Connexin43在糖尿病肾病领域的研究进展

间隙连接(gap junction,GJ)是由相邻两个细胞膜上的GJ蛋白(connexin,Cx)构成的一种细胞间连接方式。GJ直接连接相邻细胞的胞浆,建立细胞间隙连接通讯(gap junctional inter-cellular communication,GJIC),介导细胞间信息、能量及物质的交换,对细胞的新陈代谢、内环境稳定、生长、增殖和分化等生     

Connexin40基因多态性与心房颤动的相关性研究

目的探讨Connexin40基因多态性与心房颤动的相关性。方法采用等位基因聚合酶链反应（PCR）的方法，对北京地区110例研究对象进行两种Connexin40单核苷酸多态性的检测，其中孤立性房颤组50例，高血压房颤组18例，单纯高血压组12例，健康对照组30例。结果孤立性房颤、高血压房颇、单纯高血压与健康对照组之间Connexin40启动子区域—44位点（G→A）单核苷酸多态等位基因频率和基因型频率存在分布差异显著（p〈0．05）；房颤组与非房颤组相比，在Connexin40启动子区域-44位点A基因型频率及A等位基因频率高于非房颤组（p〈0．05）；孤立性房颤、高血压房颤、单纯高血压与健康对照组之间Connexin40启动子区域（＋71）的基因型频率及等位基因频率的分布差异没有显著性（p〉0．05）.结论Connexin40基因多态性（-44G→A）与心房颤动发生具有相关性。     

Connexin37基因C1019T多态性与冠心病的关联研究

目的研究缝隙连接蛋白Connexin37基因C1019T多态性与冠心病危险性的关系。方法采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法检测了173例冠心病患者和148例对照的Connexin37基因C1019T基因型和等位基因的分布。结果两组基因型频率符合Hardy-Weinberg平衡。冠心病组的Connexin37基因TT和TC基因型的频率显著高于对照组(54.9%比39.2%,P<0.05),同样冠心病组T等位基因的频率显著高于对照组(35.3%比23.6%,P<0.01)。经Logistic回归分析,T等位基因携带者(TT+TC)患冠心病的危险性是CC基因型的2.34倍(95%的CI为1.03-5.32,P<0.05)。结论Connexin37基因C1019T多态性与冠心病的危险性相关,其中T等位基因可能是冠心病的遗传危险因素。     

肿瘤相关基因Cap43在胰腺癌中的表达及意义

目的研究肿瘤相关基因Cap43在胰腺癌组织中的表达情况,探讨其对胰腺癌的诊断价值。方法收集1999年4月～2002年8月长海医院外科手术切除胰腺癌标本和癌旁正常组织33例,诊断均由病理证实。男性22例,女性11例,年龄30～73岁,平均58．1岁。所有组织标本按肿瘤、癌旁(正常)配对。采用RT-PCR和Northern杂交方法研究Cap43 mRNA表达情况。结果 RT-PCR结果显示,Cap43在肿瘤组织中表达显著上调,其在肿瘤组织和癌旁正常组织的扫描值分别为4 033±1 986和2 244±1 145,有显著差异(P<0．001)。Northern杂交亦显示Cap43在肿瘤组织中表达显著上调,相同病例的RT-PCR结果与Northern杂交的结果有较好的一致性,经回归分析,没有显著性差异(P>0．1)。结论 Cap43在胰腺癌组织中呈显著高表达,其有可能成为胰腺癌早期诊断的重要标志物。     

肿瘤相关基因Cap43在胰腺癌中的表达及意义

目的研究肿瘤相关基因Cap43在胰腺癌组织中的表达情况,探讨其对胰腺癌的诊断价值.方法收集1999年4月～ 2002年8月长海医院外科手术切除胰腺癌标本和癌旁正常组织33例,诊断均由病理证实.男性22例,女性11例,年龄30 ～ 73岁,平均58.1岁.所有组织标本按肿瘤、癌旁(正常)配对.采用RT-PCR和Northern杂交方法研究Cap43 mRNA表达情况.结果 RT-PCR结果显示,Cap43在肿瘤组织中表达显著上调,其在肿瘤组织和癌旁正常组织的扫描值分别为4 033 ± 1 986和2 244 ± 1 145,有显著差异(P < 0.001).Northern杂交亦显示Cap43在肿瘤组织中表达显著上调,相同病例的RT-PCR结果与Northern杂交的结果有较好的一致性,经回归分析,没有显著性差异(P > 0.1).结论 Cap43在胰腺癌组织中呈显著高表达,其有可能成为胰腺癌早期诊断的重要标志物.     

