Modeling method for influence of GDF-15 on ischemic injury of heart tissue of mouse

Abstract

The invention relates to the technical field of ischemia model construction, in particular to a modeling method for influence of GDF-15 on ischemic injury of mouse heart tissue, which comprises the following steps: ligation of coronary artery anterior left descending branch of a healthy mouse from which GDF-15 gene is knocked out to ischemic the heart of the mouse; determining the eligibility of the ischemia process based on the myocardial artery blood supply association relationship and the ischemia sequence; implanting an osmotic pump into the plurality of mice, and respectively refilling recombinant GDF-15 and refilling normal saline at an initial rate; obtaining a plurality of echocardiograms of the reperfusion process to determine an adjustment to the initial rate based on a plurality of sets of recovery conditions of the blood supply recovery areas of the heart with the matching degree reaching the standard; determining the qualification of the model according to the contour feature change uniformity of the blood supply recovery areas of the reperfusion recombinant GDF-15 group and the reperfusion normal saline group after adjustment; and determining the influence of the GDF-15 on the ischemic injury of the heart tissue of the mouse according to the myocardial cell function of the qualified model and a molecular detection result. The accuracy of the exploration result is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of the construction of ischemic models, and particularly to a modeling method for the effect of GDF-15 on ischemic injury of mouse heart tissue. Background Art

[0002] Myocardial infarction is one of the major cardiovascular diseases causing death and disability globally, characterized by the necrosis of heart muscle tissue due to long-term ischemia (insufficient blood supply). Although significant progress has been made in reperfusion therapies aimed at restoring blood flow (such as thrombolysis and interventional procedures), the complex pathophysiological mechanisms of myocardial infarction and its subsequent cardiac remodeling process are still not fully understood.

[0003] Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor-β (TGF-β) superfamily and plays a regulatory role in various physiological and pathological processes such as inflammation and cancer. However, its specific function in myocardial infarction has not been fully elucidated. Current research on GDF-15 mainly focuses on the fields of cancer and inflammatory diseases, and it is known to regulate immune responses and cell survival.

[0004] In the context of myocardial infarction, research on GDF-15 expression and its function is still relatively limited. Preliminary evidence suggests that GDF-15 may exert a protective effect on heart tissue through anti-inflammatory and anti-fibrotic properties, but the specific molecular mechanisms underlying its impact on the progression of myocardial infarction and cardiac remodeling are not clear.

[0005] Chinese Patent Publication No.: CN115777629A discloses a method for establishing and evaluating a myocardial ischemia-reperfusion model, including steps such as preoperative weighing and anesthesia fixation, tracheal intubation, double-needle ligation of the left anterior descending coronary artery, postoperative suture, and removing the tracheal intubation after the animal resumes spontaneous breathing. The modeling success rate and model stability of the myocardial ischemia-reperfusion model provided by this invention are improved, the required sample size is significantly reduced, and the model evaluation indicators are more comprehensive and accurate.

[0006] The following problems exist in the prior art: The criteria for determining the success of left anterior descending branch ligation in mice in the prior art are macroscopic observation and electrocardiogram monitoring. If it is observed macroscopically that the myocardial tissue distal to the ligation rapidly turns pale, and the ST segment of the electrocardiogram is significantly elevated, and the Q or S wave is wide and deformed, it can be determined that the ligation is successful. There is a lack of refined control over the ischemic process and the reperfusion process, resulting in poor uniformity of the ischemic model and a lack of pertinence in the reperfusion process, which will thus interfere with the exploration results of the effect of GDF-15 on ischemic injury of mouse heart tissue. Summary of the Invention

[0007] To this end, the present invention provides a method for modeling the effect of GDF-15 on ischemic injury of mouse heart tissue, so as to overcome the problem in the prior art that the ischemic process and the reperfusion process lack refined control, and it is impossible to ensure that several ischemic models with consistent ischemic conditions are obtained, resulting in low accuracy in the study of the effect of GDF-15 on ischemic injury of heart tissue.

[0008] To achieve the above object, the present invention provides a method for modeling the effect of GDF-15 on ischemic injury of mouse heart tissue, including: Ligate the left anterior descending coronary artery of several healthy mice with the GDF-15 gene knocked out to cause ischemia to the hearts of the mice; For the heart of a single mouse, obtain several echocardiograms during the ischemic process to determine several ischemic regions, and determine the ischemic sequence of the heart based on the image features of several ischemic regions within a preset time period; Determine the qualification of the ischemic process based on the blood supply correlation relationship of several myocardial arteries and the ischemic sequence; Implant osmotic pumps into several of the mice and reperfuse recombinant GDF-15 and normal saline at an initial rate respectively; Obtain several echocardiograms during the reperfusion process to determine the matching degree between the recovery sequence of the blood supply recovery region and the ischemic sequence; Determine the adjustment of the initial rate based on the recovery status of the blood supply recovery regions of several hearts with qualified matching degrees; Determine the model qualification according to the uniformity of the contour feature changes of the blood supply recovery regions of the recombinant GDF-15 reperfusion group and the normal saline reperfusion group after adjustment, so as to adjust the determination process of whether the matching degree is qualified according to the difference in the uniformity characteristic values under the condition that the model is determined to be unqualified; Determine the effect of GDF-15 on ischemic injury of mouse heart tissue according to the myocardial cell function and molecular detection results of the qualified model.

