腺苷对肺缺血再灌注大鼠NOS活性的影响

目的:观察腺苷(Adenosine)对大鼠肺缺血再灌注损伤(I/R)的保护作用及一氧化碳合酶(NOS)活性的影响。方法:将60只清洁级SD大鼠随机分为假手术组(Sham组,开胸但不阻断肺门)、I/R组(开胸后左肺门阻断60min+开放再灌注120min)、Adenosine预处理组,每组20只。A-denosine预处理组术前30min开始由股静脉持续静脉滴入Adenosine(0.1mg/kg/min)。实验结束前测定各组平均肺动脉压(mPAP)、动脉血氧分压(PaO2)、肺湿/干重比(W/D)及血清和肺组织匀浆中内皮型一氧化碳合酶(eNOS)、诱导型一氧化碳合酶(iNOS)活性。结果:I/R组mPAP、W/D均高于Sham组(P<0.01),PaO2低于Sham组(P<0.01)。与I/R组比较,Adenosine预处理组mPAP、W/D明显降低,PaO2显著增高。与Sham组比较,I/R组血清及肺组织匀浆中eNOS活性明显降低,iNOS活性显著增加(P<0.01);Adenosine预处理能明显升高I/R大鼠eNOS活性,降低iNOS活性(与I/R组比较,P<0.01)。结论:Adenosine可通过减轻再灌注后肺水肿、降低肺动脉压及改善肺组织氧合功能,从而有效实现对I/R肺组织的保护,其保护作用可能与NOS不同亚型的表达变化有关。     

参附注射液对肝脏缺血再灌注大鼠iNOS和NO水平的影响

目的探讨参附注射液(SF)对大鼠肝脏缺血再灌注损伤肝组织中诱导型一氧化氮合酶(iNOS)和血清一氧化氮(NO)的影响。方法32只Wistar大鼠随机分为参附实验组(SF组)和肝缺血再灌注组(IR组)。SF组腹腔注射参附注射液(10ml·kg-1),IR组大鼠给予相同剂量的生理盐水。两组均采用Pringle's法阻断肝门缺血15min再灌注1h、3h,测定血清谷丙转氨酶(ALT)、谷草转氨酶(AST)以及NO水平,并应用免疫组织化学法测定肝组织中iNOS表达。结果大鼠肝脏缺血15min再灌注1h和3h,SF组血清ALT、AST以及NO水平低于IR组(P<0.05),SF组肝组织iNOS阳性产物平均吸光度值、阳性面积百分率(%)明显低于IR组(P<0.05)。结论参附注射液抑制iNOS的表达,减少过量NO的生成,可能是其对肝脏缺血再灌注损伤的保护作用机制之一。     

脂联素心肌保护效应的研究进展

脂联素(Adiponectin,ADPN)是一种主要由脂肪组织分泌的脂肪因子,具有增加胰岛素敏感性、抗动脉粥样硬化、抗炎等效应。ADPN作为心脏疾病的重要保护因子,其在心肌肥厚、心肌缺血-再灌注损伤、糖尿病心肌病中的保护机制研究受到广泛关注,有望为相关疾病的诊断和防治提供新策略。     

