新型钾通道开放剂对心血管ATP-敏感性钾通道基因表达的调节作用

目的：研究脂肪胺类的新型钾通道开放剂（KCO）埃他卡林（Ipt）和氰胍类的KCO吡那地尔（Pin）对大鼠心血管ATP-敏感性钾通道（KATP）的亚基SUR1、SUR2、Kir6．1和Kir6．2等在mRNA水平的调节作用。方法：SD大鼠给药1周后处死并取组织,提取总RNA,利用反转录-聚合酶链式反应（RT-PCR）研究以上基因在mRNA水平的改变。结果：与正常对照相比,心脏组织中,Ipt和Pin对KATP的4个亚基在mRNA水平均无显著影响;主动脉平滑肌上,Ipt对4个亚基的mRNA表达无显著影响,但Pin可显著上调SUR2的mRNA表达;尾动脉平滑肌上,Ipt对Kit6．1／Kit6．2、Pin对SUR2／Kir6．1均有显著下调的作用。结论：心肌、大动脉平滑肌和小动脉平滑肌KATP基因表达的调控不同,Ipt选择性调节小动脉平滑肌Kit6．1／Kit6．2;Ipt对心血管KATP基因表达的调节作用不同于Pin。     

KATP通道基因在大鼠组织中的表达

为检测大鼠肺动脉平滑肌及支气管平滑肌中KATP．通道亚基的表达情况,应用RT-PCR技术．从原代培养的Wistar大鼠肺动脉平滑肌及支气管平滑肌细胞第3—5代提取总RNA,逆转录．并进行PCR扩增鉴定．发现肺动脉平滑肌细胞有Kir6.1、SUR1和SUR2B的表达,其中SUR1表达量较弱,支气管平滑肌细胞有Kir6.1和SUR2B的表达。     

Syn-1A在抑制弱酸性pH诱导的KATP通道活化过程中的作用

目的：观察Syn-1A在抑制弱酸性pH诱导的KATP通道活化过程中的作用及机制。方法：用稳定表达Kir6.2/SUR2A KATP通道的HEK-293细胞构建细胞膜内面向外的记录方式,并给细胞膜片连续灌流含或不含Syn-1A的pH7.4,7.0,6.8,6.5和6.0的溶液,观察弱酸性pH对通道的活化作用及Syn-1A对上述作用的抑制,并采用体外结合实验分析不同pH对Syn-1A与SUR2A亚单位结合的影响。结果：Syn-1A可抑制pH 6.5,6.8和7.0时诱发的通道活化作用,Syn-1A与SUR2A的结合在pH7.4至6.0范围内,随pH下降逐渐增加。结论：Syn-1A对KATP通道的抑制作用可缓冲pH波动引起的KATP通道开放,从而抑制折返性心律失常的发生。     

低氧高二氧化碳对大鼠肺动脉平滑肌细胞K_(ATP)通道表达的影响及其与p38MAPK通路的关系

本文旨在探究ATP敏感性钾通道(KATP)在大鼠低氧高二氧化碳性肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)中的表达及其与p38 MAPK信号通路的关系。体外培养SD大鼠PASMCs,复制低氧高二氧化碳模型,采用RTPCR技术及免疫印迹法检测KATP亚基磺酰脲类受体2B(sulfonylurea receptor 2B,SUR2B)和内向整流钾通道6.1(Kir6.1)mRNA及蛋白的表达水平。结果显示:(1)与常氧组(N组)、低氧高二氧化碳组(H组)、低氧高二氧化碳+溶剂DMSO对照组(HD组)、低氧高二氧化碳+p38 MAPK通路抑制剂SB203580组(HS组)相比,低氧高二氧化碳+p38 MAPK通路激动剂茴香霉素(Anisomycin)组(HA组)Kir6.1 mRNA与蛋白表达均明显降低(P0.01),N组、H组、HD组、HS组间Kir6.1 mRNA与蛋白表达差异不显著(P0.05);(2)与N组相比,H组、HD组、HS组、HA组SUR2B mRNA与蛋白表达均明显上升(P0.05),H组、HD组、HS组、HA组间SUR2B mRNA与蛋白表达差异不显著(P0.05)。以上结果提示:(1)低氧高二氧化碳、SB203580均没有引起Kir6.1 mRNA与蛋白表达变化,而茴香霉素下调Kir6.1 mRNA与蛋白表达,Kir6.1可能受其它类型的MAPK通路的调节;(2)低氧高二氧化碳明显上调SUR2B mRNA与蛋白表达,而SB203580、茴香霉素不影响低氧高二氧化碳所引起的SUR2B表达上调,低氧高二氧化碳引起的SUR2B表达的升高可能是非p38 MAPK通路依赖性的。     

