CN112913776B - Mouse model construction method for improving acute myocardial infarction prognosis - Google Patents
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Abstract
The invention relates to a construction method of a mouse model for improving acute myocardial infarction prognosis, belonging to the technical field of medical animal models. The invention provides a method for constructing a mouse model for improving acute myocardial infarction prognosis by enriching the survival environment EE, constructs a mouse model for reducing adverse remodeling after myocardial infarction injury by enriching the survival environment EE, and solves the technical problem of how to verify that enriching the survival environment EE can be used as a method for promoting injury repair after myocardial infarction; the constructed model provides an experimental choice for evaluating the myocardial infarction treatment drug and the treatment method. Provides a subject for experimental research for the prevention and treatment of myocardial infarction for people with high risk factors of ischemic heart disease in the future clinically; the invention can provide more theoretical and experimental bases for the research of preventing and treating cardiovascular diseases aiming at high risk groups of ischemic heart diseases, and has clinical transformation medical value.
Description
Technical Field
The invention relates to a mouse model construction method for improving acute myocardial infarction prognosis, and belongs to the technical field of medical animal models.
Background
At present, about 3.3 hundred million people suffering from cardiovascular diseases in China exist, and about 1100 million patients suffering from Ischemic Heart Disease (IHD) exist. Acute myocardial infarction is the most severe form of IHD and is one of the leading causes of increased morbidity and mortality worldwide. Previous studies have focused on treatment after myocardial infarction, and lack prevention or intervention in the early phase of myocardial infarction. Despite great progress in drug therapy and reperfusion therapy, complications such as heart failure and arrhythmia caused by severe left ventricular remodeling after myocardial infarction still seriously affect the quality of life and prognosis of patients. Therefore, those skilled in the art hope that the influence of the animal model on the post-myocardial infarction injury repair can be researched by constructing an animal model based on the recognition and intervention of early risk factors, and finally a method strategy is provided for the prevention and treatment of the high risk group with ischemic heart disease clinically.
As the medical model shifts to the bio-psycho-socio-medical model, myocardial infarction is also increasingly recognized as a psychosomatic related disease, the onset of which is closely related to psychosocial factors. Among them, stress is an important research direction for psychosomatic diseases. Stresses are generally classified into malignant stresses (distress) and benign stresses (eutress). The research on malignant stress and cardiovascular diseases is very common, and factors such as pressure, hostility, sadness and the like often cause hypertension and myocardial infarction to be aggravated; the study of benign stress and cardiovascular disease is rare. In 2010, the Enriched mouse living Environment (EE) was first reported in the journal of cells to significantly reduce the progression of melanoma and colon cancer, wherein EE is an ideal model of benign stress.
Since then, more and more researchers have worked on the different diseases as benign stress models, EE, as it can better simulate human sports, social and moderate survival challenges. Research shows that EE participates in various diseases such as occurrence and development of various solid tumors, retinal injury repair, acute liver injury and the like besides nervous system related diseases such as cerebral apoplexy injury repair, learning memory and neurodegenerative diseases. Through the research and analysis, one of the important mechanisms for regulating and controlling the diseases by EE is mediated immune inflammation, the immune inflammation plays a very important role in the whole process of the occurrence and development of acute myocardial infarction, including an acute inflammation stage, a fiber proliferation stage and a chronic stable stage, and the research on the role and the mechanism of the EE model in the injury repair after the acute myocardial infarction is not reported at present. The technical field needs to be verified whether EE can be used as a model for promoting the injury repair after myocardial infarction.
Disclosure of Invention
The invention aims to solve the technical problem of how to verify that abundant mouse living environment EE can be used as a method for promoting the repair of injury after myocardial infarction.
In order to solve the problems, the technical scheme adopted by the invention is to provide a method for constructing a mouse model for improving acute myocardial infarction prognosis; the method comprises the following steps:
step 1: constructing a rich environment model, feeding the experimental mouse in the environment, and preliminarily verifying that the EE model is successfully constructed;
and 2, step: performing acute myocardial infarction intervention operation, and performing myocardial infarction model operation on the experimental mouse;
and step 3: observing the survival rate of the myocardial infarction model mouse after EE pre-adaptation, and evaluating the influence of the EE model on the survival rate of the mouse after myocardial infarction;
and 4, step 4: observing the index of the cardiac function of the post-myocardial infarction model mouse after EE pre-adaptation; evaluating the influence of the EE model on the cardiac function of the mouse after myocardial infarction;
and 5: observing the morphological change of the infarct zone of the post-myocardial infarction model mouse after EE pre-adaptation; the effect of the EE model on the morphology of the post-infarct area of mice was evaluated.
Preferably, the successful construction of the EE model in the step 1 is preliminarily verified by detecting the expression level of the BDNF protein of the mouse hypothalamus.