庚醇预处理对兔缺血再灌注心肌线粒体结构、功能及线粒体Cx43的影响

目的:观察庚醇预处理对心肌缺血再灌注时线粒体的结构、功能和缝隙连接蛋白43(Cx43)影响,以探讨庚醇预处理心肌保护的可能机制。方法:兔64只,建心肌缺血再灌注模型,随机分4组(每组16只):假手术组(sham组)、缺血再灌注组(IR组)、缺血预处理组(IP组)、庚醇预处理(HT组)。测定心肌梗死面积,电镜观测线粒体超微结构改变,检测线粒体膜电位、Ca2+浓度、丙二醛(MDA)和超氧化物歧化酶(SOD)的改变。Westernblotting检测线粒体Cx43蛋白变化。结果:IP组和HT组心肌梗死面积分别为(18.97±2.80)%、(19.97±3.80)%,均明显低于IR组(35.67±5.80)%,P0.01。电镜检测发现,与sham组比较,其它组线粒体损伤明显(P0.01);与IR组比较,HT组和IP组线粒体损伤明显减轻(P0.05)、线粒体跨膜电位明显升高、线粒体Ca2+浓度明显下降(P0.01);与IR组比较,IP组SOD活性明显升高、MDA含量显著下降(P0.01)。与sham组比较,IR组线粒体Cx43蛋白显著下降(P0.05);与IR组比较,HT组和IP组心肌线粒体Cx43明显升高(P0.05)。结论:庚醇预处理可保护缺血再灌注心肌,其机制可能与提高线粒体跨膜电位、减轻线粒体钙超载和提高线粒体Cx43表达有关。     

防治心律失常的新靶点——缝隙连接蛋白43

缝隙连接是介导相邻细胞间离子和小分子信号物质直接交换的跨膜通道,连接蛋白43（connexin43,Cx43）是心肌细胞最主要的连接蛋白。研究表明,Cx43的表达和空间分布的变化与心律失常的发生密切相关,提示Cx43可能成为心律失常治疗的新靶点。本文综述Cx43与心律失常之间的关系及以Cx43为靶点的干预措施的研究进展。     

脂联素和脂联素受体1在不同病程糖尿病大鼠心肌缺血预适应中的变化

目的:探讨脂联素(APN)和脂联素受体1(Ad-R1)在不同病程糖尿病大鼠离体心肌缺血再灌注(IR)损伤和缺血预适应(IPC)保护作用中的变化。方法:分别用链脲佐菌素和高脂饮食+链脲佐菌素制备1型糖尿病(TIDM)和2型糖尿病(T2DM)大鼠模型,并分别分为4周和8周2个病程组,采用Langendorff法建立离体心脏灌流模型,每组进一步分为正常对照(Con)组、IR组和IPC组3个亚组,检测冠脉流出液中乳酸脱氢酶(LDH)及肌酸激酶(CK)活性,采用ELISA法检测血清和心肌组织APN水平,采用Western blot法检测心肌组织Ad-R1蛋白表达,测定心肌梗死面积,透射电镜观察心室乳头肌超微结构,并比较上述指标在各组中的差异。结果:与对应的IR组相比,4周时IPC组LDH和CK活性显著降低(P0.01),心肌梗死区域面积明显减少,而8周时DM组的各项检测指标无明显改变。血清APN含量在糖尿病大鼠减低,尤以T2DM降低为甚(P0.05),正常大鼠心肌组织APN含量和Ad-R1表达水平在Con、IR和IPC组之间无差异;T1DM模型大鼠心肌组织APN含量在各亚组间无变化,4周和8周心肌组织Ad-R1表达量IR组较Con组明显增加(P0.01),IPC组较对应IR组Ad-R1表达量降低(P0.01);T2DM模型大鼠心肌组织APN含量4周和8周的IR组较Con组明显减少(P0.05),IPC组较对应IR组,4周组的APN显著升高,8周组无显著差异,心肌Ad-R1表达量4周和8周的IR组较Con组明显增加(P0.05),4周的IPC组较对应IR组Ad-R1表达量降低(P0.05),8周的IPC组较对应IR组Ad-R1表达量无改变。结论:T1DM和T2DM模型大鼠4周组存在IPC保护作用,8周组IPC保护作用基本消失;心肌组织APN和Ad-R1可能参与了T2DM大鼠的IPC保护作用。     