[0009] Further, the process of determining several ischemic regions includes: Compare the gray values of several regions in several echocardiograms with a preset gray value; Determine the regions with gray values greater than or equal to the preset gray value as ischemic regions.

[0010] Further, the process of determining the ischemic sequence of the heart based on the image features of several ischemic regions includes: Determine the ischemic starting region and the diffusion area of the ischemic starting region; Determine the ischemic rate of the corresponding ischemic process when the diffusion area reaches the area threshold; Sort several ischemic rates in sequence to obtain the ischemic sequence.

[0011] Further, the process of determining the eligibility of the ischemia process includes: Pre - construct the blood supply association relationship between each cardiac region and each myocardial artery; Determine that the ischemia process is qualified based on the judgment result that the ischemia sequence is consistent with the blood supply association relationship.

[0012] Further, randomly divide several mice with qualified ischemia processes into two groups, and reperfuse recombinant GDF - 15 and reperfuse normal saline at the initial rate respectively. The process of determining the recovery sequence of the blood supply recovery region includes: Determine the blood supply starting region and the diffusion area of the blood supply starting region; Determine the blood supply rate of the corresponding blood supply process when the diffusion area reaches the area threshold; Sort several blood supply rates in sequence to obtain the recovery sequence.

[0013] Further, the process of determining whether the matching degree between the recovery sequence of the blood supply recovery region and the ischemia sequence meets the standard includes: Determine the proportion of the same regions in the recovery sequence and the ischemia sequence, and record the proportion as the matching degree; Compare the matching degree with the preset matching degree; Based on the comparison result that the matching degree is greater than or equal to the preset matching degree, determine that the matching degree between the recovery sequence and the ischemia sequence meets the standard.

[0014] Further, the process of determining the recovery status of the blood supply recovery region includes: Obtain a number of echocardiograms during the reperfusion process to determine a number of perfusion grayscales of the blood supply recovery region; Taking the gray scale of the ischemic region before the start of reperfusion as a reference, subtract the gray scale from any one of the perfusion gray scale values to obtain a series of gray scale gradients; Establish a gray scale change curve with time as the abscissa and the gray scale gradient as the ordinate; Determine the blood supply recovery speed as the ratio of the area enclosed by the gray scale change curve and the coordinate axis to the corresponding time; Compare the blood supply recovery speed with the preset speed; Based on the comparison result that the blood supply recovery speed is less than the preset speed, determine that the recovery status of the blood supply recovery region does not meet the standard.

[0015] Further, the process of adjusting the initial rate includes: Determine the real - time oxygen consumption of the mouse, and subtract the real - time oxygen consumption from the preset consumption respectively; Set the corresponding relationship between the corresponding difference and increasing or decreasing the initial rate to increase or decrease the initial rate.

[0016] Further, the process of determining whether the model is qualified based on the uniformity of the contour feature changes includes: Determine the ratio of the diffusion area of the blood supply starting region to the corresponding time as the uniformity of the contour feature changes of the blood supply recovery region; Subtract the uniformity of the contour feature changes between the recombinant GDF-15 group during reperfusion and the normal saline group during reperfusion to obtain the uniformity difference; Determine that the model is unqualified based on the comparison result that the uniformity difference is less than or equal to the preset difference.

[0017] Further, the process of adjusting the determination process of whether the matching degree meets the standard under the condition that the model is determined to be unqualified includes: Subtract the uniformity difference from the preset difference to obtain the corresponding difference; Set a threshold adjustment coefficient corresponding to the corresponding difference to increase the preset matching degree based on the threshold adjustment coefficient.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows. The present invention dynamically captures the image features of the ischemic region at multiple time points through echocardiography, and combines the analysis of the image features within a preset time period, so as to accurately determine the ischemic sequence of different myocardial regions, such as "extending from the endocardium to the epicardium" or "regional preferential ischemia", avoiding the one-sidedness of traditional single-time-point evaluation, revealing the spatio-temporal law of ischemic progression, and providing a dynamic target for subsequent reperfusion research; determining the qualification of the ischemic process according to the consistency of the blood supply correlation relationship and the ischemic sequence can effectively verify the physiological authenticity of the model and exclude operation errors, such as vascular ligation deviation or compensatory abnormalities caused by gene knockout, ensuring that the experimental data reflects the real pathological progression; at the same time, revealing the spatio-temporal dynamic law of ischemic propagation, distinguishing reversible and irreversible damage regions, and providing a basis for targeted intervention; by judging whether the matching degree between the recovery sequence of the blood supply recovery region and the ischemic sequence meets the standard, the spatio-temporal consistency of ischemic propagation and blood flow recovery can be verified, and abnormal interference of the model, such as vascular compensatory abnormalities or operation errors, can be excluded, improving the reliability of experimental data; based on the recovery status of the cardiac recovery region where the matching degree meets the standard, dynamically adjust the reperfusion rate of recombinant GDF-15, avoiding individual differences caused by weight fluctuations and metabolic differences due to fixed doses, and improving the success rate of the model; by comparing the recovery region contour uniformity between the recombinant GDF-15 group and the normal saline group, reliable models meeting the preset standards are screened out, abnormal data caused by individual pathological heterogeneity or operation errors are eliminated, the comparability between groups is enhanced, so as to obtain an ischemic model with consistent ischemic conditions, and further improve the accuracy of the research results on the impact of GDF-15 on ischemic injury of cardiac tissue.

