, protein38 mitogenic-activated protein kinase (p38 MAP kinase) inhibitor] or the MEK1 (mitogenic-activated protein kinase/extracellular signal-regulated kinase kinase) inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; 10(-5) M]. Connexin43, connexin40, and connexin45 expressions were analysed using Western blot analysis, immunohistology, and polymerase chain reaction (PCR) studies (connexin43 and connexin40). TNFalpha induced a concentration-dependent increase in connexin43 (by 2.9+/-0.6, P<0.05, n=5) but not in connexin40 or connexin45 expressions. Both connexins (40 and 45) showed a very low expression near the detection limit. The increases in connexin43 expression could be completely suppressed by SB203580 (0.9+/-0.4, P<0.05, n=5) but not by PD98059. In absence of a stimulating drug, these inhibitors (SB203580 or PD98059) did not affect connexin43 content. Additional PCR experiments revealed increases in connexin43 mRNA under the influence of 100 pg/ml TNFalpha (211+/-38%, P<0.05, n=5), which could be completely suppressed by SB203580. In contrast, the connexin40 expression remained unchanged. From these results, we conclude that TNFalpha can differentially regulate cardiac connexin expression via p38 MAP kinase pathway and thus may alter intercellular communication. This may contribute to the changes observed in heart failure with regard to the formation of an arrhythmogenic substrate.     

关白附总碱盐抗大鼠房颤作用

目的观察关白附总碱盐抗房颤作用。方法雄性SD大鼠,随机分组,CaCl210mg/mL+ACh 66μg/mL混合液iv诱发大鼠房颤,连续7d,于大鼠造模第4天起,关白附治疗组po 5、15、45mg/kg,绝奈达隆1mg/kg ip,每天1次,正常组静脉注射生理盐水7d。心电图监测大鼠房颤持续时间及QTc。7d后,测定大鼠心房肌有效不应期,western-blot法测定心房肌缝隙连接蛋白Cx40的表达。结果 Ach-CaCl2连续静脉注射引起大鼠房颤持续时间逐渐延长,与正常组比,心房有效不应期缩短(P<0.01),心房肌Cx40含量下降(P<0.05)。关白附能有效抑制房颤持续时间,延长心房ERP,保护心房Cx40表达。结论关白附能有效对抗房颤,改善房颤引起的电重构。