旋毛虫Ts43基因的克隆与原核表达

目的构建旋毛虫Ts43基因原核表达载体，并且在大肠埃希菌中表达。方法将构建的pMD-Ts43克隆质粒酶切回收目的片段定向克隆到pET-28a（＋）载体上，构建原核表达载体pET-28a—Ts43，酶切鉴定正确后，导入菌株Rosetta中，用IPTG诱导表达，并经SDS-PAGE及Western blot鉴定。结果成功构建了含目的基因的原核表达质粒pET-28a—Ts43，IPTG诱导表达分子质量单位约为43ku的融合蛋白，与预测值相符。以0．9mmol／LIPTG诱导4．5h后表达量最高，薄层扫描表达的融合蛋白占菌体蛋白总量的20％。Western blot显示表达产物可被抗旋毛虫的多克隆血清识别。结论原核细胞融合表达旋毛虫Ts43基因获得成功，为旋毛虫重组苗的研究奠定了基础。     

卵巢癌组织中MDR-1、Wnt-1、FZD-1基因的表达变化及其与化疗耐药的关系

目的观察化疗及非化疗卵巢癌组织中多药耐药基因-1（MDR-1）及Wnt-1、FZD-1基因的表达变化,探讨其与卵巢癌化疗耐药的关系。方法卵巢癌患者60例,包括紫杉醇化疗组（Ⅰ组）和未化疗组（Ⅱ组）各30例,Ⅰ组分为先期化疗有效组（ⅠA组）22例和化疗后复发组（ⅠB组）8例。采用实时荧光定量RT-PCR技术检测各组卵巢癌组织中的MDR-1、Wnt-1、FZD-1基因。结果Ⅰ组MDR-1基因阳性表达率为70%（21/30）,Ⅱ组为86%（26/30）,两组相比,P〉0.05;ⅠB组MDR-1阳性6例（6/8）,较ⅠA组的15例（15/22）略高,但两组相比,P〉0.05。Wnt-1基因在MDR-1表达阳性的卵巢癌组织中的Ct值为4.742 9±0.756 9,与MDR-1阴性者的3.298 1±1.489 7相比,P〈0.01。FZD-1基因在MDR-1表达阳性的卵巢癌组织中的Ct值为3.677 3±1.220 8,与MDR-1阴性者的4.898 0±0.633 9相比,P〈0.01。在Ⅰ组中,MDR-1和Wnt-1、FZD-1基因的表达呈正相关（r分别为0.525、0.678,P均〈0.05）;但在Ⅱ组中,MDR-1和Wnt-1、FZD-1基因的表达无相关性（r分别为-0.091、0.031,P均〉0.05）。结论卵巢癌组织中MDR-1基因有较高的阳性表达率,与卵巢癌的化疗耐药有关,MDR-1基因可能通过抑制Wnt-1、FZD-1基因的表达而发挥作用。     

旋毛虫抗原基因Ts43原核表达及重组蛋白鉴定

目的原核表达旋毛虫相对分子质量(Mr)43 000抗原基因(Ts43)并对重组蛋白进行抗原性鉴定。方法将旋毛虫抗原基因Ts43克隆入原核表达载体pET30a(+),构建重组表达质粒pET30a(+)-Ts43,经测序分析正确后转化大肠埃希菌BL21,以异丙基-β-D硫代半乳糖苷(IPTG)诱导表达。十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹分析(Western blotting analysis)鉴定重组蛋白的抗原性。结果SDS-PAGE结果显示表达产物为Mr 41 000的重组融合蛋白,IPTG诱导4 h时表达量最大,薄层凝胶光密度扫描显示表达的融合蛋白占菌体蛋白总量的14.7%。Western blotting结果显示该重组蛋白可被旋毛虫、乡土旋毛虫及纳氏旋毛虫感染小鼠血清及旋毛虫病患者血清识别,与人芽囊原虫、微小隐孢子虫感染小鼠和正常小鼠血清和正常人血清无交叉反应,但与棘球蚴病、囊尾蚴病、日本血吸虫病、并殖吸虫病及华支睾吸虫病患者血清均有交叉反应。结论旋毛虫Ts43基因的重组蛋白具有较好的抗原性,但与某些寄生虫病患者血清有交叉反应。     