脂联素通过减轻氧化应激治疗动脉粥样硬化的研究

观察不同滴度的脂联素腺病毒载体对高脂饮食载脂蛋白E基因敲除小鼠动脉粥样硬化模型的影响,以探讨脂联素抗动脉粥样硬化（AS）是否与减轻氧化应激有关。将48只健康成年12周龄载脂蛋白E基因敲除小鼠随机分为4组,对照组,模型组,低剂量脂联素组,高剂量脂联素组。对照组给予正常饮食8周;模型组给予高脂饮食8周;低剂量脂联素组每两周经尾静脉注射脂联素腺病毒载体1．0×108p．f．u,实验期间高脂饮食;高剂量脂联素组每两周经尾静脉注射脂联素腺病毒载体5．0×108p．f．u．实验期间高脂饮食。实验结束后,摘眼球采血取血清,测定总胆固醇（TC）,甘油三酯（TG）,低密度脂蛋白（LDL-C）,高密度脂蛋白（HDL-C）,以及血清脂联素（APN）含量,超氧化物歧化酶（SOD）活性,丙二醛（,MDA）含量,取小鼠主动脉血管。实时荧光定量扩增法（RT-PCR）测定小鼠主动脉血管APN,内皮型一氧化氮合酶（eNOS）的表达。与模型组比较,外源性的脂联素降低了血清TG,TC,LDL-C,MDA的含量,血清SOD活性增加,上调动脉血管中APN和eNOS的表达。AS损伤面积明显减少,分别降低了26％和31％（P＜0．05）,脂纹区的脂滴直径降低极显著（P＜0．01）,随着APN剂量的增加,AS损伤逐步减轻,但是低剂量脂联素组和高剂量脂联素组的差异没有统计学意义。减轻氧化应激是脂联素抑制动脉粥样硬化的一种保护机制和途径。     

大鼠肢体缺血-再灌注后肾脏iNOS表达的变化及意义

目的:探讨大鼠肢体缺血-再灌注(I-R)致肾脏损伤时肾内iNOS表达的变化及意义。方法:夹闭、再开放大鼠双侧股动脉,复制肢体I-R模型。RT-PCR检测肾组织iNOSmRNA表达的变化;免疫组化染色法观察iNOS蛋白及硝基酪氨酸(NT)在肾内的生成及分布;比色法测定肾组织MDA含量及SOD活性;应用氨基胍抑制大鼠体内iNOS活性后观察其肾组织的病理学变化。结果:肢体I-R后肾内iNOSmRNA表达显著高于对照组(P<0.01),肾小管上皮细胞内出现大量iNOS及NT阳性产物;肢体I-R后肾组织MDA含量显著升高,SOD活性显著降低(P<0.01);应用氨基胍后肾组织损伤减轻。结论:肢体I-R后肾内iNOS表达显著上调,由其诱生的高浓度NO可能参与介导了肾脏损伤。     

脂联素受体表达及影响因素研究进展

脂联素（adiponectin）作为近年来发现的一种由脂肪细胞分泌的特异性蛋白，可通过与靶细胞膜上的脂联素受体（adiponectin receptor，AdipoR）结合而激活腺苷酸活化激酶（AMPK）、过氧化物酶体增殖物活化受体α（PPAR-α），促进脂肪酸氧化和葡萄糖摄取，参与葡萄糖、脂肪代谢调节，从而发挥其抗炎，抗糖尿病，抗动脉粥样硬化和增敏胰岛素等作用。故脂联素受体在介导脂联素的作用中起着至关重要的作用，从现在研究我们可知不同组织脂联素受体的分布不同，脂联素受体的种类与脂联素的结合力和敏感性密切相关，现就脂联素受体在不同组织的表达及影响因素的有关研究进展作一综述。     

