Modeling method based on GDF-15 defect

Abstract

The invention relates to the field of biological medicine, in particular to a GDF-15 defect-based modeling method which comprises the following steps: ligating left coronary anterior descending branches of a plurality of GDF-15 knockout mice, and respectively establishing myocardial ischemia reperfusion models based on recombinant GDF-15 and normal saline; the method comprises the following steps: taking a myocardial tissue sample, and treating the sample at a pre-plating temperature and a pre-plating duration to obtain myocardial cells; detecting an AMPK signal channel expressed by the myocardial tissue protein; determining whether the activity of the AMPK signal path is inhibited or not based on the phosphorylation state value, and determining the osmotic pump rate; or, detecting the ROS level of the myocardial cells to obtain a captured image, determining the state of the myocardial cells based on the gray value of the image, and determining the stability of the state of the myocardial cells according to the oxidative stress index of the captured image; carrying out an anoxic experiment to obtain a fluorescence image; and determining the myocardial cell purity based on the fluorescence signal intensity, adjusting the osmotic pump rate, or adjusting the pre-plating time length or the pre-plating temperature, so that the accuracy of GDF-15 defect modeling drug administration is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a modeling method based on GDF-15 deficiency. Background Art

[0002] Myocardial infarction is a leading cause of death and disease worldwide, characterized by the death of myocardial tissue due to a prolonged lack of blood supply, a condition known as ischemia. Growth differentiation factor-15, a member of the transforming growth factor-15 superfamily, is involved in a variety of physiological and pathological processes, including inflammation and cancer. Research on GDF-15 has primarily focused on its function in cancer and inflammatory diseases, where it can regulate immune responses and cell survival.

[0003] Chinese patent application publication number: CN118922721A discloses a cMyBPC marker combination for early discrimination between type 2 and type 1 acute myocardial infarction, comprising the following steps: determining the amount of a first biomarker in a sample from a subject, wherein the first biomarker is cMyBPC; determining the amount of a second biomarker in the sample from the subject, wherein the second biomarker is selected from the group consisting of: BMP10-type peptide (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23), BNP-type peptide (brain natriuretic peptide-type peptide), GDF-15 (growth differentiation factor 15), ANG2 (angiopoietin 2), CRP (C-reactive protein), ESM1 (endothelial cell-specific molecule 1) or lipid biomarkers such as cholesterol, LDL (low-density lipoprotein) or APOAT (apolipoprotein A-1); comparing the amount of the biomarker with a reference for the biomarker and / or calculating a score for assessing myocardial infarction based on the amount of the biomarker; and assessing the subject based on the comparison and / or calculation. The present invention also relates to the use of the following items for assessing myocardial infarction: a first biomarker and a second biomarker, wherein the first biomarker is cMyBPC, and the second biomarker is selected from the group consisting of the following items: BMP10-type peptide, FGF23, BNP-type peptide, GDF15, ANG2, CRP (C-reactive protein), ESM1 or lipid biomarkers such as cholesterol or LDL; or at least one detection agent for the first biomarker and at least one detection agent for the second biomarker.

[0004] However, the existing technology has the following problems: the existing technology does not accurately regulate the drug administration in the GDF-15 deficiency modeling process, especially the osmotic pump rate, resulting in poor uniformity of the ischemia model and lack of specificity in the reperfusion process, thus leading to the problem of low drug administration accuracy in GDF-15 deficiency modeling. Summary of the Invention

[0005] To this end, the present invention provides a modeling method based on GDF-15 deficiency, which is used to overcome the problem that the existing technology does not accurately regulate the drug administration in the GDF-15 deficiency modeling process, especially the osmotic pump rate, resulting in poor uniformity of the ischemia model and lack of specificity in the reperfusion process, thereby leading to low drug administration accuracy in GDF-15 deficiency modeling.

[0006] To achieve the above objectives, the present invention provides a modeling method based on GDF-15 defects, comprising:

[0007] Several GDF-15 knockout mice were ligated at the left anterior descending coronary artery, and myocardial ischemia-reperfusion models were established using recombinant GDF-15 and normal saline, respectively.