Wnt3a信号通路的调控及在糖尿病中的作用

Wnt3a属于Wnt1类蛋白,通过调控Wnt经典信号通路各组成蛋白的作用实现其在细胞中的功能.在细胞信号网路调控中,Wnt3a信号通路主要与骨形态发生蛋白(BMP)/Smads、转化生长因子-β(TGF-β)/Smad3和丝裂原活化蛋白激酶(MAPKs)家族信号分子之间存在交互(crosstalk)调控.近年来研究表明,Wnt3a信号通路不仅调控胰岛β细胞的代谢和功能,而且参与糖尿病并发症的发生、发展,显示其在糖尿病中具有越来越重要的作用.     

STAT3调控胰腺癌侵袭转移相关基因的筛选

目的 应用基因芯片筛选信号转导与转录激活因子3(STAT3)调控胰腺癌侵袭转移的相关基因.方法 以慢病毒感染获得稳定STAT3沉默人胰腺癌细胞株SW1990,以空质粒病毒组、未感染组为对照.应用基因芯片筛选3组细胞与肿瘤侵袭转移相关的差异表达基因.用实时PCR及蛋白质印迹法检测STAT3 mRNA及蛋白表达,并验证所选取的3个差异表达基因(MMP-7、IL-1β、IgTα7).结果 靶向STAT3的病毒感染SW1990细胞后,STAT3 mRNA表达水平为0.391±0.037,显著低于空质粒病毒组的1.002±0.015和未感染组的1.206 ±0.042(P ＜0.05);STAT3蛋白表达水平为182.38±65.32,亦明显低于空质粒病毒组的223.40±58.40和未感染组的212.33±53.69(P＜0.05).STAT3沉默的SW1990细胞有8个肿瘤侵袭转移相关基因表达上调,3个表达下调,经验证,沉默组细胞MMP-7、IL-1β mRNA表达水平明显低于空质粒病毒组(0.287±0.115比1.010±0.124,t =19.45,P=0.000;0.490±0.010比1.002±0.002,t=13.83,P=0.000),而IgTα7 mRNA表达水平无明显变化(1.173±0.280比0.998±0.003,t=4.236,P=0.094).同时沉默组细胞MMP-7蛋白表达亦显著降低.结论 STAT3沉默可导致人胰腺癌SW1990细胞多个与肿瘤侵袭转移相关基因表达的改变,其中MMP-7可能是受STAT3调控的主要靶基因.     

缺氧对肥大心肌细胞凋亡和缝隙连接蛋白43表达的影响

目的探讨缺氧对正常心肌细胞和肥大心肌细胞缝隙连接蛋白43(Cx43)表达的影响。方法将培养的乳鼠心肌细胞或诱导肥大后的心肌细胞分别缺氧24 h,并以Hoechst33258染色法检测缺氧对心肌细胞凋亡的影响,Western blot和免疫荧光法检测Cx43的表达。结果培养的心肌细胞经血管紧张素Ⅱ诱导48 h后出现肥大,肥大心肌细胞缺氧24 h比正常心肌细胞出现更明显的凋亡,缺氧明显下调心肌细胞Cx43的表达,而肥大心肌细胞Cx43的表达下调更为明显。结论缺氧导致肥大心肌细胞Cx43的表达显著下调,细胞凋亡更明显,可能与缺氧时肥大心肌的电生理重构和恶性心律失常的产生机制有关。     