吡那地尔对缺血缺氧PC12细胞凋亡及Bcl-2蛋白表达的影响

目的探讨吡那地尔对缺血缺氧PC12细胞凋亡及对Bcl-2蛋白表达的影响。方法取传代后3d PC12细胞,分为A（对照组）,B（缺血缺氧组）,C（KATP通道开放剂）,D（KATP通道开放剂＋阻断剂组）。采用Annexin—v FITC／PI双染流式细胞分析仪检测凋亡率,应用免疫荧光染色和Western blot检测Bcl-2蛋白表达水平。结果缺血缺氧后B,C,D组细胞凋亡率随时间点增加而增加,24h达高峰。B,C,D组与A组比较均有显著性差异（P〈0．01）,C组和B,D组比较有统计学意义（P〈0．01）,B,C,D组细胞Bcl-2蛋白表达随时间点增加而增加,12h达高峰。B,C,D组均显著高于对照组（P〈0．01或P〈0．05）。C组表达显著高于B和D组（P〈0．01）。B与D组各时间点细胞凋亡及Bcl—2蛋白表达均无显著性差异（P〉0．05）。结论KATP通道开放剂能抑制缺血缺氧PC12细胞凋亡,这一作用机制可能与增加Bcl—2蛋白表达有关。     

钠氢交换体1在病理性心肌肥厚大鼠心肌组织及血浆中的表达

目的：研究异丙肾上腺素诱导的病理性心肌肥厚大鼠心肌组织及血浆中钠氢交换体1（sodium—hydrogen exchanger1,NHE—1）的表达,探讨NHE1在心肌肥厚发生和发展中的作用。方法：30只雄性SD大鼠随机并平均分为2组：病理性心肌肥厚组和对照组,每组15只,病理性心肌肥厚组（以下简称ISO组）予以ISO（异丙肾上腺素）连续每日以20、10和5mg／kg的剂量递减皮下注射,再以3mg／kg的剂量维持皮下注射7d,对照组予相同剂量生理盐水皮下注射。给药结束后进行心脏超声检测左室舒张末径（LVEDD）、左室收缩末径（LVESD）、室间隔厚度（IVST）、短轴缩短率（FS）、左室射血分数（LVEF）。分别测定各组大鼠体重（Bw）、心室重量（VW）、左心室重量（LVW）,计算心室重量指数VWI（VW／BW）、左心室重量指数LVWI（LVW／BW）。取血检测血浆中NHE．1的浓度,并取心肌组织观察病理形态学特征,用免疫组化法检测心肌组织中NHE—1的表达量。结果：与对照组相比,ISO组大鼠LVEF、IVST显著增加（P〈0．05）,LVESD明显降低（P〈0．05）,VWI、LVWI明显增加（P〈0．01）,血浆NHE—1浓度明显升高（P〈0．01）,心肌组织NHE-1表达增多（P〈0．01）。结论：NHE-1可能在病理性心肌肥厚的发生和发展过程中起着重要作用。     

