[0066] Example 2 Preparation and identification of mouse myocardial infarction model
[0067] In this example, a mouse myocardial infarction model was prepared and identified.
[0068] 1) The left coronary artery occlusion method was used to induce myocardial infarction in mice, thereby establishing a mouse myocardial infarction model.
[0069] After anesthesia, the mice were fixed on the experimental wooden board. ECG monitoring. Cut the trachea in the middle of the neck and intubate, connect the animal artificial ventilator, open the chest in the fourth intercostal space, carefully lift the pericardium and cut open, fully expose the heart and blood vessels. Between the pulmonary artery cone and the left atrial appendage, using the trunk of the left coronary vein as a mark, insert the needle below the root of the left atrial appendage with a depth of 0.5mm; pass the 6/0 suture through the surface of the myocardium and exit next to the pulmonary artery Needle; After the ECG is stable, the left anterior descending coronary artery (LAD) is ligated. The ST segment arch dorsal elevation in leads I and aVL is greater than 0.1mV and lasts for more than 0.5h as a sign of successful ligation. Clear the blood in the chest cavity and close the chest cavity with aspirating gas with a needle-removing syringe.
[0070] After a certain period of time (in this experiment, postoperative ischemia 45min reperfusion was used) reperfusion was used to prepare a mouse myocardial infarction model (mouse ischemia-reperfusion model referred to as I/R model).
[0071] 2) Methods to identify and detect cardiac function in mouse models of myocardial infarction:
[0072] (1) Determine the area of myocardial infarction:
[0073] Cut the heart specimen, cut the left ventricle into thin slices (7μm) evenly along the longitudinal axis of the heart, and place it in NBT solution (nitrotetrazolium blue chloride: 0.06133gNBT with phosphate buffer solution to make the volume to 100ml, store in the dark) 37 ℃ water bath for 15 minutes. The necrotic area is not stained because there is no dehydrogenase, and the non-necrotic area is stained blue because it contains dehydrogenase. Under a dissecting microscope, carefully separate the stained area from the unstained area and weigh them with an electronic balance. The size of the infarct area is expressed as the percentage of the weight of the infarct area and the weight of the left ventricle.
[0074] (2) Determine the apoptosis of cardiomyocytes:
[0075] TUNEL and Cell Death Detection ELISA were used to detect cardiomyocyte apoptosis.
[0076] TUNEL method to detect cardiomyocyte apoptosis: digestion with proteinase K (digested samples are tissue sections taken from the heart of myocardial infarction model mice to observe cardiomyocyte apoptosis. The concentration of proteinase K is 20μg/mL, and 10mM Tris- Dissolved in HCl, pH 7.4-8.0, purchased from Hoffmann-La Roche Co., Ltd. After deparaffinization of the tissue section, digestion with proteinase K, and the sample is placed at 21℃-37℃ for 15min-30min) to expose the DNA. Terminal transferase (terminal transferase: Terminal transferase, TdT (purchased from Biyuntian Institute of Biotechnology), is a DNA polymerase that does not require a template, catalyzes the binding of deoxynucleotides to the 3'hydroxyl end of DNA molecules. Protruding, recessed or smooth-ended single- and double-stranded DNA molecules can be used as the substrate of TdT) under the mediation of dUTP, in which fluorescein-labeled dUTP is incorporated into the 3'OH of the double-stranded or single-stranded DNA of apoptotic cells End (use the TUNEL cell apoptosis detection kit, follow the product instructions provided by the kit manufacturer, the kit is used to detect the nuclear DNA breakage of the tissue cells in the early process of apoptosis. The principle is fluorescein labeling Under the action of deoxyribonucleotide terminal transferase, the dUTP can be connected to the 3'-OH end of broken DNA in apoptotic cells and specifically bind to the luciferin antibody linked to horseradish peroxidase, which in turn It reacts with HRP substrate diaminobenzidine to produce a strong color reaction, which is dark brown, and specifically and accurately locates apoptotic cells, so apoptotic cells can be observed under an optical microscope; due to normal or proliferating cells There is almost no DNA fragmentation, so there is no 3'-OH formation, and it is rarely stained.), observe the cells labeled with fluorescein-dUTP under a fluorescence microscope (the laser confocal microscope was used in the experiment, and the excitation wavelength of the photo was taken. It is 790nm, magnified 100 times). The apoptotic cell nucleus is green fluorescence, and the normal cell nucleus is blue fluorescence.
[0077] Calculate the apoptosis index (AI) of each group, AI=number of apoptotic cells/total number of cells (each high-powered field of view), take 5 high-powered field of view for each slide, and take the average value. Prepare tissue sections, and then perform TUNEL staining. Use a laser confocal microscope to observe and photograph the apoptosis status, perform statistics, and calculate the apoptosis index according to AI=number of apoptotic cells/total number of cells.
[0078] (3) Determination of heart function:
[0079] There are two methods of detecting heart function:
[0080] 1. Determination of echocardiographic parameters: M-mode echocardiography examines the fundus and ventricular complexes to determine the left atrial diameter (LA), left ventricular end diastolic diameter (LVIDd), left ventricular end systolic diameter (LVIDs), ventricular Interval thickness (IVSd). Calculate stroke volume (SV), volume per minute (CO), left ventricular ejection fraction (EF) and short axis shortening rate (FS%), left ventricular short axis shortening rate (FS%) is left ventricular contraction Function index parameters). The short axis shortening rate of the left ventricle, FS%, is directly proportional to the left ventricular systolic function of the heart, which can directly indicate the strength of the heart.
[0081] FS%=[(LVIDd-LVIDs)/LVIDd]×100
[0082] 2. On the apical four-chamber view, place the sampling volume at the mitral valve orifice and the left ventricular outflow tract respectively, measure the early diastolic blood flow velocity E peak of the mitral valve orifice, and the late diastolic blood flow velocity A peak and calculate the E peak /A peak ratio. The tested sample was injected with 1×10 6 A myocardial infarction model mouse with mi-iPS cells/64mg/kg microRNA-421 inhibitor was measured with echocardiography for peak E and peak A. Combined with electrocardiogram to measure left ventricular isovolumic relaxation time (IVRT), left ventricular isovolumic contraction time (IVCT), left ventricular ejection time (ET). Calculate Tei index=IVRT+IVCT/ET. The measurement data is measured for 3 consecutive cardiac cycles, and the average value is taken. Tei index, also known as Myocardial Performance Index (MPI), can comprehensively reflect the diastolic function of the ventricular systolic machine, and is mainly used to study the left ventricular function of dilated cardiomyopathy.
[0083] In this paper, the left ventricular end-diastolic diameter (LVIDd) and left ventricular end-systolic diameter (LVIDs) were used to calculate the left ventricular short axis shortening rate FS% to detect the degree of repair of myocardial infarction and cardiac function in mice before and after administration.