大鼠肢体缺血再灌注后肺组织一氧化氮合酶的变化

目的:研究正常大鼠肺组织内一氧化氮合酶(NOS)的分布及肢体缺血再灌注(LIR)后肺组织内NOS分布及活性的变化。方法:用止血带复制肢体缺血再灌注模型,利用β-NADPH-d组织化学方法、计算机图像分析系统及分光光度法,观察对照组大鼠肺内NOS的分布及LIR组肺内NOS分布及活性的变化。结果:组织学上显示,对照组大鼠呼吸道包括支气管、细支气管、终末细支气管、肺泡管的上皮细胞和血管内皮细胞NOS表达均阳性,肺泡上皮细胞NOS表达阴性;LIR组上述肺组织阳性部位NOS表达增强,且出现血管平滑肌细胞、肺泡上皮细胞NOS表达阳性;生化测定结果显示,LIR组与对照组比较,NOS活性增强,NO₂^-/NO₃^-水平增多。结论:一氧化氮不仅参与肺的生理过程,而且在LIR后急性肺损伤(ALI)病理生理过程中可能发挥重要作用。     

Altered expression of connexin43 and phosphorylation connexin43 in glioma tumors

In this study, we aim to evaluate the connexin (Cx43) and phosphorylation Cx43 (p-Cx43) expression of human glioma tumors and correlate their expression with degrees of malignancy and proliferation, apoptosis, and migration activity of tumors. Cx43 and p-Cx43 expression were examined by Western blot analysis and immunohistochemical staining. The U251 cell viability was measured by MTT analysis. The apoptosis and migration were also evaluated by flow cytometric analysis and fluoroblok transwell chambers, respectively. We found that the Cx43 expression were significantly downregulated in in malignant glioma (WHO grade III and IV), compared to the malignant glioma (WHO grade I and II) and the p-Cx43 expression levels of malignant glioma (WHO grade III and IV) were significantly increased (P<0.05), compared to the malignant glioma (WHO grade I and II) at immunohistochemical analysis. After treatment of cells with a specific inhibitor of PKC, MAPK, and PTK inhibitors, the cell viability and migration were significantly decreased, while the apoptosis was slightly induced. In conclusion, the Cx43 expression level is inversely correlated with the tumor grade and proliferation and migration activity of tumor. Higher p-Cx43 expression level in high tumor grade suggests that a complex mechanism is involved in the suppression of tumor growth by connexins.     

实验性病毒性心肌炎组织中连接蛋白43和结蛋白的表达

目的 了解病毒性心肌炎时心肌细胞连接蛋白４３和结蛋白表达情况,以探讨病毒性心肌炎时心律失常的机制。方法 应用免疫组织化学ＳＡＢＣ法,对实验性病毒性心肌炎小鼠心肌细胞连接蛋白４３和结蛋白的表达进行了观察。结果 连接蛋白４３和结蛋白在正常小鼠心肌闰盘中定位分布,均匀存在,后者还在肌节横纹中显示阳性;病毒性心肌炎时两者的表达明显减弱甚至阴性。结论 病毒性心肌炎时受累的心肌细胞连接蛋白４３和结蛋白的表达受     

miR-301a-3p对大鼠星形胶质细胞Cx43转录后的靶向调控作用

目的:研究大鼠星形胶质细胞中微小RNA-301a-3p(miR-301a-3p)对缝隙连接蛋白43(Cx43)表达的靶向调控作用及其作用位点。方法:合成miR-301a-3p agomir和miR-301a-3p antagomir,转染至星形胶质细胞,Western blot检测各组细胞中Cx43蛋白的表达情况;构建重组载体wt-pEZX-MT05-Cx43和mut-pEZX-MT05-Cx43,采用双萤光素酶报告基因实验验证miR-301a-3p的靶基因;构建表达载体pcDNA3.1-Cx43,通过回复实验分析miR-301a-3p对细胞凋亡的影响。结果:将miR-301a-3p agomir转染到星形胶质细胞后,Western blot检测显示,与对照组相比,Cx43蛋白表达显著降低(P<0.05)。双萤光素酶报告基因实验结果表明,miR-301a-3p能够与Cx43的3′-UTR结合,对其表达产生负调控;将不含Cx43 3′-UTR的重组载体pcDNA3.1-Cx43转染星形胶质细胞后,能够回复miR-301a-3p对Cx43蛋白表达的负调控作用,引起细胞凋亡。结论:Cx...     

