心肌缝隙连接蛋白43在拟交感心房颤动模型中表达水平及分布变化的研究

发布时间：2018-06-15 09:06

本文选题：交感神经 + 心房颤动　；参考：《广西医科大学》2017年硕士论文

【摘要】：目的本课题旨在通过建立拟交感心房颤动模型,研究心房缝隙连接蛋白43(connexin 43,Cx43)表达水平及分布的变化,从而探讨交感性房颤发生与维持的可能的内在机制。方法建立交感性心房颤动模型,健康幼犬15只,雌雄不分,将其随机分成3组,每组5只,分别为:1.对照组(Control组),将实验犬心脏取出后,快速连接至langendorff心脏离体灌流仪,予台氏液灌流,持续约1h。快速心房起搏组(rapid atrial pacing,RAP组),灌流方法同Control组,仅在灌流时用电生理刺激仪以800次/min频率起搏30s,共30次。异丙肾上腺素灌流组+快速心房起搏组(isoprenaline+rapid atrial pacing,ISO+RAP组),以含0.1umol/L异丙肾上腺素(isoprenaline,ISO)的改良台氏液灌流心脏,余同RAP组。(1)在离体灌流状态下,通过快速心房起搏构建拟交感性房颤模型。各组分别测量心房有效不应期(atrial effective refractory period,AERP)和记录各组的房颤诱发率的情况。实验结束后,取左心房组织固定,通过免疫组化检测心房神经生长因子(NGF)和酪氨酸羟化酶(TH)的表达和分布变化。观察拟交感效应对快速起搏离体心脏的影响。(2)取部分左心房组织,采用western blotting方法检测各组心房肌中总Cx43的含量及磷酸化Cx43的含量,Q-PCR检测Cx43mRNA表达量,免疫荧光(immunofluorescence,IF)检测心房肌组织Cx43的分布变化,凋亡试剂盒检测心房肌组织的细胞凋亡率,透射电镜检测心房肌线粒体形态的变化,荧光比色法检测心房肌线粒体ROS的生成量。结果(1)各组所测得的AERP中,Control组和RAP组无显著差别(P0.05),ISO+RAP组AERP则显著缩短(P0.05);ISO+RAP组可成功诱发房颤,而Control组及RAP组均无法诱发房颤;与Control组比较,RAP组及ISO+RAP组NGF和TH的含量及分布均增多,ISO+RAP组高于RAP组。(2)与Control组比较,RAP组及ISO+RAP组Cx43及磷酸化Cx43含量呈逐渐降低趋势(P0.05);与Control组比较,RAP组Cx43分布呈明显侧向化,线粒体轻度肿胀,基质完整,ISO+RAP组Cx43呈点状散在分布,线粒体明显肿胀,部分基质透明;ISO+RAP组细胞凋亡指数(apoptosis index,AI)及线粒体ROS生成量均显著高于RAP组及Control组(P0.05),而RAP组则高于Control组(P0.05)。结论1.通过异丙肾上腺素灌流模拟交感神经兴奋效应,可以显著缩短心房肌的动作电位时程,表现为AERP时程缩短。2.单纯的高频心房起搏在本实验持续的时间内无法诱发房颤,而在拟交感效应下则可以成功诱发,成功构建拟交感性房颤模型。3.在交感性心房颤动中,交感神经可能通过氧化应激效应,引起Cx43的重构与下调,从而介导心房颤动的发生。
[Abstract]:Objective to study the changes of the expression and distribution of atrial gap junction protein (43(connexin 43) by establishing a sympathetic atrial fibrillation model, and to explore the possible mechanism of occurrence and maintenance of sympathetic atrial fibrillation. Methods A model of sympathetic atrial fibrillation was established. Fifteen healthy puppies were randomly divided into 3 groups, 5 in each group: 1: 1. After the heart was removed from the experimental dog, the control group was quickly connected to the langendorff heart perfusion apparatus in vitro and perfused with Tyrtle fluid for about 1 hour. In the rapid atrial pacing group, the perfusion method was the same as that in the control group. Isoprenaline rapid atrial pacing group (IPA) was perfused with modified Tyrna solution containing 0.1 umol / L isoproterenol (isoproterenol) and isoprenaline isoprenalineiso (ISO), and the rest was the same as that in rap group under isolated perfusion. A sympathetic atrial fibrillation model was constructed by rapid atrial pacing. Atrial effective refractory AERP (AERP) and atrial fibrillation evoked rate were measured in each group. The expression and distribution of nerve growth factor NGF and tyrosine hydroxylase were detected by immunohistochemistry. To observe the effect of pseudo sympathetic effect on the isolated heart of rapid pacing, some left atrial tissues were taken. The contents of total Cx43 and phosphorylated Cx43 were detected by western blotting method and the expression of Cx43 mRNA was detected by Q-PCR. The distribution of Cx43 in atrial myocytes was detected by immunofluorescence fluorescence assay, apoptosis rate in atrial muscle tissue was detected by apoptosis kit, mitochondrial morphology of atrial muscle was detected by transmission electron microscope, Ros production in mitochondria of atrial muscle was detected by fluorescence colorimetry. Results 1) there was no significant difference in AERP between control group and rap group. AERP in ISO rap group was significantly shorter than that in ISO rap group, but atrial fibrillation could not be induced in control group and rap group. The contents and distribution of NGF and th in rap group and ISO rap group were higher than those in rap group.) compared with control group, the contents of Cx43 and phosphorylated Cx43 in rap group and ISO rap group decreased gradually (P 0.05), and the distribution of Cx43 in rap group was more lateral than that in control group. The mitochondria were slightly swollen, the distribution of Cx43 in ISO rap group was dotted, the mitochondria was obviously swollen, and the apoptosis index (AI) and Ros production of mitochondria in ISO rap group were significantly higher than those in rap group and Control group, while that in rap group was higher than that in Control group. Conclusion 1. By using isoproterenol perfusion to simulate sympathetic excitatory effect, the action potential duration of atrial muscle can be significantly shortened, as shown by the shortening of AERP duration by .2. High frequency atrial pacing alone could not induce atrial fibrillation during the duration of the experiment, but could be induced successfully under the pseudo sympathetic effect, and successfully constructed the pseudo sympathetic atrial fibrillation model. 3. In the process of sympathetic atrial fibrillation, sympathetic nerve may induce the remodeling and down-regulation of Cx43 through oxidative stress, thus mediating the occurrence of atrial fibrillation.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.75;R-332

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