[0008] taking a number of myocardial tissue samples from the perfused mouse heart, and processing the myocardial tissue samples under pre-plating temperature and pre-plating time conditions to obtain a number of myocardial cells;

[0009] detecting the AMPK signaling pathway of myocardial tissue protein expression in several of the myocardial tissue samples;

[0010] determining whether the activity of the AMPK signaling pathway is inhibited based on the phosphorylation state value of the AMPK signaling pathway, and determining the osmotic pump rate according to the ratio of the phosphorylation state value to a preset phosphorylation state value;

[0011] or, detecting the ROS level of the cardiomyocytes to obtain a captured image, determining the cardiomyocyte state based on an image grayscale value of the captured image, and determining the stability of the cardiomyocyte state according to an oxidative stress index of the captured image;

[0012] taking at least one cardiomyocyte to perform a hypoxia experiment, and detecting the mitochondrial membrane potential of the cardiomyocyte to obtain a fluorescence image;

[0013] The purity of the cardiomyocytes is determined based on the fluorescence signal intensity of the fluorescence image, and the osmotic pump rate is adjusted according to the relative difference or ratio between the oxidative stress index and the preset oxidative stress index, or the pre-plating time or pre-plating temperature is adjusted according to the difference between the fluorescence signal intensity and the preset fluorescence signal intensity.

[0014] Furthermore, the process of determining the myocardial tissue protein expression of the myocardial tissue sample includes:

[0015] The protein concentration was determined using the BCA protein assay kit;

[0016] Equal amounts of protein were separated using 8% to 12.5% SDS-PAGE and transferred to a nitrocellulose membrane;

[0017] The cell membrane was blocked with TBS containing 0.1% Tween20 and 5% skim milk for 1 h at room temperature;

[0018] After incubation with primary antibodies overnight at 4°C, the membranes were washed three times with TBST, each wash lasting 5 minutes;

[0019] Protein detection was performed using horseradish peroxidase-conjugated secondary antibodies;

[0020] The phosphorylation status values of the AMPK signaling pathway expressed in myocardial tissue proteins were obtained.

[0021] Furthermore, based on the comparison result that the phosphorylation state value of the AMPK signaling pathway is greater than the preset phosphorylation state value, it is determined that the activity of the AMPK signaling pathway is not inhibited, and based on the comparison result of the ratio of the preset phosphorylation state value to the phosphorylation state value and the preset ratio, it is determined that the osmotic pump rate is reduced by the first preset rate adjustment coefficient or the second preset rate adjustment coefficient.

[0022] Furthermore, the process of detecting the mitochondrial membrane potential of the cardiomyocytes includes:

[0023] The cardiomyocytes were incubated with JC-1 reagent at 37° C. for 20 minutes;

[0024] Fluorescence images after incubation were obtained using a fluorescence microscope;

[0025] The captured fluorescence images were analyzed to obtain the mitochondrial membrane potential.

[0026] Furthermore, myocardial cell damage is determined based on the comparison result that the image grayscale value of the captured image is greater than the image grayscale threshold, the myocardial cell state is determined to be stable based on the comparison result that the oxidative stress index of the captured image is less than or equal to the preset oxidative stress index, and the myocardial cell purity is determined to be unqualified based on the comparison result that the fluorescence signal intensity of the fluorescence image is less than or equal to the preset fluorescence intensity.

[0027] Furthermore, myocardial cell damage is determined based on the comparison result that the image grayscale value of the captured image is greater than the image grayscale threshold, the myocardial cell state is determined to be stable based on the comparison result that the oxidative stress index of the captured image is less than or equal to the preset oxidative stress index, and the myocardial cell purity is determined to be unqualified based on the comparison result that the fluorescence signal intensity of the fluorescence image is less than or equal to the preset fluorescence intensity.

[0028] Furthermore, under the condition that the myocardial cell state is determined to be unstable and the myocardial cell purity is qualified, based on the comparison result of the ratio of the oxidative stress index to the preset oxidative stress index and the ratio threshold, it is determined to reduce the preset rate adjustment coefficient by the first preset optimization adjustment coefficient or the second preset optimization adjustment coefficient.

[0029] Furthermore, based on the comparison result that the oxidative stress index of the captured image is greater than the preset oxidative stress index, it is determined that the myocardial cell state is unstable, and based on the comparison result that the fluorescence signal intensity of the fluorescence image is less than or equal to the preset fluorescence intensity, it is determined that the myocardial cell purity is unqualified.

[0030] Furthermore, under the condition that it is determined that the state of the myocardial cells is unstable and the purity of the myocardial cells is unqualified, based on the comparison result of the difference between the fluorescence signal intensity and the preset fluorescence signal intensity and the preset difference, it is determined to increase the pre-plating time by the preset pre-plating time adjustment coefficient or to increase the pre-plating temperature by the preset pre-plating temperature adjustment coefficient.