缝隙连接蛋白43在老年大鼠缺血性室性心律失常中的作用

Objective To investigate the expression of connexin43 ( Cx43 ) and effect of vagal nerve stimulation (VNS) on ventricular tachyarrhythmias during acute myocardial ischemia( MI )in aged rats. Methods Male Sprague-Dawley rats[Adult group ( ≤ 4 months) and Aged group ( ≥24 months)]:MI (n = 15 ):ligated left anterior descending coronary for 30 minutes; MI-vagal nerve stimulation(VNS) ( n = 15 ); MI-VNS-atropine (0. 5 mg/kg, n = 13 ); MI-VNS-carbenoxolone ( 10 mg/kg, n = 11 ); sham operation (SO, n = 10):without coronary ligation. Ventricular arrhythmias were monitored by an electrocardiogram. Cx43 protein expression was analyzed by Western blot. Results During the 30 minutes ligation,incidences of ventricular tachycardia (VT) and ventricular fibrillation(VF) in aged rats increased significantly compared to those of adult rats ( P ＜ 0. 05 ). VNS did not affect the occurrence of VT and VF ( both P ＞ 0.05 ); however, VNS suppressed the occurrence of irreversible VF ( P ＜ 0. 05 ); both atropine and carbenoxolone ( a gap junction inhibitor) could abolish the effect of VNS on ischemia-induced irreversible VF ( both P ＜0. 05). Ischemia did not result in changes of total Cx43 amount in adult and aged rats compared to that of SO group,respectively. The amount of nonphosphorylated Cx43 was increased markedly in adult and aged rats compared to that of SO group,respectively.Cx43 dephosphorylation induced by ischemia was significantly suppressed by VNS in adult and aged rats( P ＜0. 05 ). However,the amount of total Cx43 of SO group in aged rats was significantly decreased by 50% compared to that of SO group in adult rats ( P ＜ 0. 05 ). Conclusion The present study suggested that the incidence of ischemia-induced ventricular tachyarrhythmias increased markedly and the anti-arrhythmic effect of VNS was decreased significantly in aged rats, which may be associated with reduction of Cx43 protein of ventricle in aged rats.     

缝隙连接蛋白43在老年大鼠缺血性室性心律失常中的作用

Objective To investigate the expression of connexin43 ( Cx43 ) and effect of vagal nerve stimulation (VNS) on ventricular tachyarrhythmias during acute myocardial ischemia( MI )in aged rats. Methods Male Sprague-Dawley rats[Adult group ( ≤ 4 months) and Aged group ( ≥24 months)]:MI (n = 15 ):ligated left anterior descending coronary for 30 minutes; MI-vagal nerve stimulation(VNS) ( n = 15 ); MI-VNS-atropine (0. 5 mg/kg, n = 13 ); MI-VNS-carbenoxolone ( 10 mg/kg, n = 11 ); sham operation (SO, n = 10):without coronary ligation. Ventricular arrhythmias were monitored by an electrocardiogram. Cx43 protein expression was analyzed by Western blot. Results During the 30 minutes ligation,incidences of ventricular tachycardia (VT) and ventricular fibrillation(VF) in aged rats increased significantly compared to those of adult rats ( P ＜ 0. 05 ). VNS did not affect the occurrence of VT and VF ( both P ＞ 0.05 ); however, VNS suppressed the occurrence of irreversible VF ( P ＜ 0. 05 ); both atropine and carbenoxolone ( a gap junction inhibitor) could abolish the effect of VNS on ischemia-induced irreversible VF ( both P ＜0. 05). Ischemia did not result in changes of total Cx43 amount in adult and aged rats compared to that of SO group,respectively. The amount of nonphosphorylated Cx43 was increased markedly in adult and aged rats compared to that of SO group,respectively.Cx43 dephosphorylation induced by ischemia was significantly suppressed by VNS in adult and aged rats( P ＜0. 05 ). However,the amount of total Cx43 of SO group in aged rats was significantly decreased by 50% compared to that of SO group in adult rats ( P ＜ 0. 05 ). Conclusion The present study suggested that the incidence of ischemia-induced ventricular tachyarrhythmias increased markedly and the anti-arrhythmic effect of VNS was decreased significantly in aged rats, which may be associated with reduction of Cx43 protein of ventricle in aged rats.     