[0019] Furthermore, by comparing the gray - scale value of the region in the echocardiogram with the preset gray - scale value and determining the ischemic region, the present invention can achieve an objective quantitative assessment of myocardial ischemia, effectively reduce the subjective error of manual interpretation, and improve the consistency and repeatability of experimental data. At the same time, through standardized threshold setting, unified analysis can be carried out across different time points, different mouse models or experimental groups, enhancing the comparability between groups. In addition, the dynamic change of the gray - scale value can directly reflect the process of myocardial injury, providing a highly sensitive imaging basis for determining the ischemic sequence and optimizing the reperfusion strategy, thereby further screening the model to improve the consistency of the ischemic condition of the ischemic model, and further improving the accuracy of the research results on the impact of GDF - 15 on ischemic injury of cardiac tissue.

[0020] Furthermore, by determining the blood - supply starting region and its diffusion area, and quantifying the blood - supply rate when the diffusion area reaches the threshold, and then sorting to obtain the recovery sequence, the present invention can dynamically analyze the spatio - temporal law of ischemic progression, clarify the path and speed of ischemia spreading from the core area to the edge, and accurately capture the critical time window of blood - flow recovery. By sorting the recovery sequence, the "rapid - recovery area" and the "delayed - recovery area" can be distinguished, revealing the differences in hemodynamic characteristics of different regions, providing a basis for optimizing the reperfusion strategy, thereby further improving the accuracy of the research results on the impact of GDF - 15 on ischemic injury of cardiac tissue.

[0021] Furthermore, by calculating the blood - supply recovery speed according to the change of the gray - scale gradient of the echocardiogram and comparing it with the preset speed threshold to determine the recovery status, the present invention can achieve a dynamic quantitative assessment of blood - flow recovery, overcome the subjectivity of traditional naked - eye observation, and quantify the blood - perfusion efficiency in the initial stage of reperfusion through the gray - scale gradient, thereby further improving the accuracy of the research results on the impact of GDF - 15 on ischemic injury of cardiac tissue.

[0022] Furthermore, by real - time monitoring the oxygen consumption of mice and dynamically adjusting the initial rate, the present invention can achieve precise matching of metabolic demand and intervention intensity, overcome the limitation that fixed - rate drug administration cannot adapt to individual metabolic fluctuations, dynamically optimize the balance between tissue oxygen supply and energy metabolism through the corresponding relationship between the oxygen - consumption difference and the rate, and reduce the secondary injury caused by anaerobic metabolism in the myocardial ischemic region, thereby improving the model stability and further improving the accuracy of the research results on the impact of GDF - 15 on ischemic injury of cardiac tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a flowchart of the modeling method for the impact of GDF - 15 on ischemic injury of mouse cardiac tissue according to an embodiment of the present invention; Figure 2 is a flowchart of determining the eligibility of the ischemic process according to an embodiment of the present invention; Figure 3 Flow chart for determining whether the matching degree of the restoration order and the ischemia order meets the standard in the embodiments of the present invention; Figure 4 Flow chart for determining whether the restoration status of the blood supply restoration area meets the standard in the embodiments of the present invention. Detailed implementation manners

[0024] In order to make the objectives and advantages of the present invention clearer, the present invention will be further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] The preferred implementation manners of the present invention will be described below with reference to the accompanying drawings. Those skilled in the art should understand that these implementation manners are only used to explain the technical principles of the present invention and do not limit the protection scope of the present invention.

[0026] Please refer to Figures 1 - 4 as shown Figure 1 Flow chart of a modeling method for the effect of GDF-15 on ischemic injury of mouse heart tissue in the embodiments of the present invention; Figure 2 Flow chart for determining the qualification of the ischemia process in the embodiments of the present invention; Figure 3 Flow chart for determining whether the matching degree of the restoration order and the ischemia order meets the standard in the embodiments of the present invention; Figure 4 Flow chart for determining whether the restoration status of the blood supply restoration area meets the standard in the embodiments of the present invention.

