小鼠心肌肥厚或心衰时心肌横管结构及其相关蛋白JP-2和Cav-3表达水平的变化

发布时间：2018-04-15 02:18

本文选题：胸主动脉缩窄 + Junctophilin-2　；参考：《安徽医科大学》2011年硕士论文

【摘要】：目的应用胸主动脉缩窄(TAB)手术建立小鼠心肌肥厚或心肌衰竭(简称:心衰)模型,以此模型为基础,我们致力于研究心肌疾病中横管的结构是否改变,以及探索横管结构相关蛋白junctophilin-2(JP-2)和caveolin-3(Cav-3)水平的变化。方法 1小鼠分组及模型建立雄性约8周的C57BL/6小鼠,体重20-25 g,清洁级。手术前随机将动物分假手术对照组(n = 6)和手术组(n = 12),将做手术的小鼠氯胺酮/甲苯噻嗪(剂量分别是40 mg/kg和5 mg/kg)腹腔注射麻醉,用镊子将一段3/0的手术线放置于主动脉下。在头臂动脉和左颈动脉分支之间的主动脉上放入27号针头,丝线绕过主动脉和针头打结,然后移走针头,这样就把主动脉内径固定为针头的直径大小。假手术对照组仅观察主动脉,但不结扎。 2心脏超声手术后5周利用Sonos 5500给小鼠做心脏超声,氯胺酮盐酸(25 mg/kg)皮下注射使所有存活小鼠处于镇静状态,通过8 MHz的扇形探头在左心室的长轴和短轴得到高分辨率的二维图像,每秒可产生1000张图。根据心脏超声资料将手术组分为心肌肥厚组和心衰组。 3体外整体心脏外膜心肌细胞横管的共聚焦成像完整的小鼠心脏室温下langendorf灌流含有2.5μM的脂溶性的膜结构荧光指示剂FM4-64的无钙台式液20-30分钟,之后将心脏置于激光共聚焦显微镜的灌流腔内,同时灌流无FM4-64的无钙台式液。分别在左右心室的自由壁的不同部位采集10-15张图片,图片的平均幂频谱代表每个心室的横管结构。 4 Western blot冰上收集小鼠左心室,?80°C保存。利用心脏裂解液提取左心室全蛋白,利用BCA法测蛋白浓度。取样本于10%十二烷基磺酸钠-聚丙烯酰胺凝胶电泳后,蛋白恒流湿转入PVDF膜上,5%新鲜脱脂牛奶室温封闭1 h,孵一抗4°C过夜,0.1% PBS-T洗膜3×15 min,室温共同孵育二抗和HRP-GAPDH抗体1.5 h,0.1% PBST洗膜3×15 min,暗室中ECL显色后曝光。利用image J软件分析蛋白条带。 5统计学分析采用实验结果以(X|-)±s表示,两组均数之间比较用非配对t检验,三组均数的比较采用Origin软件做方差分析。P 0.05代表有统计学显著性差异。结果 1心脏超声结果小鼠大约在TAB手术后5周形成心肌肥厚或心衰,心肌肥厚的小鼠与假手术对照组相比在心脏重量、心重与体重的比值和左室重量均有显著性的增加,但是肺重,舒张末期容积和射血分数没有明显的改变。心衰组的小鼠不仅心脏重量、心重与体重的比值和左室重量变化更显著,而且肺的重量和舒张末期容积明显增加,射血分数显著性的下降。 2整体心脏的离体横管成像在左心室,横管结构的紊乱开始于心肌肥厚阶段,心衰小鼠的横管遭到更严重的破坏。同样横管的幂频谱的左移趋势也起于心肌肥厚阶段。然而,右心室和左心室不同,只是在心衰的小鼠发现横管结构的改变和横管的幂频谱左移。 3 Western blot与假手术对照组比较,心肌肥厚组、心衰组的JP-2和Cav-3表达呈现逐渐下降的趋势,心衰时JP-2和Cav-3表达的下降有统计学意义。结论 1心脏形态和功能的变化和心肌横管的改变有着密切的相关性。 2心肌横管的重塑可能与兴奋-收缩耦联相关蛋白JP-2和Cav-3表达下降相关。
[Abstract]:Purpose

To establish a model of myocardial hypertrophy or myocardial failure ( heart failure ) in mice by thoracic aortic constriction ( TAB ) operation , we are focused on the study of whether the structure of transverse tube in myocardial diseases has changed , and to explore the change of the level of related protein junctionilin - 2 ( JP - 2 ) and Cav - 3 ( Cav - 3 ) .

method

The rats were randomly divided into two groups : control group ( n = 6 ) and operation group ( n = 12 ) . The rats were randomly divided into sham operation control group ( n = 6 ) and operation group ( n = 12 ) .

Two - dimensional images with high resolution were obtained by subcutaneous injection of ketamine hydrochloride ( 25 mg / kg ) at 5 weeks after 2 - cardiac ultrasound , and a high - resolution two - dimensional image was obtained in the long axis and the short axis of the left ventricle through a 8 MHz fan - shaped probe .

3 . A total of 2 . 5 . m u.M fat - soluble film - structured fluorescent indicator FM4 - 64 was injected into the perfusion cavity of a laser confocal microscope for 20 - 30 minutes , and then the heart was placed in the perfusion cavity of the laser confocal microscope , and the free calcium - free desktop solution without FM4 - 64 was injected .

The left ventricle of mice was collected by Western blot . The left ventricle was collected by Western blot . Left ventricular whole protein was extracted with cardiac lysates . The protein concentration was measured by BCA method . After the sampling was performed on 10 % sodium dodecyl sulfate - polyacrylamide gel electrophoresis , the protein constant flow was transferred to PVDF membrane . After incubation for 1 h at room temperature , incubated for 1 h at room temperature , incubated for 1 h at room temperature , incubated at room temperature for 3 脳 15 min , and exposed in dark room ECL color . The protein bands were analyzed by image J software .

The results of statistical analysis were as follows : ( X 卤 - ) 卤 s , the comparison between the two groups was performed by non - paired t - test , and the comparison of the mean numbers of the three groups was performed by Origin software . P 0.05 represents a statistically significant difference .

Results

Heart weight , heart weight , weight ratio and left ventricular weight were significantly increased in mice with heart failure group , but the weight of heart weight , heart weight , weight and left ventricular weight were significantly increased , and the weight and end diastolic volume of lung increased significantly , and the ejection fraction decreased significantly .

However , the right ventricle and the left ventricle are different , but only in the heart failure mice find the change of the transverse tube structure and the left shift of the power spectrum of the horizontal tube .

The expressions of JP - 2 and Cav - 3 in the myocardial hypertrophy group and heart failure group were gradually decreased compared with the sham - operated control group , and the decrease of JP - 2 and Cav - 3 expression in heart failure group was statistically significant .

Conclusion

Changes in morphology and function of heart and changes of myocardial transverse tube were closely related .

remodeling of the myocardium transverse tube may be associated with a decrease in the expression of the excitation - shrink - coupled - associated protein jp - 2 and cav - 3 .

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R542.2;R-332

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