ATP敏感钾通道参与大鼠前脂肪细胞增殖和分化

为了探讨ATP敏感钾通道在前脂肪细胞增殖分化中作用,本实验用逆转录实时定量PCR方法检测大鼠前脂肪细胞和诱导5d获得的脂肪细胞中该通道磺脲类受体2（sulphonylurea receptor2,SUR2）mRNA表达,探讨该通道阻滞剂格列本脲和激动剂二氮嗪对前脂肪细胞中SUR2mRNA表达的影响;MTT检测前脂肪细胞增殖;流式细胞仪检测细胞周期;油红O染色法检测细胞内脂质含量;Image-Pro Plus5．0软件测量细胞直径;逆转录PCR检测过氧化物酶体增殖物激活受体-γ（peroxisome proliferator-activatedreceptor-γ PPAR-γ）mRNA表达。结果显示：前脂肪细胞及诱导5d获得的脂肪细胞均有SUR2mRNA表达,且后者明显高于前者;格列本脲抑制前脂肪细胞SUR2mRNA表达,剂量依赖性地促进前脂肪细胞增殖,增加G2／M＋S期细胞百分比,增加细胞脂质含量,使脂肪细胞直径增大,增加PPAR-γ mRNA的表达;二氮嗪在这些方面的作用与格列本脲相反。以上结果提示,ATP敏感钾通道在前脂肪细胞增殖和分化中可能起调节作用,PPAR-γ可能参与这些作用。     

NaHS对ATP诱导大鼠小胶质细胞P2X受体表达的影响

目的：观察硫化氢（H₂S）供体硫氢化钠（NaHS）对ATP致伤的大鼠小胶质细胞细胞活力、细胞膜通透性及P2X7受体表达的影响。方法：实验取对数期形态结构及生长分化良好的大鼠小胶质细胞,随机分4组,每组设3个复孔。①正常对照组：常规培养,不进行ATP处理。②ATP组：接种细胞24 h后ATP处理。③NaHS+ATP组：NaHS预先孵育30 min后再用ATP处理,并且NaHS始终存在于反应体系中。④KN-62（P2X₇受体阻断剂）+ATP组：KN-62预先孵育30 min,其余同NaHS+ATP组。MTT检测各组细胞活力,荧光染料YO-PRO-1检测各组相对荧光单位（RFU）反映膜的通透性,Western blot检测各组P2X₇受体表达水平。结果：①与对照组相比,不同浓度的ATP （1、3、5、10 mmol/L）作用3 h均可明显降低大鼠小胶质细胞活力,NaHS （200 μmol/L）干预后大鼠小胶质细胞活力较ATP组明显增加（P<0.01）,但NaHS达400 μmol/L浓度时,其保护作用未进一步增加。②随着ATP浓度的增加,大鼠小胶质细胞内YO-PRO-1的荧光强度显著增加,NaHS预处理可明显减少细胞对YO-PRO-1的摄取（P<0.01）。③ATP （3 mmol/L）能上调P2X₇受体蛋白表达水平,而NaHS （200 μmol/L）预孵育则可明显抑制ATP引起的P2X₇受体蛋白表达的上调（P<0.01）。结论：NaHS可减少ATP致伤的大鼠小胶质细胞的P2X₇受体表达、降低通透性、增加细胞活力,提示调控P2X₇受体的表达和功能可能是H₂S神经保护作用的重要环节。     