Preferably, the indices of the central function of step 4 above include the left ventricular end systolic diameter, the end diastolic diameter, the ventricular septum and the thickness of the posterior wall, the end diastolic volume, the end systolic volume, the stroke volume and the ejection fraction.
Preferably, the morphological change of the infarct area in step 5 above comprises a change in the area of the mouse infarct area, infarct compartment wall thickness and extracellular matrix ECM synthesis composition of the infarct area.
Preferably, the infarct area extracellular matrix ECM synthesis components include α -SMA, collagen i and collagen iii proteins.
The invention provides application of a mouse model construction method for improving acute myocardial infarction prognosis in evaluating drugs for treating myocardial infarction.
The invention provides application of a mouse model construction method for improving acute myocardial infarction prognosis in evaluating a treatment method for treating myocardial infarction.
Compared with the prior art, the invention has the following beneficial effects:
the invention constructs a mouse model capable of reducing adverse remodeling after myocardial infarction injury, so that the mouse model has clinical transformation medical value and provides an experimentable object for theoretical support of prevention and treatment of myocardial infarction of population with high risk factors of ischemic heart disease in future.
The EE model is widely used in neoplastic diseases and brain injury diseases, but is still applied to myocardial infarction for the first time. Because EE participates in the occurrence and development of other diseases by regulating and controlling immune inflammation, and acute myocardial infarction is closely related to the immune inflammation, the invention provides a construction condition of EE as a mouse model capable of reducing the severity of acute myocardial infarction, provides a construction method of the mouse model for improving the prognosis of acute myocardial infarction, and is expected to provide more theoretical and experimental bases for the research of preventing and treating cardiovascular diseases of high risk groups of ischemic heart diseases.
Drawings
FIG. 1 is a diagram: western Blot results and their quantitative analysis of mouse hypothalamic BDNF protein levels after 4 weeks of EE pre-adaptation were p <0.05.
FIG. 2 is a diagram of: results of mouse hypothalamic BDNF mRNA levels after 4 weeks of EE pre-adaptation were p <0.05.
FIG. 3 is a diagram of: results of immunofluorescence assay of mouse hypothalamic BDNF after 4 weeks of EE pre-adaptation were p <0.05.
FIG. 4 shows: after 4 weeks of EE pre-adaptation, myocardial infarction intervention was performed, survival rates of control group mice and EE group mice were compared and analyzed, and p was <0.05 for 40 mice per group of initial myocardial infarction group.
FIG. 5 is a diagram: after EE pre-adapts for 4 weeks, myocardial infarction intervention is implemented, and the results of ultrasonography of the control group mice and the EE group mice are p <0.05.
FIG. 6 is a diagram of: after 4 weeks of EE pre-adaptation, myocardial infarction intervention was performed, and the histological examination results of control group mice and EE group mice were p <0.05.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings:
as shown in fig. 1 to 6, the present invention provides a method for constructing a mouse model for improving acute myocardial infarction prognosis; the method comprises the following steps:
step 1: constructing a rich environment model, feeding experimental mice in the environment, and preliminarily verifying that the EE model is successfully constructed;
step 2: performing acute myocardial infarction intervention operation, and performing myocardial infarction model operation on the experimental mouse;
and step 3: observing the survival rate of the myocardial infarction model mouse after EE pre-adaptation, and evaluating the influence of the EE model on the survival rate of the mouse after myocardial infarction;
and 4, step 4: observing the index of the cardiac function of the post-myocardial infarction model mouse after EE pre-adaptation; evaluating the influence of the EE model on the cardiac function of the mouse after myocardial infarction;
and 5: observing the morphological change of the infarct zone of the post-myocardial infarction model mouse after EE pre-adaptation; and (3) evaluating the influence of the EE model on the morphology of the post-myocardial infarction area of the mouse.
The successful construction of the EE model in the step 1 is preliminarily verified by detecting the expression level of the BDNF protein of the hypothalamus of the mouse.
The central function indexes in the step 4 comprise the left ventricle end systolic internal diameter, the end diastolic internal diameter, the ventricular interval and the thickness of the back wall, the end diastolic volume, the end systolic volume, the stroke volume and the ejection fraction.
The morphological change of the infarct zone in the step 5 comprises the area of the infarct zone of the mouse, the wall thickness of the infarct zone and the change of the extracellular matrix ECM synthesis component of the infarct zone.
The ECM synthesis components in the infarct area comprise alpha-SMA, collagen I and collagen III proteins.
The invention provides application of a mouse model construction method for improving acute myocardial infarction prognosis in evaluating a medicament for treating myocardial infarction.
The invention provides application of a mouse model construction method for improving acute myocardial infarction prognosis in evaluating a treatment method for treating myocardial infarction.