[0031] Compared with the prior art, the beneficial effect of the present invention lies in that, by constructing a myocardial ischemia-reperfusion model in GDF-15 knockout mice, the present invention studies the protective effect of GDF-15 on cardiomyocytes through the regulation of the AMPK signaling pathway and the oxidative stress response, adjusts the osmotic pump rate, pre-plating temperature and pre-plating time, and reveals the mechanism by which GDF-15 inhibits cardiomyocyte apoptosis and maintains mitochondrial functional homeostasis by activating the AMPK pathway. In addition, an oxidative stress damage evaluation system is established by quantifying ROS levels and membrane potential changes. The osmotic pump rate is dynamically fed back by AMPK activity to avoid excessive or insufficient administration, thereby optimizing the protective effect of the damage model.

[0032] Furthermore, the present invention detects the phosphorylation status value of the AMPK signaling pathway in myocardial tissue to determine whether the activity of the AMPK signaling pathway is inhibited. When the pathway is not inhibited, the osmotic pump administration rate is gradually reduced with a preset rate adjustment coefficient, thereby avoiding metabolic imbalance caused by excessive activation of the AMPK pathway, accurately regulating the AMPK signaling pathway, and accurately controlling the release amount of recombinant GDF-15 to avoid excessive stimulation of the AMPK pathway, thereby improving the maintenance of metabolic homeostasis of myocardial cells, reducing cell apoptosis and functional disorders, improving model survival rate, optimizing drug delivery efficiency, and improving the accuracy of drug release through dynamic control strategy.

[0033] Furthermore, the present invention determines whether myocardial cells are damaged by comparing the image grayscale value with a preset image grayscale threshold, and determines the stability of the myocardial cell state by comparing the oxidative stress index with a preset oxidative stress index, taking into account both damage judgment and state warning, monitoring the oxidative stress level, optimizing the culture environment of myocardial cells, improving the repeatability and scientificity of the experiment, enhancing the detection accuracy, and quickly and accurately evaluating the degree of damage and stability of myocardial cells.

[0034] Furthermore, the present invention determines that the state of myocardial cells is stable and the purity of myocardial cells is unqualified, adjusts the osmotic pump rate, and intelligently links the osmotic pump rate with the oxidative stress state to accurately maintain cell homeostasis, improve cell purification efficiency and survival rate, inhibit the proliferation of contaminating cells and enhance the survival advantage of target cells, optimize the cell environment, ensure the purity of myocardial cells and the reliability of experimental results, improve the construction of high-purity myocardial cell models, and provide a more reliable basis for the study of myocardial ischemia-reperfusion injury, thereby improving the accuracy and efficiency of the model.

[0035] Furthermore, the present invention determines that the state of myocardial cells is unstable and the purity of myocardial cells is qualified, adopts a progressive adjustment strategy, reduces the osmotic pump rate in stages, and realizes a gradual decrease in the osmotic pump rate, which not only prevents the imbalance of the cellular environment caused by a sudden drop in the rate, but also specifically alleviates oxidative stress, gradually reduces oxidative damage, promotes the recovery of mitochondrial function, reduces the impact of sudden changes in drug concentration on cell metabolism, slowly releases and regulates the balance of cell metabolic pressure, maintains the purity advantage, and improves the dynamic stability of the in vitro culture system.

[0036] Furthermore, the present invention determines that the state of cardiomyocytes is unstable and the purity of cardiomyocytes is unqualified, adjusts the pre-plating time or pre-plating temperature, thereby enhancing the adhesion selectivity of target cells, improving the mildness and specific expression of the enzymatic hydrolysis reaction, fine-tuning the cell attachment conditions to improve the purity and state, and specifically improving the attachment efficiency of cardiomyocytes, reducing the impact of mechanical damage on cell integrity, improving the repairability and subsequent culture plasticity of unqualified cardiomyocytes, and improving the accuracy and reliability of modeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a flow chart of a modeling method based on GDF-15 defects according to an embodiment of the present invention;

[0038] Figure 2 A flow chart for determining whether the activity of the AMPK signaling pathway is inhibited according to an embodiment of the present invention;

[0039] Figure 3 A flowchart for determining the state of a myocardial cell according to an embodiment of the present invention;

[0040] Figure 4 This is a flow chart for determining the purity of cardiomyocytes according to an embodiment of the present invention. DETAILED DESCRIPTION

[0041] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention.