MiRNA-125a-5p对于脊髓损伤后炎症反应、细胞凋亡及促进神经再生的作用机制研究

摘要 目的：探讨MiRNA-125a-5p对于脊髓损伤（SCI）后炎症反应、细胞凋亡及促进神经再生的作用机制。方法：建立6周龄大鼠SCI模型,将60只大鼠随机分为对照组（n=20）、SCI组（n=20）和MiRNA-125a-5p组（n=20）,对照组仅行椎板切除术,SCI组大鼠鞘内注射等量生理盐水,MiRNA-125a-5p组大鼠鞘内注射MiRNA-125a-5p agomir。采用定量实时聚合酶链反应（qRT-PCR）检测各组大鼠MiRNA-125a-5p和核因子kB（NF-kB）的相对RNA表达水平。酶联免疫吸附法（ELISA）检测炎症因子白细胞介素1（IL-1）、趋化因子受体-4（CXCR-4）和单核细胞趋化因子-1（MCP-1）水平的表达。采用蛋白免疫印迹试验检测细胞凋亡蛋白和神经调节因子蛋白的表达情况。采用Basso-Beattie-Bresnahan（BBB）运动能力评定量表评价大鼠神经运动功能。结果：与对照组比较,SCI组MiRNA-125a-5p表达均降低,NF-kB表达均升高,差异均有统计学意义（P＜0.05）;与SCI组比较,MiRNA-125a-5p组MiRNA-125a-5p表达均升高,NF-kB表达均降低,差异均有统计学意义（P＜0.05）。与对照组比较,SCI组IL-1、CXCR-4、MCP-1水平均升高,差异有统计学意义（P＜0.05）。与SCI组比较,MiRNA-125a-5p组IL-1、CXCR-4、MCP-1水平均降低,差异有统计学意义（P＜0.05）。与对照组比较,SCI组半胱氨酸天冬氨酸蛋白酶3（caspase-3）、聚腺苷二磷酸-核糖聚合酶（cleaved PARP）蛋白和原癌基因（bcl-2）蛋白表达均升高,神经细胞粘附分子1（NCAM1）、神经胶质蛋白1（NL1）和神经调节蛋白-1（NRG1）蛋白表达均降低,差异有统计学意义（P＜0.05）;与SCI组比较,MiRNA-125a-5p组caspase-3和cleaved PARP蛋白表达均降低,bcl-2、NCAM1、NL1和NRG1蛋白表达均升高,差异均有统计学意义（P＜0.05）。与对照组比较,SCI组大鼠神经运动功能评分在第7 d以后显著降低,差异有统计学意义（P＜0.05）;与SCI组比较,MiRNA-125a-5p组大鼠神经运动功能评分在第7 d以后均升高,差异有统计学意义（P＜0.05）。结论：MiRNA-125a-5p抑制NF-kB信号通路,降低炎症反应和细胞凋亡,促进神经再生,改善大鼠神经运动功能恢复。     

苏格木勒-3汤对异丙肾上腺素诱导的大鼠心肌肥厚后Caspase3/9表达的影响

目的：探讨苏格木勒-3汤（SD-3）对异丙肾上腺素（ISO）所致大鼠心肌肥厚的保护作用并探讨其机制。方法：32只Wistar大鼠随机分为空白对照组、模型组、SD-3治疗组和SD-3对照组（n=8）。模型组和SD-3治疗组皮下注射85 mg/（kg·d） ISO连续2 d致大鼠心肌肥厚模型;造模后,SD-3治疗组和SD-3对照组灌胃给予2 g/kg的SD-3,空白对照组、模型组灌以等体积的蒸馏水,干预14 d。于16 d末,观察各组大鼠心脏外观、心脏指数形态学变化;检测Caspase-3/9的活性和表达情况。结果：与正常对照组相比,模型组心脏变大,缺血,心脏指数升高,病理损伤严重,SD-3汤改善上述现象。进一步发现SD-3汤降低因ISO诱导的Caspase-3/9的活性并下调Caspase-3/9的表达。SD-3组大鼠心脏没有变化,与空白对照组无明显差异。结论：SD-3对ISO诱导的大鼠心肌肥厚具有保护作用,并通过下调Caspase-3/9表达缓解心肌肥厚转为心衰。     