The invention comprises the following steps:
(1) Constructing a rich environment model: c57BL/6 mice were purchased from Beijing Wittingle, and divided into 2 groups of 50 mice each; one group was bred under normal laboratory standard conditions to set SE groups, 4 per cage; the other group is bred and set as an EE group in the rich environment constructed by people, the cage space of the EE group is larger, and the EE group is provided with rich and diverse toy facilities such as wooden houses, rollers, building blocks, climbing platforms, pipelines, cotton, sand baths and the like, and the number of the mouse members is more than 13-15. They were induced in each environment for 4 weeks. And preliminarily verifying the success of the EE model construction by detecting the expression level of the BDNF protein of the mouse hypothalamus after 4 weeks. In addition, it can also be further confirmed by using mouse behavioral assay method.
(2) Intervention of acute myocardial infarction: subsequently, each group of mice was further divided into 2 groups, one group being a sham group (sham group) and one group being a myocardial infarction group (MI group), i.e., a myocardial infarction model was established by permanently ligating the left anterior descending branch of the mice. Specifically, the method comprises the following steps: the mouse is anesthetized by 2% isoflurane and fixed on an operating table in a supine position, the precordial region is unhaired, a 1.2cm longitudinal incision is made on the left lower part of a sternum, subcutaneous tissues and pectoral muscles are separated bluntly, a fourth intercostal space is fully exposed and spread, the chest is slightly squeezed to enable the heart to slide out from the intercostal space, a 6-0 silk thread is used for permanently ligating a left anterior descending branch at a position which is about 3mm away from a left auricle, the successful basis is that the anterior wall of a ventricle becomes pale, and the electrocardiogram of the mouse is lead to have ST-segment elevation. Immediately after ligation, the heart was repositioned, the muscles were closed layer by layer and the skin was sutured with 4-0 silk, after which the mice were placed on a 37 ℃ thermostatic hot plate until they recovered. The sham operation group was only threaded and not ligated.
(3) To determine whether the EE model can improve cardiac function and ventricular remodeling:
observing the influence of an EE pre-adaptation model on survival rate after myocardial infarction: the 4 groups of mice: the quantities of the sham + SE group, the sham + EE group, the MI + SE group and the MI + EE group are respectively 10,10, 40 and 40. Pre-exposing for 4 weeks in SE or EE environment, then performing myocardial infarction surgical intervention, observing for one month, and counting the survival condition of the mice within one month;
(ii) observe the effect of EE pre-adaptation on cardiac function after myocardial infarction: a myocardial infarction model is established according to the method, the mouse precordial area is depilated by depilatory cream one day before 0, 3, 7 and 14 days after operation, the mouse is slightly anesthetized by 0.5 percent isoflurane on the next day, is placed on a constant-temperature ultrasonic operation flat plate at 37 ℃ in a supine position, the four limbs and the head of the mouse are fixed by adhesive tapes, and a little alcohol is dripped between electrode plates of the four limbs to conduct electricity. A proper amount of ultrasonic couplant is smeared on the precordial region of the mouse, the heart rate of the mouse after anesthesia is maintained to be 480-520bpm/min, and ultrasonic image information is acquired on long-axis and short-axis views beside the left sternum of the mouse by an ultrasonic probe. The acquired cardiac ultrasound data were analyzed with the Vevo2100 ultrasound system: cardiac function indices such as left ventricular end systolic diameter, end diastolic diameter, ventricular septum and thickness of the posterior wall, end Diastolic Volume (EDV), end Systolic Volume (ESV), stroke Volume (SV) and Ejection Fraction (EF).
(iii) observation of the effect of EE pre-adaptation on infarct zone morphology:
the model was established according to the above method, and after weighing surviving mice 7 days and 14 days after the myocardial infarction, the mice were sacrificed by cervical dislocation, the chest was opened rapidly, the heart was exposed, the right auricle was punctured, and 20mL of pre-cooled PBS solution was injected into the heart through the apex of the heart to sufficiently drain blood. The heart was cut from the bottom of the heart, the connective tissue, left and right atria were trimmed, the left and right ventricles were left intact, and rinsed thoroughly with PBS solution. The trimmed heart tissue was quickly dried on paper towels, fixed in 4% paraformaldehyde for 24h, rinsed 1 time with PBS solution, and then paraffin-embedded and sectioned to a thickness of about 5 μm. HE staining was then performed to count the area of infarcted regions of different mice. In addition, different levels are selected to calculate the thickness of the infarcted area, and finally, the mean value is taken to analyze the influence of EE pre-adaptation on the wall thickness of the infarcted area.
(iv) observing the effect of EE preconditioning on infarct area fibrosis: paraffin sections were prepared according to the above method, and Masson staining and sirius red staining were performed, and positive staining areas of infarct areas of different treatment groups were observed and counted under a microscope, masson staining was a blue area, and sirius red staining was a red area. The calculation formula is as follows: area ratio of positively colored area to infarcted area.
