小鼠心肌Hsp25基因缺失致心肌梗死后心脏破裂的机制研究

发布时间：2018-06-26 03:26

本文选题：热休克蛋白25 + 心肌梗死　；参考：《南京师范大学》2014年硕士论文

【摘要】：背景和目的小分子热休克蛋白作为一类在进化上高度保守的蛋白,其在哺乳动物中生物学作用的重要性早已引起广泛关注。该家族中的Hsp25(鼠源)分子量为25kDa；而其人源的同源蛋白为Hsp27,分子量为27kDa。研究表明,Hsp25/Hsp27与心血管疾病发生发展之间存在重要关系。本课题组以往研究表明,过表达Hsp27能减轻急性心肌缺血/再灌注损伤,缩小心肌梗死面积,提示Hsp25/Hsp27具有潜在的心脏保护功能。但是Hsp25/27对心肌梗塞后的心肌重塑、中长期存活率的影响,都不清楚。因此,本课题组前期在构建Hsp25心肌特异性敲除小鼠基础上,发现Hsp25基因缺失显著增加心肌梗塞小鼠的死亡率,其死亡原因主要是心脏破裂。本实验将从细胞外基质重塑的角度,探讨Hsp25基因对心梗后心肌间质重塑的影响,以期为揭示Hsp25对心梗后疤痕修复的调节作用提供理论依据。实验方法 1动物模型：本实验室通过与南京大学模式动物研究所合作构建了Hsp25基因心肌特异性敲除小鼠(Hsp25knockout mice,Hsp25KO),饲养在南京大学模式所的无特定病原体(Specific Pathogen Free,SPF)动物房中。对照组为具有同样背景的野生型小鼠(Wild Type,WT) 2小鼠基因型的鉴定： 1)用聚合酶链式反应(Polymerase Chain Reaction, PCR)确定小鼠的基因型； 2)用蛋白质免疫印迹法(Western Blot)检测小鼠心肌中Hsp25的蛋白表达水平。 3小鼠心肌梗死(Myocardial Infarction,MI)模型的构建：戊巴比妥纳麻醉小鼠后,取仰卧位,呼吸机机械通气。备皮、开胸、暴露出心脏,撕开心包膜,在冠状动脉左前降支三分之一处进行永久性结扎,关胸,缝皮,30min之后撤下呼吸机,让小鼠自然苏醒。 4心功能评价：分别对假手术(sham)、MI后1天、3天的KO小鼠和WT小鼠进行异氟烷气体麻醉,取仰卧位,使用超声心动图测定心脏多项心功能指标,包括左心室射血分数(EF%)、心室短轴缩短率(FS%)、舒张期左室内径(LVIDd)、收缩期左室内径(LVIDs)等。 5小鼠心脏组织分区分离：断颈处死小鼠,迅速开胸,将心脏取出置于4℃的PBS中,首先截取结扎线以上的组织为梗死远端区(Infarct remote left ventricle;Infarct remote LV)；剩下的组织按照梗死界限进行分离,梗死的区域称为(Infar leftventricle;Infarct LV);另一部分组织称为梗死周围区(Infarct border left ventricle; Infarct border LV)。 6细胞外基质的胶原检测：将梗死区的心肌置于戊二醛固定,送至南京医科大学电镜室进行制片,透射电子显微镜(Transmission electron microscope,TEM)观察心脏组织超微结构变化。7统计学方法：使用SPSS17.0进行数据处理。数据以均数士标准差(x士s)表示,两组数据之间的比较用两样本均数检验(t检验),多组数据之间的比较用单因素方差分析(ANOVA),P0.05表示有显著性差异,P0.01表示有极显著性差异。实验结果 1WT鼠心肌梗死后,其心肌中的Hsp25蛋白表达量会随着时间的延长而逐渐上调。 2构建Hsp25心肌特异敲除的小鼠模型,其野生型(WT)小鼠的心肌中的Hsp25表达水平正常,而基因敲除型(KO)小鼠心肌中的Hsp25表达水平与之相比明显下降。 3Hsp25基因敲除后,不影响小鼠的正常发育以及生殖功能。 4小鼠MI后,在统计的两周时间内WT小鼠的存活率在75%,而KO小鼠与之相比,其存活率明显下降,并且在造模后的五天时间内全部死亡,尸体解剖发现90%的小鼠是因为心脏破裂而死。 5MI后1天和3天的心功能追踪显示,KO小鼠心功能较WT小鼠显著降低。 6通过对MI后1天后3天的心脏梗死区的的细胞外基质研究发现,KO小鼠的Ⅰ型和Ⅲ型胶原较WT小鼠在蛋白水平有明显下降。 7通过对MI后1天后的心脏梗死区的的细胞外基质研究发现,KO小鼠的基质金属酶(matrix metalloproteinases,MMP)2和9较WT小鼠在蛋白水平有明显升高。结论 Hsp25基因对于心肌梗死的创伤修复存在保护作用,该作用与调节细胞外机制重塑有关。
[Abstract]:Background and purpose
As a class of highly conserved proteins, small molecular heat shock proteins have been widely concerned in the importance of biological action in mammals. The molecular weight of Hsp25 (Shu Yuan) in this family is 25kDa, and the homologous protein of the human source is Hsp27, and the molecular weight of 27kDa. shows that Hsp25/Hsp27 and cardiovascular disease occur and develop. Previous studies have shown that overexpression of Hsp27 can reduce acute myocardial ischemia / reperfusion injury and narrow the area of myocardial infarction, suggesting that Hsp25/Hsp27 has potential cardiac protective function. However, the effect of Hsp25/27 on myocardial remodeling after myocardial infarction and the effect of medium and long term survival is not clear. Therefore, this research group On the basis of the early construction of Hsp25 specific knockout mice, we found that Hsp25 gene deletion significantly increased the mortality of myocardial infarction mice. The main cause of death was cardiac rupture. The effect of Hsp25 gene on myocardial interstitial remodeling after myocardial infarction was discussed from the angle of extracellular matrix remodeling, in order to reveal the scar of Hsp25 after myocardial infarction. The regulation of scar repair provides a theoretical basis.
Experimental method
1 animal model: in this laboratory, the Hsp25 gene Hsp25knockout mice (Hsp25KO) was constructed in collaboration with the Nanjing University model animal research institute, which was raised in the Specific Pathogen Free, SPF animal room of the Nanjing University model. The control group was a wild type mouse with the same background (Wild T). Ype, WT)
2 Identification of the genotypes of mice:
1) polymerase chain reaction (Polymerase Chain Reaction) (PCR) was used to identify the genotype of mice.