[0027] The embodiments of the present invention provide a modeling method for the effect of GDF-15 on ischemic injury of mouse heart tissue, including: Step S1, ligating the left anterior descending coronary artery of a number of healthy mice with the GDF-15 gene knocked out to cause ischemia in the hearts of the mice; Step S2, for the heart of a single one of the mice, obtaining a number of echocardiograms during the ischemia process to determine a number of ischemic regions, and determining the ischemia order of the heart based on the image features of the number of ischemic regions within a preset time period; Step S3, determining the qualification of the ischemia process based on the blood supply association relationship of a number of myocardial arteries and the ischemia order; Step S4, implanting osmotic pumps into a number of the mice and respectively reperfusing recombinant GDF-15 and reperfusing normal saline at an initial rate; Step S5, obtaining a number of echocardiograms during the reperfusion process to determine the matching degree of the restoration order and the ischemia order of the blood supply restoration area; Step S6, determining the adjustment of the initial rate based on the restoration status of the blood supply restoration area of a number of hearts with the matching degree meeting the standard; Step S7, determine the qualification of the model according to the uniformity of the contour feature changes in the blood supply recovery areas of the recombined GDF-15 group and the saline reperfusion group after adjustment, so as to adjust the determination process of whether the matching degree meets the standard for the difference in the uniformity feature values under the condition that the model is determined to be unqualified; Step S8, determine the effect of GDF-15 on ischemic injury of mouse heart tissue according to the myocardial cell function and molecular detection results of the qualified model.

[0028] Specifically, the value range of the preset duration is set to [3 min, 5 min], and 3 min is preferably selected in the embodiments of the present invention.

[0029] Specifically, the process of ligating the left anterior descending branch of the coronary artery of the mouse includes: Fix the mouse after isoflurane anesthesia and remove the hair in the left anterior chest area; Bluntly separate the chest wall muscles layer by layer through a longitudinal incision at the left edge of the sternum; Use a hemostatic forceps to spread the ribs through the 3rd or 4th intercostal space to expose the heart; Tear open the pericardium to expose the left anterior descending branch, and use a suture to ligate the left anterior descending branch; Among them, the healthy mice are male mice aged 8-10 weeks.

[0030] Specifically, when anesthetizing the mouse, continuously inhale a mixed gas of 4-5% isoflurane and oxygen through a nasal cone or tracheal intubation, and adjust the maintenance stage to 1.5-2% isoflurane. The anesthesia depth is confirmed by toe pinch reflex test to prevent the mouse from waking up during the operation; the longitudinal incision at the left edge of the sternum is located 1-2 mm from the left edge of the sternum, and the incision is about 1.5 cm long.

[0031] Specifically, the suture is an 8-0 or 6-0 suture with a needle. The needle tip needs to be blunt to avoid piercing the blood vessel. The ligation position is 1-2 mm from the left atrial appendage. Insert the needle 0.5 mm beside the pulmonary artery conus, with a depth of 0.5-1 mm and a needle width of about 2 mm to avoid misligating the pulmonary artery branch.

[0032] Specifically, the process of determining several of the ischemic regions includes: Compare the gray values of several regions in several echocardiograms with a preset gray value; Determine the regions where the gray value is greater than or equal to the preset gray value as ischemic regions.

[0033] It is understandable that ischemia can lead to myocardial cell edema, microcirculation disorders, and tissue necrosis. These pathological changes will significantly alter the acoustic properties of the myocardium (such as density and echo intensity). In ultrasonic images, normal myocardium shows medium gray values due to its uniform cell structure and water content, while ischemic areas show increased gray values, that is, enhanced echo, due to structural damage or increased exudate.

[0034] Specifically, the threshold of the preset gray value needs to be determined through preliminary experiments in combination with equipment parameters and the characteristics of the animal model. The gray value range of the normal myocardium of healthy mice is usually 40 - 60. The ischemia threshold, that is, the preset gray value, is taken as the mean of the gray values of the healthy control group plus twice the standard deviation. For example, if the mean of the healthy group is 50 and the standard deviation is 5, then the preset gray value is 60.

[0035] Specifically, the process of determining the ischemia sequence of the heart based on the image features of several ischemic areas includes: Determine the ischemia starting area and the diffusion area of the ischemia starting area; Determine the ischemia rate corresponding to the ischemia process when the diffusion area reaches the area threshold; Sort several ischemia rates in sequence to obtain the ischemia sequence.

[0036] Specifically, the identification of the ischemia starting area needs to combine dynamic sequence analysis of echocardiogram and gray change characteristics. The specific steps are as follows: Track the gray values of consecutive multiple frames of ultrasonic images after ligation, 1 - 2 frames per second, to locate the local area where the gray value first increases, that is, the ischemic core area. For example, it is found through frame - by - frame comparison that the gray value of a certain area is continuously higher than that of the surrounding tissues within 5 seconds after ligation.

[0037] Taking the initial gray - value abnormal point as the center, set the gray - gradient threshold, for example, the gray - gradient of adjacent pixels ≥5, gradually expand the region boundary to form the initial ischemic area, and determine the diffusion area of the initial ischemic area.

[0038] Specifically, the setting of the area threshold is determined by calculating the standard deviation of the area from the data of the healthy group in preliminary experiments. The set threshold is the sum of the average area of the healthy group and three times the standard deviation. For example, the average area of a certain ischemic area is 5% of the left - ventricular area, about 1mm 2 , and the standard deviation is 0.2, then the area threshold is 1.6mm 2 .

[0039] Specifically, the process of determining the eligibility of the ischemia process includes: Pre - construct the blood - supply correlation between each heart area and each myocardial artery; Determine that the ischemia process is qualified based on the judgment result that the ischemia sequence is consistent with the blood - supply correlation.