[0042] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0043] It should be pointed out that the data in this embodiment are obtained by comprehensive analysis and evaluation of the historical test data and the corresponding historical test results of the three months before this test. It can be understood by those skilled in the art that the present invention can determine the above parameters for a single item by selecting the value with the highest proportion as the preset standard parameter based on the data distribution, using weighted summation to use the obtained value as the preset standard parameter, substituting each historical data into a specific formula and using the value obtained by the formula as the preset standard parameter or other selection methods, as long as the present invention can clearly define the different specific situations in the single determination process through the obtained values.

[0044] See also Figure 1 As shown, it is a flow chart of the modeling method based on GDF-15 defects according to an embodiment of the present invention.

[0045] The modeling method based on the GDF-15 defect in the embodiment of the present invention includes:

[0046] Step S1, ligating the left anterior descending coronary artery in several GDF-15 knockout mice, and establishing myocardial ischemia-reperfusion models based on recombinant GDF-15 and normal saline, respectively;

[0047] Step S2, taking a plurality of myocardial tissue samples from the perfused mouse heart, and processing the plurality of myocardial tissue samples under pre-plating temperature and pre-plating time conditions to obtain a plurality of myocardial cells;

[0048] Step S3, detecting the AMPK signaling pathway of the myocardial tissue protein expression in the myocardial tissue samples;

[0049] Step S4, determining whether the activity of the AMPK signaling pathway is inhibited based on the phosphorylation state value of the AMPK signaling pathway, and determining the osmotic pump rate according to the ratio of the phosphorylation state value to a preset phosphorylation state value;

[0050] Step S5, or, detecting the ROS level of the cardiomyocytes to obtain a captured image, determining the cardiomyocyte state based on the image grayscale value of the captured image, and determining the stability of the cardiomyocyte state according to the oxidative stress index of the captured image;

[0051] Step S6, taking at least one cardiomyocyte to perform a hypoxia experiment, and detecting the mitochondrial membrane potential of the cardiomyocyte to obtain a fluorescence image;

[0052] Step S7, determining the purity of the cardiomyocytes based on the fluorescence signal intensity of the fluorescence image, and adjusting the osmotic pump rate according to the relative difference or ratio between the oxidative stress index and the preset oxidative stress index, or adjusting the pre-plating time or pre-plating temperature according to the difference between the fluorescence signal intensity and the preset fluorescence signal intensity.

[0053] In the examples of the present invention, the normal saline group served as a negative control to exclude nonspecific interference of surgical manipulation and osmotic pump carriers on the experimental results; GDF-15 knockout was used to reveal the baseline effect of endogenous GDF-15 deficiency on myocardial ischemia-reperfusion injury and serve as a comparative benchmark for the intervention effect of exogenous recombinant GDF-15; the myocardial ischemia-reperfusion group was used to simulate the pathological environment of clinical ischemia-reperfusion injury, and by comparing the differences before and after recombinant GDF-15 intervention, its therapeutic potential to inhibit oxidative stress and maintain mitochondrial function through the AMPK pathway was verified.

[0054] Specifically, the present invention constructs a myocardial ischemia-reperfusion model in GDF-15 knockout mice, studies the protective effect of GDF-15 on cardiomyocytes through regulation of the AMPK signaling pathway and oxidative stress response, adjusts the osmotic pump rate, pre-plating temperature and pre-plating time, and reveals the mechanism by which GDF-15 inhibits cardiomyocyte apoptosis and maintains mitochondrial functional homeostasis by activating the AMPK pathway. It also establishes an oxidative stress damage assessment system by quantifying ROS levels and membrane potential changes, and dynamically feeds back the osmotic pump rate through AMPK activity to avoid excessive or insufficient administration, thereby optimizing the protective effect of the damage model.

[0055] Specifically, in step S1, an Alzet osmotic pump was implanted to establish an ischemia-reperfusion model, and recombinant GDF-15 or saline was administered at a rate of 12 μg / day. The myocardial ischemia-reperfusion model was established by ligating the left anterior descending coronary artery (LAD). It is understood that mice were anesthetized with 2% isoflurane, intubated, and the chest area was shaved. The chest cavity was accessed through an incision between the fourth and fifth intercostal spaces and secured with a chest retractor. The LAD was ligated with a 7-0 propylene suture, using a 2 mm long 4-0 propylene suture as a cushion. Successful ligation was confirmed by electrocardiogram showing blanching of the ischemic area and ST-segment elevation. After the chest was closed, the mouse was extubated and placed on a heating pad. After 90 minutes of ischemia, the chest was reopened and the ligature was removed to allow reperfusion of the heart, which was manifested by reddening of the previously pale ischemic area. Finally, the chest was closed and the mouse was allowed to recover. It is understood that animals that did not survive the procedure were not included in the analysis.