(v) observation of the effect of EE preconditioning on the extracellular matrix ECM in the infarct zone: myocardial infarction tissue was harvested 7 and 14 days after MI into 2mL centrifuge tubes, lysed routinely, and centrifuged to obtain protein supernatants. Western blot is used for detecting the expression changes of alpha-SMA, collagen I and collagen III proteins in the infarct area. Comparing whether EE pre-adaptation can affect synthesis of infarct zone ECM.
Examples
The following examples are presented to enable one of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.
In the following examples, mouse cages were purchased from von willebrand laboratory animal facilities, inc; genotypic tool mice were purchased from shanghai square model biotechnology, ltd, and the Jackson laboratory, usa.
1.EE model induction success establishment:
in order to study whether the induction of the mouse with rich environment is successful, the expression levels of the hypothalamic BDNF protein and RNA of the mouse pre-adapted for 4 weeks and the normal control group mouse are detected, and the detection results are shown in figures 1-3.
The EE model improves the survival rate and the cardiac function after myocardial infarction:
on the basis of successful induction of EE mice, acute myocardial infarction intervention is further carried out on related mice, and the influence of EE on myocardial infarction stress is observed. The results show that: compared with SE mice, the survival rate of myocardial infarction mice can be obviously improved after EE is pre-adapted for 4 weeks, and the result is shown in figure 4; echocardiography examination showed EE induction to improve left cardiac function in infarct mice including increasing left ventricular ejection fraction and left ventricular shortening rate and decreasing left ventricular end diastolic/systolic volume and internal diameter, with the results shown in figure 5.
The EE model slows down the remodeling of the ventricle of the infarct area:
histomorphic staining including HE staining, masson staining and sirius red staining compared infarct area, infarct area thickness and infarct area collagen fiber deposition in two groups of mice. The detection result shows that compared with an SE mouse, EE pre-adaptation can obviously reduce the area of an infarct area, increase the wall thickness of the infarct area and increase fibrin deposition of the infarct area. The results are shown in FIG. 6.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalents to the disclosed technology without departing from the spirit and scope of the present invention, and all such changes, modifications and equivalents are intended to be included therein as equivalents of the present invention; meanwhile, any equivalent changes, modifications and evolutions of the above embodiments according to the essential technology of the present invention are still within the scope of the technical solution of the present invention.
Claims (7)
1. A mouse model construction method for improving acute myocardial infarction prognosis; the method is characterized in that: the method comprises the following steps:
step 1: constructing a rich environment model, feeding experimental mice in the environment, and preliminarily verifying the success of the EE model construction by using a mouse behavioral determination method; or the success of the EE model construction is preliminarily verified by detecting the expression level of the mouse hypothalamus BDNF protein;
step 2: performing acute myocardial infarction intervention operation, and performing myocardial infarction model operation on the experimental mouse;
and step 3: observing the survival rate of the myocardial infarction model mouse after EE pre-adaptation, and evaluating the influence of the EE model on the survival rate of the mouse after myocardial infarction;
and 4, step 4: observing the index of the cardiac function of the post-myocardial infarction model mouse after EE pre-adaptation; evaluating the influence of the EE model on the cardiac function of the mice after myocardial infarction;
and 5: observing the morphological change of the infarct zone of the post-myocardial infarction model mouse after EE pre-adaptation; and (3) evaluating the influence of the EE model on the morphology of the post-myocardial infarction area of the mouse.
2. The method of claim 1, wherein the method comprises the following steps: the success of the construction of the EE model in the step 1 is preliminarily verified by detecting the expression level of the BDNF protein of the mouse hypothalamus.
3. The method of claim 1, wherein the method comprises the following steps: the indices of the central function of step 4 include left ventricular end systolic diameter, end diastolic diameter, ventricular septum and thickness of the posterior wall, end diastolic volume, end systolic volume, stroke volume and ejection fraction.
4. The method of claim 1, wherein the method comprises the steps of: the morphological change of the infarct area in the step 5 comprises the change of the area of the infarct area of the mouse, the wall thickness of the infarct compartment and the ECM synthesis component of the extracellular matrix of the infarct area.
5. The method of claim 4, wherein the method comprises the steps of: the ECM synthetic components of the extracellular matrix in the infarct area comprise alpha-SMA, collagen I and collagen III proteins.
6. Use of the method of any one of claims 1 to 5 for the evaluation of a medicament for the treatment of myocardial infarction in the construction of a mouse model for improving the prognosis of acute myocardial infarction.
7. Use of a mouse model construction method for improving acute myocardial infarction prognosis as claimed in any one of claims 1 to 5 in evaluating a therapeutic method for treating myocardial infarction.
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