建立小鼠心肌梗死模型的研究

目的建立心肌梗死小鼠模型。方法40只昆明小鼠,PTCA导丝逆行引导下气管插管,人工呼吸,采用开胸结扎左冠状动脉前降支方法造成心肌梗死。结果40只小鼠成功行左前降支结扎术;术后1天存活小鼠38只,术后2周存活32只。结论此方法可有效模拟心肌梗死的发生。     

Therapeutic silencing miR-146b-5p improves cardiac remodeling in a porcine model of myocardial infarction by modulating the wound reparative phenotype

Fibrotic remodeling is an adverse consequence of immune response-driven phenotypic modulation of cardiac cells following myocardial infarction(Ml).MicroRNA-146b(miR-146b)is an active regulator of immunomodulation,but its function in the cardiac inflammatory cascade and its clinical implication in fibrotic remodeling following Ml remain largely unknown.Herein,miR-146b-5p was found to be upregulated in the infarcted myocardium of mice and the serum of myocardial ischemia patients.Gain-and loss-of-function experiments demonstrated that miR-146b-5p was a hypoxia-induced regulator that governed the pro-fibrotic phenotype transition of cardiac cells.Overexpression of miR-146b-5p activated fibroblast proliferation,migration,and fibroblast-to-myofibroblast transition,impaired endothelial cell function and stress survival,and disturbed macrophage paracrine signaling.Interestingly,the opposite effects were observed when miR-146b-5p expression was inhibited.Luciferase assays and rescue studies demonstrated that the miR-146b-5p target genes mediating the above phenotypic modulations included interleukin 1 receptor associated kinase 1(IRAKI)and carcinoembryonic antigen related cell adhesion molecule 1(CEACAM1).Local delivery of a miR-146b-5p antagomir significantly reduced fibrosis and cell death,and upregulated capillary and reparative macrophages in the infarcted myocardium to restore cardiac remodeling and function in both mouse and porcine Ml models.Local inhibition of miR-146b-5p may represent a novel therapeutic approach to treat cardiac fibrotic remodeling and dysfunction following Ml.     

A computational method for developing hierarchical large deformation viscoelastic models of the contracting heart

In this study, a new computational method for modelling the contracting heart is described. Using this method, the cardiac wall is constructed from basic, repeating contractile units that represent individual myocardial units and collagen, each with its own set of parameters, including orientation, passive and active behaviour and stimulation propagation. The method allows individual control of each structural unit (e.g. at the level of a single myocardial unit). Feasibility of modelling dynamic heart contraction using this method is demonstrated using 2D cross-sections and simplified 3D geometries. Effects of non-contractile scar and myopathic tissue were also tested in these geometrical configurations. Results from the 2D and 3D simulations were, overall, in agreement with well-established physiological data. The present method holds promise for modelling complex heart pathologies, abnormal mechanical properties (e.g. myocardial infarcts) and electrical conduction properties (branch blocks), and their spatial distributions across the myocardial tissues.     

The effect of hydrogel injection on cardiac function and myocardial mechanics in a computational post-infarction model

An emerging therapy to limit adverse heart remodelling following myocardial infarction (MI) is the injection of polymers into the infarcted left ventricle (LV). In the few numerical studies carried out in this field, the definition and distribution of the hydrogel in the infarcted myocardium were simplified. In this computational study, a more realistic biomaterial distribution was simulated after which the effect on cardiac function and mechanics was studied. A validated finite element heart model was used in which an antero-apical infarct was defined. Four infarct models were created representing different temporal phases in the progression of a MI. Hydrogel layers were simulated in the infarcted myocardium in each model. Biomechanical and functional improvement of the LV was found after hydrogel inclusion in the ischaemic models representing the early phases of MI. In contrast, only functional but no mechanical restitution was shown in the scar model due to hydrogel presence.     