[0056] Specifically, the time point before ischemia was 0-1h, that is, 1 hour before the onset of ischemia, and the time points after reperfusion were 0h, 2h, 6h, 12h, 12h, 1d, 3d, and 7d.

[0057] Specifically, during the ischemia-reperfusion surgery, the ventilator parameters were maintained at: inspiration / expiration ratio 1:1, respiratory rate 90–100 breaths / min, and tidal volume 1–2 mL.

[0058] Specifically, in step S2, a plurality of myocardial tissue samples are obtained from the perfused mouse heart, and the plurality of myocardial tissue samples are processed under the pre-plating temperature and pre-plating time conditions to obtain a plurality of myocardial cells. The process is as follows: the mouse heart is cut into small pieces of preset size in pre-cooled HBSS to obtain myocardial tissue samples, the myocardial tissue samples are placed in a mixture of preset proportions, the myocardial tissue samples are enzymatically cut for a preset time at the pre-plating temperature and preset carbon dioxide concentration, and then washed with HBSS and non-myocardial cells are removed for the pre-plating time, and non-adherent myocardial cells are collected.

[0059] In the embodiment of the present invention, the pre-plating temperature and the pre-plating time are processing parameters for performing the differential adherent growth method on the myocardial tissue sample.

[0060] Specifically, the cardiomyocytes were stored in DMEM supplemented with 10% fetal bovine serum and 1% penicillin or streptomycin at 37°C in a humidified atmosphere with a carbon dioxide content of 5%, and the mouse heart was cut into small pieces of a preset size to obtain tissue fragments, wherein the preset size was 1 to 3 mm. 2 .

[0061] Specifically, the mixture is enzyme 1 and enzyme 2 in a preset ratio of 20:1, the preset temperature is 37°C, the preset carbon dioxide concentration is 5%, the preset time is 30 minutes, and after washing with HBSS, the cardiomyocytes are placed in a 15 cm culture dish containing 10% fetal bovine serum and 1% penicillin or streptomycin at a pre-plating temperature of 37°C for a pre-plating time of 90 minutes to remove non-cardiomyocytes.

[0062] In the embodiment of the present invention, the recombinant GDF-15 is obtained by cloning the target gene into an expression vector, inducing expression, and then undergoing multiple purification steps such as affinity chromatography and ion exchange, and finally obtaining a high-purity, biologically active protein through enzymatic digestion and activity verification. The HBSS (Hank's Balanced Salt Solution) and the recombinant GDF-15 are both existing technologies and will not be described in detail.

[0063] In the embodiment of the present invention, the enzyme 1 is collagenase and the enzyme 2 is trypsin.

[0064] Specifically, in step S3, the process of detecting the AMPK signaling pathway expressed by myocardial tissue proteins in the myocardial tissue samples includes:

[0065] Step S201, using a BCA protein detection kit to determine the protein concentration;

[0066] Step S202, using 8% to 12.5% SDS-PAGE to separate equal amounts of proteins, and transferring the separated proteins to a nitrocellulose membrane;

[0067] Step S203, blocking the cell membrane with TBS containing 0.1% Tween 20 and 5% skim milk at room temperature for 1 hour;

[0068] Step S204, after incubation with the primary antibody at 4°C overnight, wash three times with TBST, each wash lasting 5 minutes;

[0069] Step S205, protein detection using horseradish peroxidase-coupled secondary antibody;

[0070] Step S206: obtaining the phosphorylation status value of the AMPK signaling pathway expressed by myocardial tissue proteins.

[0071] In the embodiment of the present invention, the SDS-PAGE is an electrophoresis technique for separating proteins, the Tween 20 is a non-ionic surfactant, the primary antibody is the first antibody, the secondary antibody is the second antibody, the primary antibody is a specific antibody against the target protein, and the secondary antibody is an antibody against the primary antibody.

[0072] See also Figure 2 As shown, it is a flow chart of determining whether the activity of the AMPK signaling pathway is inhibited according to an embodiment of the present invention.