[0040] Specifically, the establishment of the blood supply correlation between each heart region and each myocardial artery in the present invention is well-known to those skilled in the art. From an anatomical perspective, the blood supply correlation regions corresponding to the anterior descending branch of the left coronary artery are the anterior wall and anterior septum of the left ventricle, the blood supply correlation regions corresponding to the circumflex branch of the left coronary artery are the lateral wall and posterior lateral wall of the left ventricle, and the blood supply correlation regions corresponding to the right coronary artery are the right ventricle, the inferior wall and posterior wall of the left ventricle. This is the prior art and will not be elaborated here.

[0041] Specifically, a number of mice qualified in the ischemia process are randomly divided into two groups, and recombinant GDF-15 and saline for reperfusion are reperfused at an initial rate respectively. The process of determining the restoration order of the blood supply restoration region includes: Determining the blood supply starting region and the diffusion area of the blood supply starting region; Determining the blood supply rate of the corresponding blood supply process when the diffusion area reaches the area threshold; Sequentially sorting a number of blood supply rates to obtain the restoration order.

[0042] Specifically, the initial rate is 12 μg / d. The determination of the blood supply starting region needs to combine the dynamic sequence analysis of echocardiogram and the gray-scale change characteristics. The specific steps are as follows: For consecutive multiple frames of ultrasonic images after reperfusion, 1-2 frames per second, perform gray-scale value tracking to locate the local region where the gray-scale reduction first appears, that is, the blood supply core region. For example, it is found through frame-by-frame comparison that the gray-scale value of a certain region is continuously lower than that of the surrounding tissues within 5 seconds after reperfusion; Taking the initial gray-scale abnormal point as the center, setting a gray-scale gradient threshold, for example, the gray-scale gradient of adjacent pixels ≥ 10, gradually expanding the region boundary to form an initial blood supply region, and determining the diffusion area of the blood supply initial region.

[0043] Specifically, the process of determining whether the matching degree between the restoration order of the blood supply restoration region and the ischemia order meets the standard includes: Determining the proportion of the same regions in the restoration order and the ischemia order, and recording the proportion as the matching degree; Comparing the matching degree with a preset matching degree; Based on the comparison result that the matching degree is greater than or equal to the preset matching degree, determining that the matching degree between the restoration order and the ischemia order meets the standard; Based on the comparison result that the matching degree is less than the preset matching degree, determining that the matching degree between the restoration order and the ischemia order does not meet the standard.

[0044] It can be understood that the restoration order of the ischemic region should be highly consistent with the ischemic occurrence order, and the early ischemic region is preferentially restored. If the matching degree between the two is low, it indicates that the reperfusion strategy is ineffective or there is secondary injury, and the qualification of the model is low.

[0045] Specifically, the value range of the matching degree is set to [70%, 80%], and 75% is preferably selected in the embodiments of the present invention.

[0046] Specifically, the process of determining the recovery status of the blood supply recovery area includes: Obtaining a plurality of echocardiograms during the reperfusion process to determine a plurality of perfusion grayscales of the blood supply recovery area; Taking the grayscale of the ischemic area before the start of reperfusion as a reference, subtracting the grayscale from any one of the perfusion grayscale values to obtain a series of grayscale gradients; Taking time as the abscissa and the grayscale gradient as the ordinate to establish a grayscale change curve; Determining the blood supply recovery speed as the ratio of the area enclosed by the grayscale change curve and the coordinate axis to the corresponding time; Comparing the blood supply recovery speed with a preset speed; Based on the comparison result that the blood supply recovery speed is less than the preset speed, determining that the recovery status of the blood supply recovery area does not meet the standard; Based on the comparison result that the blood supply recovery speed is greater than or equal to the preset speed, determining that the recovery status of the blood supply recovery area meets the standard.

[0047] Specifically, the preset speed is determined according to experiments. The grayscale recovery rates of the heart images of several mice with qualified reperfusion processes are determined, and the average value obtained by summing up each grayscale recovery rate is the preset speed.

[0048] Specifically, the process of adjusting the initial rate includes: Determining the real-time oxygen consumption of the mouse, and subtracting the real-time oxygen consumption from the preset consumption respectively; Setting the corresponding relationship between the corresponding difference value and increasing or decreasing the initial rate to increase or decrease the initial rate.

[0049] Specifically, under the condition of determining that the perfusion rate is unqualified, determining the real-time oxygen consumption of the mouse, and comparing the real-time oxygen consumption with the first preset oxygen consumption and the second preset oxygen consumption respectively; Based on the comparison result that the real-time oxygen consumption is less than the first preset oxygen consumption, determining to decrease the initial rate; Based on the comparison result that the real-time oxygen consumption is greater than the second preset oxygen consumption, determining to increase the initial rate.

[0050] Specifically, the first preset oxygen consumption and the second preset oxygen consumption are respectively determined according to experiments, and are the minimum real-time oxygen consumption and the maximum real-time oxygen consumption of the mouse during the qualified perfusion process. Before determining the value range of the oxygen consumption threshold, the oxygen consumption baseline and fluctuation range of the myocardial ischemia model are clarified through preliminary experiments.