Connexin 43 in ischemic pre- and postconditioning

Connexin 43 (Cx43) is the predominant protein forming gap junctions and non-junctional hemichannels in ventricular myocardium, but Cx43 is also localized at the inner membrane of cardiomyocyte mitochondria. In cardiomyocytes, Cx43 is involved in the formation of reactive oxygen species, which are central to the signal transduction cascade of ischemic preconditioning’s protection. Accordingly, genetically-induced or age-related loss of Cx43 abolishes infarct size reduction by ischemic preconditioning. Similarly, mitochondrial import inhibition of Cx43 completely blocks infarct size reduction by pharmacological preconditioning with diazoxide. In contrast to its importance for preconditioning-induced cardioprotection, Cx43 is not important for infarct size reduction by ischemic postconditioning. In summary, Cx43––especially Cx43 localized in mitochondria––appears to be one key element of the signal transduction cascade of the protection by preconditioning. The authors’ studies were supported by the German Research Foundation (Schu843/7-1).     

Effect of Beta-Carotene on Oxidative Stress and Expression of Cardiac Connexin 43

Background

Intervention studies have shown an increased mortality in patients who received beta-carotene. However, the mechanisms involved in this phenomenon are still unknown.

Objective

Evaluate the influence of beta-carotene on oxidative stress and the expression of connexin 43 in rat hearts.

Methods

Wistar rats, weighing approximately 100 g, were allocated in two groups: Control Group (n = 30), that received the diet routinely used in our laboratory, and Beta-Carotene Group (n = 28), which received beta-carotene (in crystal form, added and mixed to the diet) at a dose of 500 mg of beta carotene/kg of diet. The animals received the treatment until they reached 200-250g, when they were sacrificed. Samples of blood, liver and heart were collected to perform Western blotting and immunohistochemistry for connexin 43; morphometric studies, dosages of beta carotene by high performance liquid chromatography as well as reduced glutathione, oxidized glutathione and lipids hydroperoxides were performed by biochemical analysis.

Results

Beta-carotene was detected only in the liver of Beta-Carotene Group animals (288 ± 94.7 μg/kg). Levels of reduced/ oxidized glutathione were higher in the liver and heart of Beta-Carotene Group animals (liver - Control Group: 42.60 ± 1.62; liver - Beta-Carotene Group: 57.40 ± 5.90; p = 0.04; heart: - Control Group: 117.40 ± 1.01; heart - Beta-Carotene Group: 121.81 ± 1.32 nmol/mg protein; p = 0.03). The content of total connexin 43 was larger in Beta-Carotene Group.

Conclusion

Beta-carotene demonstrated a positive effect, characterized by the increase of intercellular communication and improvement of anti-oxidizing defense system. In this model, mechanism does not explain the increased mortality rate observed with the beta-carotene supplementation in clinical studies.     

参松养心胶囊改善心力衰竭大鼠QT离散度及缝隙连接蛋白43的表达

目的观察参松养心胶囊对压力超负荷所致心力衰竭大鼠的QT离散度及缝隙连接蛋白43表达的干预作用。方法采用腹主动脉缩窄法建立大鼠心力衰竭模型,经参松养心胶囊治疗8周。用皮下插入自制电极检测心室电生理变化,经HE染色观察其形态结构变化,Masson染色判断心肌纤维化沉积,免疫组织化学法观察缝隙连接蛋白43在左心室心肌组织的分布。结果心力衰竭大鼠QT离散度明显延长(37.20±9.94 ms,P<0.05),心肌细胞排列紊乱,心肌组织纤维沉积面积明显增加(101217.30±33970.02μm2,P<0.05),心肌组织缝隙连接蛋白43表达明显减少(55.93±11.61,P<0.05)。参松养心胶囊可缩短心力衰竭大鼠的QT离散度(25.50±8.21 ms),减少心肌组织纤维沉积面积(13580.64±8213.73μm2)并增加缝隙连接蛋白43的表达(69.09±16.59)。结论参松养心胶囊可以缩短心力衰竭大鼠的QT离散度,增加心肌组织缝隙连接蛋白43表达并减少纤维化面积。     