连接蛋白43在培养心肌肥大时的表达及其意义

目的研究培养心肌连接蛋白４３（Ｃｘ４３）的表达及与心肌肥大的关系。方法分离培养大鼠心肌细胞,用去甲肾上腺素（ＮＥ）或苯肾上腺素（ＰＥ）诱导心肌肥大,用免疫组化方法观察心肌细胞Ｃｘ４３、增殖细胞核抗原（ＰＣＮＡ）及ｃｄｃ２表达,以图像分析系统检测Ｃｘ４３表达量的变化。结果在培养中加ＮＥ或ＰＥ的心肌细胞,Ｃｘ４３表达明显低于对照组,而其ＰＣＮＡ的阳性率明显高于对照组。ｃｄｃ２阳性率不变。结论在ＮＥ或ＰＥ作用下,培养大鼠心肌细胞中Ｃｘ４３表达下降,与细胞进入Ｓ期有关,这可能是两者促进心肌肥大的机制     

Ischaemia-induced temporal expression of connexin43 in rat heart

 To investigate the regulation of cell-to-cell coupling in myocardial ischaemia, the three-dimensional expression of connexin43 (Cx43) during experimental ischaemia was examined using a confocal laser scanning microscope. After induction of myocardial infarction in rats, serial optical sections were obtained from the left ventricular myocardium at various times (3 h to 60 days after ligation). The expression of Cx43 was detected immunohistochemically with FITC-labelled anti-rat Cx43 antibody. Fluorescent dots of Cx43 remained along the intercalated disc and decreased in number around the infarct up to 12 h after ligation. Cx43-expression disappeared completely within 48 h after ligation. After day 4, and especially on days 8 and 15 after ligation, the edges of the cardiomyocytes bordering the infarcted area manifested numerous sarcoplasmic tentacles that reacted positively to anti-desmin antibody. Distinct expression of Cx43 was observed extensively on the tentacles, although no cardiomyocytes remained viable around them. By day 60 after ligation, atypical expression of Cx43 had disappeared. These findings suggest that ischaemia induces temporally abnormal expression of Cx43, which might be responsible for abnormal conduction around the infarct. Received: 4 April 1997 / Accepted: 19 June 1997     

大鼠骨骼肌成肌细胞与乳鼠心肌细胞共培养对缝隙连接蛋白43表达的影响

目的 观察体外共培养的乳鼠心肌细胞与大鼠骨骼肌成肌细胞(简称L6细胞)以及乳鼠心肌细胞与转染外源性缝隙连接蛋白43(Cx43)基因的L6细胞(简称L6-Cx43细胞)间Cx43蛋白的表达情况,从而探讨共培养时心肌环境对成肌细胞的作用.方法 将乳鼠心肌细胞与4′,6-二脒基-2-苯基吲哚标记的大鼠骨骼肌成肌细胞进行共培养...     

Immunohistochemical localization of connexin 43 in the developing tooth germ of rat