[0051] It is understandable that a large real-time oxygen consumption of the mouse indicates a large oxygen supply demand of cardiomyocytes, and the perfusion rate should be appropriately increased to provide the oxygen demand. A small real-time oxygen consumption indicates a small oxygen supply demand of cardiomyocytes, and the perfusion rate should be appropriately decreased to avoid damage caused by excessive oxygen.

[0052] Specifically, the process of decreasing the initial rate includes: Subtracting the real-time oxygen consumption from the first preset oxygen consumption to obtain a first percentage, and comparing the first percentage with a preset percentage; Based on the comparison result that the first percentage is greater than the preset percentage, it is determined to decrease the initial rate with a first adjustment coefficient; Based on the comparison result that the first percentage is less than or equal to the preset percentage, it is determined to decrease the initial rate with a second adjustment coefficient.

[0053] Specifically, the value range of the preset percentage is set to [3%, 10%], and 5% is preferably selected in the embodiments of the present invention; the value range of the first adjustment coefficient is set to [0.975, 0.984], and 0.98 is preselected in the embodiments of the present invention; the value range of the second adjustment coefficient is set to [0.985, 0.998], and 0.99 is preselected in the embodiments of the present invention.

[0054] Specifically, the process of increasing the initial rate includes: Subtracting the second preset oxygen consumption from the real-time oxygen consumption to obtain a second percentage, and comparing the second percentage with a preset percentage; Based on the comparison result that the second percentage is greater than the preset percentage, it is determined to increase the initial rate with a third adjustment coefficient; Based on the comparison result that the second percentage is less than or equal to the preset percentage, it is determined to increase the initial rate with a fourth adjustment coefficient.

[0055] Specifically, the value range of the third adjustment coefficient is set to [1.015, 1.02], and 1.018 is preselected in the embodiments of the present invention; the value range of the fourth adjustment coefficient is set to [1.005, 1.014], and 1.008 is preselected in the embodiments of the present invention.

[0056] It is understandable that the adjustment of the initial rate by the adjustment coefficient is: the product of the adjustment coefficient and the initial rate. For example, when the first adjustment coefficient is selected to adjust the initial rate, the adjusted perfusion rate is the product of 0.98 and 12 μg / d.

[0057] Specifically, the process of determining whether the model is qualified based on the uniformity of the contour feature change includes: Determine the ratio of the diffusion area of the blood supply starting region to the corresponding time as the contour feature change uniformity of the blood supply recovery region; Subtract the contour feature change uniformity of the reperfusion recombinant GDF-15 group and the reperfusion normal saline group to obtain the uniformity difference; Based on the comparison result that the uniformity difference is less than or equal to the preset difference, determine that the model is unqualified; Based on the comparison result that the uniformity difference is greater than the preset difference, determine that the model is qualified.

[0058] Specifically, the uniformity difference is the percentage of the difference between the contour feature change uniformities of the two groups and the contour feature change uniformity of the reperfusion recombinant GDF-15 group.

[0059] It can be understood that GDF-15 should improve the uniformity of blood flow recovery and reduce the no-reflow area by regulating cardiomyocyte survival, inhibiting apoptosis, and promoting angiogenesis, such as activating the PI3K / Akt pathway. If the uniformity difference between the two groups is significant, it indicates that the intervention effect of GDF-15 has biological significance.

[0060] Specifically, the value range of the preset difference is set to [15%, 25%], and 20% is preferably selected in the embodiments of the present invention.

[0061] Specifically, the process of adjusting the determination process of whether the matching degree meets the standard under the condition of determining that the model is unqualified includes: Subtract the uniformity difference from the preset difference to obtain the corresponding difference; Set a threshold adjustment coefficient corresponding to the corresponding difference to increase the preset matching degree based on the threshold adjustment coefficient.

[0062] It can be understood that when the difference between the reperfusion GDF-15 group and the reperfusion normal saline group is too small, the determination criterion for meeting the matching degree is improved, thereby improving the screening criterion for qualified models.

[0063] Specifically, subtract the uniformity difference from the preset difference to obtain the corresponding difference; Compare the difference with a preset difference; Based on the comparison result that the difference is greater than the preset difference, determine to increase the preset matching degree with a first threshold adjustment coefficient; Based on the comparison result that the difference is less than or equal to the preset difference, determine to increase the preset matching degree with a second threshold adjustment coefficient.

[0064] Specifically, the value range of the preset difference is set to [3%, 8%], and 5% is preferred in the embodiments of the present invention; the value range of the first threshold adjustment coefficient is set to [1.1, 1.15], and 1.12 is preferred in the embodiments of the present invention; the value range of the second threshold adjustment coefficient is set to [1.01, 1.14], and 1.08 is preferred in the embodiments of the present invention.

[0065] It can be understood that the adjustment process of the threshold adjustment coefficient is similar to the adjustment process of the initial rate.