Cardiomyocyte-restricted deletion of connexin43 during mouse development

Although the gap junction protein Connexin43 (Cx43) is expressed in various cell types during embryonic development, mice with a global inactivation of Cx43 survive until birth but die perinatally due to an obstruction of the right ventricular outflow tract of the heart. To analyze the functional role of Cx43 gap junction channels in cardiomyocytes of the developing and early postnatal heart, we used alphaMyHC-Cre mice to ablate Cx43 expression selectively in cardiomyocytes during development. We found efficient ablation of Cx43 in cardiomyocytes during embryonic development starting at embryonic day (ED) 9.5 in the ventricular wall. Analyses of cardiac Cx43 protein at birth indicated complete loss of Cx43 expression in cardiomyocytes. All mice homozygously deficient for Cx43 in cardiomyocytes died until postnatal day (PD) 16. Heterozygous inactivation of Cx43 in cardiomyocytes neither altered atrial nor ventricular activation, but homozygous ablation led to changes in ventricular activation, i.e. significant decrease of the QRS-amplitude and prolonged QRS-duration already at PD 4. Cardiac morphology was similar to controls until PD 1, but subtle morphological changes were found in a subgroup of mutant mice at later stages. Besides narrowing of the ventricular outlet region at PD 6, hypertrophy of ventricular myocardium was found at PD 12. Our data indicate that complete inactivation of cardiac Cx43 during development predisposes hearts to develop postnatal morphological alterations, which differ from outflow tract obstructions described for Cx43 null mice. In addition, complete loss of cardiac Cx43 protein during development correlates with slowed ventricular activation at PD 4, impairs viability during development, and leads to death of all mutant mice until PD 16.     

心力衰竭大鼠心脏缝隙连接蛋白43的变化及厄贝沙坦的干预作用

目的观察压力超负荷所致心力衰竭大鼠心室肌中缝隙连接蛋白43表达的变化以及厄贝沙坦对心力衰竭时缝隙连接重构的干预作用。方法采用腹主动脉缩窄法建立大鼠心力衰竭模型,随机分为厄贝沙坦组和心力衰竭组,分别用厄贝沙坦(50 mg/kg)和安慰剂治疗,另设假手术对照组。术后16周用颈总动脉插管法测定心功能,用免疫印迹法检测心肌细胞缝隙连接蛋白43蛋白表达的变化,并以透射电镜观察缝隙连接空间重构的变化。结果心力衰竭大鼠心肌中缝隙连接蛋白43表达明显下调并出现空间重构,厄贝沙坦可明显上调缝隙连接蛋白43蛋白的表达并改善缝隙连接的空间重构。结论压力超负荷所致心力衰竭大鼠心室肌存在明显缝隙连接重构,这可能是心力衰竭时心肌电生理重构的机制之一,而此过程与血管紧张素Ⅱ的作用有关,血管紧张素受体拮抗剂厄贝沙坦可以明显改善心力衰竭时出现的缝隙连接重构。     

Connexin40 and connexin43 determine gating properties of atrial gap junction channels

While ventricular gap junctions contain only Cx43, atrial gap junctions contain both Cx40 and Cx43; yet the functional consequences of this co-expression remain poorly understood. We quantitated the expression of Cx40 and Cx43 and their contributions to atrial gap junctional conductance (g_j). Neonatal murine atrial myocytes showed similar abundances of Cx40 and Cx43 proteins, while ventricular myocytes contained at least 20 times more Cx43 than Cx40. Since Cx40 gap junction channels are blocked by 2 mM spermine while Cx43 channels are unaffected, we used spermine block as a functional dual whole cell patch clamp assay to determine Cx40 contributions to cardiac g_j. Slightly more than half of atrial g_j and ≤ 20% of ventricular g_j were inhibited. In myocytes from Cx40 null mice, the inhibition of ventricular g_j was completely abolished, and the block of atrial g_j was reduced to < 20%. Compared to ventricular gap junctions, the transjunctional voltage (V_j)-dependent inactivation of atrial g_j was reduced and kinetically slowed, while the V_j-dependence of fast and slow inactivation was unchanged. We conclude that Cx40 and Cx43 are equally abundant in atrium and make similar contributions to atrial g_j. Co-expression of Cx40 accounts for most, but not all, of the differences in the V_j-dependent gating properties between atrium and ventricle that may play a role in the genesis of slow myocardial conduction and arrhythmias.