心脏的再生性研究进展

心力衰竭(心衰)是由多种心血管疾病引起的心功能不全,最终出现心肌局部坏死和不可逆转的纤维化。传统的药物治疗、介入和外科治疗方案都存在着一定的局限性。随着转化医学的发展,心脏再生和修复无疑是解决以上临床问题的最佳方案,基础研究的成果正在为临床实践提供理论基础。文章从心脏再生性修复策略、心肌细胞来源、重编程和模式动物等几个方面的进展概括了整个心脏再生领域的发展。     

An approach to determine myocardial ischemia by hidden Markov models

A hidden Markov model (HMM) of electrocardiogram (ECG) signal is presented for detection of myocardial ischemia. The time domain signals that are recorded by the ECG before and during the episode of local ischemia were pre-processed to produce input sequences, which is needed for the model training. The model is also verified by test data, and the results show that the models have certain function for the detection of myocardial ischemia. The algorithm based on HMM provides a possible approach for the timely, rapid and automatic diagnosis of myocardial ischemia, and also can be used in portable medical diagnostic equipment in the future.     

Virtual reality of stem cell transplantation to repair injured myocardium

The search for the fountain of youth continues into the 21st century with hopes that embryonic or hematopoietic stem cells (SC) will repair injured tissues in the heart, lungs, pancreas, muscles, nerves, liver, or skin. This commentary focuses on the potential of SC for inducing cardiac regeneration after myocardial injury, the barriers to SC treatment that need to be overcome for ensuring successful cardiac repair, and the experimental approaches that can be applied to the problem.     

大鼠自主呼吸下建立急性心肌梗死模型

目的大鼠自主呼吸情况下,快捷、简便地建立大鼠急性心肌梗死模型。方法 180～220gSD大鼠60只,于胸骨左缘第4-5肋间隙切开皮层作荷包缝合,逐层钝性分离肌肉,挤出心脏,快速结扎左冠状动脉前降支(LAD)后,送回心脏同时挤压胸廓,拉紧荷包以建立心肌梗死模型。记录结扎前、结扎后3h心电图;结扎3h后取出心脏,冰冻切片TTC染色。结果 50只大鼠成功建立心肌梗死模型,模型成功率为83.33%。心电图显示结扎冠脉后出现R波峰降低,ST段拱背抬高及ST-T融合,TTC染色后左心室出现明显灰白色梗死区。结论本方法可在大鼠自主呼吸情况下,较短的时间内以简便的手术、较小的创伤建立大鼠急性心肌梗死模型。     

Adenosine and cardioprotection during reperfusion – an overview

Ischemic heart disease includes a number of entities that have been grouped in accordance with physiopathology and evolutive criteria. In recent years new ischemic syndromes have been described. Within the new ischemic syndromes, ventricular post-ischemic dysfunction – also known as stunned myocardium – is worth mentioning. In this route, several studies have suggested that reperfusion per se could cause cellular injury (reperfusion injury). In previous years, a protective effect on the injury caused by ischemia and reperfusion in the heart has been attributed to adenosine. These effects have been documented in different experimental in vivo and in vitro models. Thus, the administration of exogenous adenosine, or agonists of adenosine receptors prior to ischemia reduces the size of the infarction, improves the recovery of the ventricular function during reperfusion (attenuating stunning) and prolongs the time period to the ischemic contracture. However, focusing on a potential therapeutic application, it is of the utmost importance to find this protection and learn the mechanisms involved when procedures are applied during early reperfusion.We showed that adenosine, administered from the beginning of reperfusion, attenuated systolic and diastolic (myocardial stiffness) alterations of the stunned myocardium. This protective effect was mediated by the activation of A₁ adenosine receptors, and without modification on infarct size. According to some authors, adenosine can decrease the release of endothelin, during early reperfusion, and reduce an overload of Ca²⁺ that could cause a cellular lesion. Finally, ischemic preconditioning involves a series of intracellular events that are initiated with the activation of the A1 receptor, and end at the sensitive K⁺ ATP channels of the mitochondria. The phosphorylation and opening of these channels would cause the protective effect. Activation of this specific mechanism during reperfusion has not been studied extensively.     

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.