[0066] Specifically, the heart tissues of mice with qualified models of perfused recombinant GDF-15 and perfused normal saline are collected respectively. The heart tissues are processed to obtain cardiomyocytes. The process of processing the heart tissues includes: Cut the heart tissue into small pieces in a pre-cooled buffer to obtain tissue fragments; Use the enzyme digestion solution to enzymatically digest the tissue fragments at 37°C for 30-60 minutes; Wash the enzymatically digested tissue fragments with the termination solution and then pipette them a preset number of times with a wide-mouth pipette to obtain a single-cell suspension; Place the single-cell suspension in an incubator at 37°C and let it stand for 1 hour to collect non-adherent cardiomyocytes; After centrifuging the non-adherent cardiomyocytes for 5 minutes, resuspend them with DMEM medium containing 10% fetal bovine serum, and inoculate them in a special culture dish for culture.

[0067] Specifically, the buffer is 137 mM NaCl + 5.4 mM KCl + 1.2 mM MgSO4 + 0.33 mM NaH2PO4 + 10 mM HEPES + 10 mM Glucose. Adjust the pH value of the buffer to 7.4 and filter it to sterilize for standby.

[0068] Specifically, the termination solution is the buffer plus 10% fetal bovine serum (FBS); the enzyme digestion solution is the buffer plus collagenase II and trypsin. Among them, the concentration of collagenase II is 0.5-1.0 mg / ml, and the concentration of trypsin is 0.1-0.2 mg / ml, which is prepared before use.

[0069] Specifically, the preset number of times is 15-20 times.

[0070] Specifically, the heart tissues after reperfusion are collected respectively. The heart tissues are processed to obtain cardiomyocytes, and the functions and molecules of the cardiomyocytes are detected to determine the effect of GDF-15 on ischemic injury of mouse heart tissues. Among them, the function and molecule detection includes protein analysis, evaluation of mitochondrial membrane potential and oxygen analysis.

[0071] It is understandable that the method of performing functional and molecular detection on cardiomyocytes to determine the effect of GDF-15 on ischemic injury of mouse heart tissue is well-known to those skilled in the art, and will not be elaborated here.

[0072] Example 1: Select 100 GDF-15 knockout mice. After ligating the left anterior descending branch, echocardiograms were collected every 5 minutes for 30 minutes. During the grayscale analysis, two experienced experimenters independently marked the ischemic area and counted its grayscale value; at the same time, using the preset grayscale value of 60 (the mean value of healthy myocardium is 50±5), the area with a grayscale value ≥60 was automatically extracted as the ischemic area by ImageJ software.

[0073] According to the obtained grayscale values, the mean value, standard deviation, coefficient of variation and misjudgment rate of the grayscale values were calculated. It is understandable that the calculation methods of the mean value, standard deviation and coefficient of variation of the grayscale values are well-known to those skilled in the art and will not be elaborated here. The misjudgment rate was determined by comparing the differences between the two groups of methods and the histological results (TTC staining). The specific process is well-known to those skilled in the art and will not be elaborated here. The experimental results are shown in Table 1: Table 1 Improvement in the uniformity of the interpretation of the ischemic area by the grayscale threshold

[0074] It can be seen from Table 1 that using the grayscale threshold can significantly reduce individual differences, and the misjudgment rate drops to 3%, verifying the improvement of the objectivity and quantification on the uniformity of the model.

[0075] Example 2: Track the grayscale changes through a dynamic echocardiogram sequence (1 frame per second) to identify the initial ischemic area, for example: endocardium → epicardium. According to the anatomical criteria (the area supplied by the left anterior descending branch is the anterior wall and the septum), determine whether the ischemic sequence matches. Use the Planimetry method to calculate the area of the ischemic area and count the standard deviation. The results are shown in Table 2: Table 2 Verification of the correlation between the ischemic sequence and blood supply

[0076] It can be seen from Table 2 that the matching rate of the ischemic sequence in the qualified group is as high as 95%, and the area standard deviation is significantly lower than that in the unqualified group.

[0077] Example 3: Real-time monitor the oxygen consumption of mice using a metabolic cage, adjust the reperfusion rate according to the difference, calculate the ratio of the area under the grayscale change curve to time, and count the coefficient of variation (CV). Using the standard that the recovery area standard deviation <1.0 mm² is qualified, the results are shown in Table 3: Table 3 Effect of dynamically adjusting the initial rate on blood supply recovery

[0078] As can be seen from Table 3, the CV of the blood supply recovery speed in the dynamic adjustment group was significantly lower than that in the fixed group, and the qualified rate of the model was increased to 90%.

[0079] So far, the technical solution of the present invention has been described in conjunction with the preferred embodiments shown in the drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

Claims

1. A modeling method for the effect of GDF-15 on ischemic injury of mouse heart tissue, characterized in that, Comprising: Ligate the left anterior descending coronary artery of several healthy mice with the GDF-15 gene knocked out to cause myocardial ischemia in the mice; For the heart of a single said mouse, obtain several echocardiograms during the ischemia process to determine several ischemic regions, and determine the ischemia sequence of the heart based on the image features of several ischemic regions within a preset time period; Determine the eligibility of the ischemia process based on the blood supply correlation relationship of several myocardial arteries and the ischemia sequence; Implant osmotic pumps into several said mice and reperfuse recombinant GDF-15 and reperfuse normal saline at an initial rate respectively; Obtain several echocardiograms during the reperfusion process to determine the matching degree between the recovery sequence of the blood supply recovery region and the ischemia sequence; Determine the adjustment of the initial rate based on the recovery status of the blood supply recovery regions of several groups of hearts with the matching degree reaching the standard; Determine the model eligibility according to the uniformity of the contour feature changes of the blood supply recovery regions of the recombinant GDF-15 reperfusion group and the normal saline reperfusion group after adjustment, so as to adjust the determination process of whether the matching degree reaches the standard for the difference in the uniformity feature values under the condition that the model is determined to be unqualified; Determine the effect of GDF-15 on ischemic injury of mouse heart tissue according to the myocardial cell function and molecular detection results of the qualified model.