Distribution of gap junction protein in maxillary tooth germs of 1-day-old rats was examined by immunohistochemistry, using an affinity-purified antibody specific to residues 360–376 of rat connexin (CX) 43. In 1-day-old rats, the maxillary second molar formed the shape of the cusp, but neither dentine nor enamel was formed between the cells of the dental papilla and the inner enamel epithelium. In the tooth germ, CX 43 was expressed in the cells of the stratum intermedium and the inner enamel epithelium. Labelling in the stratum inter-medium was extensive and showed an increasing gradient from peripheral to cuspal regions. CX 43 detected in the inner enamel epithelium was at cell surfaces facing the interface between the dental papilla and the inner enamel epithelium. The cells of the dental papilla and the inner enamel epithelium began differentiation as odontoblasts and secretory ameloblasts respectively, in the cusps of the first molars, where predentine and dentine were formed but enamel matrix was not secreted. CX 43 was present in the stratum intermedium, inner enamel epithelium, preodontoblasts, odontoblasts and subodontoblasts. The incisors showed the most advanced stage of development, where the enamel matrix and calcified dentine were formed in the labial part of the teeth. The CX 43 epitope was seen in the stratum intermedium, inner enamel epithelium, preameloblasts, preodontoblasts, odontoblasts, and subodontoblasts. Immunolabelling was more extensive in the stratum intermedium and subodontoblasts than in preameloblasts, preodontoblasts, and odontoblasts. The immunolabelling in preameloblasts and preodontoblasts was accumulated at cell surfaces facing the predentine. Further, the labelling in preameloblasts and preodontoblasts disappeared or was reduced at the initiation of enamel matrix secretion and calcification of dentine matrix.The present results suggest that gap junctional cell communication has important roles in tooth development. Further, the extensive CX 43 expression in the stratum intermedium and the subodontoblast layer suggests that gap junctions have an important role in amelogenesis and dentinogenesis.     

美托洛尔对心力衰竭大鼠心肌细胞磷酸化缝隙连接蛋白43表达及心肌细胞凋亡的影响

 目的：观察美托洛尔对心力衰竭（HF）大鼠在体心肌组织磷酸化缝隙连接蛋白43（p-Cx43）表达水平和心肌细胞凋亡的影响,并探讨其可能机制。方法：SD大鼠100只随机分为5组（n=20）：假手术（sham）组、HF组、小剂量（1.25 mg·kg-1·d-1）美托洛尔治疗（MetoA）组、中剂量（5 mg·kg-1·d-1）美托洛尔治疗（MetoB）组和大剂量（20 mg·kg-1·d-1）美托洛尔治疗（MetoC）组。缩窄腹主动脉建立HF动物模型,术后第4周开始给药至第8周。术后第4周和第8周超声心动图测定血流动力学指标;术后第8周取出心脏,HE和Masson染色观察心脏结构改变和胶原纤维增生情况,Western blotting检测p-Cx43表达水平,TUNEL法检测心肌细胞凋亡,p-Cx43表达水平与心肌细胞凋亡指数进行Pearson相关分析。结果：(1) 美托洛尔治疗改善HF大鼠血流动力学,在治疗剂量范围内美托洛尔剂量增加可有效逆转HF时的心肌重塑,呈剂量依赖效应。(2) HF组中p-Cx43表达量显著高于sham组（P<001）,而随美托洛尔治疗剂量的增加,p-Cx43表达量较HF组逐渐降低,各治疗组间两两比较亦有显著差异（P<001）。(3) HF组心肌细胞凋亡指数［(51.17±6.94)%］较sham组［(4.62±1.60)%］明显增加（P<001）;MetoA组凋亡指数为(40.60±4.15)%, MetoB组凋亡指数为(30.66±4.00)%,MetoC组凋亡指数为(22.24±5.69)%,均显著低于HF组（P<001）,各治疗组间两两比较亦有显著差异（P<001）。(4) 大鼠心肌组织p-Cx43表达水平与心肌细胞凋亡指数呈显著正相关（r=0.905, P<001）。结论: 美托洛尔对抗HF诱导的心肌细胞凋亡的机制可能与其抑制p-Cx43表达有关。     

Effects of cGMP-dependent phosphorylation on rat and human connexin43 gap junction channels

The effects of 8-bromoguanosine 3:5-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (g_j) in rat Cx43-transfected cells by 24.0±3.7% (mean±SEM, n=5), whereas g_j was not affected in human Cx43-transfected cells by the same treatment. The relaxation of g_j in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (_j) of about 20, 40–45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the _j distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [³²P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [³²P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased g_j, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.