2) protein expression level of Hsp25 in murine myocardium was detected by Western Blot.
The construction of 3 Myocardial Infarction (MI) model in mice: after pentobarbital was anesthetized in mice, the supine position, ventilator mechanical ventilation, skin preparation, open chest, exposure of heart, tear film, 1/3 permanent ligation of the left anterior descending coronary artery, closed chest, suture, and 30min after 30min were removed to make the mice natural Suzhou. Wake up.
4 cardiac function evaluation: Isoflurane gas anesthesia was performed on false operation (sham), 1 days after MI, 3 days of KO mice and WT mice. Cardiac multinomial cardiac function indexes were measured by echocardiography, including left ventricular ejection fraction (EF%), short axis shortening rate (FS%), diastolic left ventricular diameter (LVIDd), systolic left ventricular diameter (LVIDs), etc.
5 division of the mouse heart tissue Division: cut the neck to death in mice, quickly open the chest, put the heart out of the PBS at 4 degrees C, first intercepted the tissue above the ligation line for the distal infarct region (Infarct remote left ventricle; Infarct remote LV); the remaining tissues were separated according to the boundary of the infarct, the infarct area called (Infar leftventricle; Infa) RCT LV); another part is called Infarct border left ventricle (Infarct border LV).
6 collagen detection of extracellular matrix: the myocardium in the infarct area was fixed by glutaraldehyde and sent to the electron microscope room of Nanjing Medical University. Transmission electron microscope (TEM) was used to observe the ultrastructural changes of the heart tissue,.7 statistical method: data processing with SPSS17.0. X s) said that the comparison between the two groups of data was tested by two samples (t test), and the comparison between the multiple groups of data was made by the single factor variance analysis (ANOVA), and the P0.05 showed significant differences, and the P0.01 indicated a very significant difference.
experimental result
After 1WT myocardial infarction, the expression of Hsp25 protein in myocardium gradually increases with time.
2 a mouse model of Hsp25 specific knockout was constructed, and the expression level of Hsp25 in the myocardium of its wild type (WT) mice was normal, while the Hsp25 expression level in the myocardium of the gene knockout (KO) mice was significantly lower than that in the myocardium.
Knockout of 3Hsp25 gene did not affect the normal development and reproductive function of mice.
After 4 mice MI, the survival rate of WT mice was 75% during the two week period of statistics, and the survival rate of KO mice decreased significantly compared with that of the mice, and all died within five days after the model. The autopsy found that 90% of the mice died of the heart rupture.
Cardiac function tracing on the 1 and 3 days after 5MI showed that the heart function of KO mice was significantly lower than that of WT mice.
6 it was found that type I and type III collagen in KO mice were significantly lower than that of WT mice in the WT mice through the study of the extracellular matrix in the 3 days after 1 days of the heart infarction.
7 it was found that the matrix metalloproteinases (matrix metalloproteinases, MMP) 2 and 9 in KO mice were significantly higher in the protein level than in the WT mice through the study of the extracellular matrix of the heart infarction area after MI 1 days later.
conclusion
Hsp25 gene plays a protective role in wound healing of myocardial infarction, which is related to remodeling of extracellular mechanism.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R542.22

【参考文献】