2. The modeling method for the effect of GDF-15 on ischemic injury of mouse heart tissue according to claim 1, wherein The process of determining several said ischemic regions includes: Compare the gray values of several regions in several echocardiograms with a preset gray value; Determine the regions with the gray value greater than or equal to the preset gray value as ischemic regions.

3. The modeling method for the effect of GDF-15 on ischemic injury of mouse heart tissue according to claim 2, wherein The process of determining the ischemia sequence of the heart based on the image features of several said ischemic regions includes: Determine the ischemia starting region and the diffusion area of the ischemia starting region; Determine the ischemia rate of the corresponding ischemia process when the diffusion area reaches the area threshold; Sort several ischemia rates in sequence to obtain the ischemia sequence.

4. The modeling method for the effect of GDF-15 on ischemic injury of mouse heart tissue according to claim 3, wherein The process of determining the eligibility of the ischemia process includes: Pre-construct the blood supply correlation relationship between each heart region and each myocardial artery; Determine that the ischemia process is qualified based on the determination result that the ischemia sequence is consistent with the blood supply correlation relationship.

5. The modeling method for the effect of GDF-15 on ischemic injury of mouse heart tissue according to claim 4, wherein, Randomly divide several mice with qualified ischemia processes into two groups, and reperfuse recombinant GDF-15 and reperfuse normal saline at an initial rate respectively. The process of determining the recovery sequence of the blood supply recovery region includes: Determine the blood supply starting region and the diffusion area of the blood supply starting region; Determine the blood supply rate of the corresponding blood supply process when the diffusion area reaches the area threshold; Sort several blood supply rates in sequence to obtain the recovery sequence.

6. The modeling method for the influence of GDF-15 on ischemic injury of mouse heart tissue according to claim 5, characterized in that The process of determining whether the matching degree between the recovery sequence of the blood supply recovery region and the ischemia sequence reaches the standard includes: Determine the proportion of the same regions in the recovery sequence and the ischemia sequence, and record the proportion as the matching degree; Compare the matching degree with a preset matching degree; Determine that the matching degree between the recovery sequence and the ischemia sequence reaches the standard based on the comparison result that the matching degree is greater than or equal to the preset matching degree.

7. The modeling method for the influence of GDF-15 on ischemic injury of mouse heart tissue according to claim 6, characterized in that The process of determining the recovery status of the blood supply recovery region includes: Obtain several echocardiograms during the reperfusion process to determine several perfusion gray levels of the blood supply recovery region; Taking the gray scale of the ischemic area before the start of reperfusion as a reference, subtracting the gray scale from any of the perfusion gray scale values to obtain a series of gray scale gradients; Taking time as the abscissa and the gray scale gradient as the ordinate to establish a gray scale change curve; Determining the ratio of the area enclosed by the gray scale change curve and the coordinate axis to the corresponding time as the blood supply recovery speed; Comparing the blood supply recovery speed with a preset speed; Based on the comparison result that the blood supply recovery speed is less than the preset speed, determining that the recovery status of the blood supply recovery area does not meet the standard.

8. The modeling method for the influence of GDF-15 on ischemic injury of mouse heart tissue according to claim 7, wherein The process of adjusting the initial rate includes: Determining the real-time oxygen consumption of the mouse and subtracting the real-time oxygen consumption from the preset consumption respectively; Setting the corresponding relationship between the corresponding difference and increasing or decreasing the initial rate to increase or decrease the initial rate.

9. The modeling method for the effect of GDF-15 on ischemic injury of mouse heart tissue according to claim 8, wherein The process of determining whether the model is qualified based on the uniformity of the contour feature changes includes: Determining the ratio of the diffusion area of the blood supply starting area to the corresponding time as the uniformity of the contour feature changes of the blood supply recovery area; Subtracting the uniformity of the contour feature changes of the reperfusion recombinant GDF-15 group and the reperfusion normal saline group to obtain the uniformity difference; Based on the comparison result that the uniformity difference is less than or equal to the preset difference, determining that the model is unqualified.

10. The modeling method for the effect of GDF-15 on ischemic injury of mouse heart tissue according to claim 9, wherein The process of adjusting the determination process of whether the matching degree meets the standard under the condition of determining that the model is unqualified includes: Subtracting the uniformity difference from the preset difference to obtain the corresponding difference; Setting a threshold adjustment coefficient corresponding to the corresponding difference to increase the preset matching degree based on the threshold adjustment coefficient.

Images