[0073] Specifically, the embodiment of the present invention determines whether the activity of the AMPK signaling pathway is inhibited based on the comparison result of the phosphorylation state value of the AMPK signaling pathway and the preset phosphorylation state value;

[0074] When the phosphorylation state value is less than or equal to a preset phosphorylation state value, it is determined that the activity of the AMPK signaling pathway is inhibited;

[0075] When the phosphorylation state value is greater than the preset phosphorylation state value, it is determined that the AMPK signaling pathway activity is not inhibited.

[0076] In the embodiment of the present invention, the preset phosphorylation state value is 800 AU. The preset phosphorylation state value is obtained by taking the average of several historical phosphorylation state values in which the activity of the AMPK signaling pathway is inhibited. However, the above value is not limited to this, and those skilled in the art can also adjust the value according to actual needs.

[0077] It is understandable that the activity of AMPK is directly regulated by the phosphorylation level of its α subunit (such as the Thr172 site), and the phosphorylation status value reflects the degree of AMPK activation. After phosphorylation, AMPK changes its conformation, activates downstream targets, and regulates energy metabolism and cell growth.

[0078] Specifically, under the condition that the activity of the AMPK signaling pathway is determined not to be inhibited, the osmotic pump rate is determined based on the comparison result of the ratio of the preset phosphorylation state value to the phosphorylation state value and the preset ratio of 0.65:

[0079] When the ratio is less than or equal to the preset ratio, it is determined to reduce the osmotic pump rate to a corresponding value using a first preset rate adjustment coefficient of 0.96;

[0080] When the ratio is greater than the preset ratio, it is determined to reduce the osmotic pump rate to a corresponding value using a second preset rate adjustment coefficient of 0.93;

[0081] The ratio is the ratio of the preset phosphorylation state value to the phosphorylation state value.

[0082] Understandably, overactivation of AMPK is accompanied by enhanced mitochondrial autophagy. Reducing the osmotic pump rate at this time can reduce the interference of exogenous drugs on mitochondrial membrane potential and maintain autophagy-energy balance. AMPK activity is negatively correlated with ROS levels. Differences in the pump rate regulation coefficient control drug release through a gradient, preventing excessive oxidative stress indexes caused by sudden changes in osmotic pressure.

[0083] In the embodiment of the present invention, the preset ratio is 0.65, but the above value is not limited thereto, and those skilled in the art may also adjust the value according to actual needs.

[0084] In an embodiment of the present invention, the reduced osmotic pump rate is the product of the pre-plating time and the preset pre-plating time adjustment coefficient. The preset rate adjustment coefficient includes a first preset rate adjustment coefficient, which is 0.96, and a second preset rate adjustment coefficient, which is 0.93.

[0085] Specifically, the present invention detects the phosphorylation status value of the AMPK signaling pathway in myocardial tissue to determine whether the activity of the AMPK signaling pathway is inhibited. When the pathway is not inhibited, the osmotic pump administration rate is gradually reduced with a preset rate adjustment coefficient, thereby avoiding metabolic imbalance caused by excessive activation of the AMPK pathway, accurately regulating the AMPK signaling pathway, and accurately controlling the release amount of recombinant GDF-15 to avoid excessive stimulation of the AMPK pathway, thereby improving the maintenance of metabolic homeostasis of myocardial cells, reducing cell apoptosis and functional disorders, improving model survival rate, optimizing drug delivery efficiency, and improving the accuracy of drug release through dynamic control strategies.

[0086] Specifically, in step S5, the process of detecting the ROS level of the cardiomyocytes includes:

[0087] Step S301, diluting DCFH-DA and Hoechst 33342 in serum-free culture medium;

[0088] Step S302, incubate the diluted DCFH-DA, Hoechst33342 and cardiomyocytes in the dark at 37°C for 15 minutes;

[0089] Step S303, obtaining a captured image after incubation using a fluorescence microscope;

[0090] Step S304 : Analyze the captured image using image analysis software to analyze the ROS level in the cardiomyocytes.

[0091] In the embodiment of the present invention, the DCFH-DA is a fluorescent probe for detecting intracellular reactive oxygen species (ROS), and the Hoechst 33342 is a blue fluorescent dye that can penetrate cell membranes.

[0092] See also Figure 3 As shown, it is a flow chart of determining the state of myocardial cells according to an embodiment of the present invention.

[0093] Specifically, the embodiment of the present invention determines the state of the myocardial cells based on the comparison result of the image grayscale value of the captured image and the image grayscale threshold;

[0094] When the image grayscale value is less than or equal to the image grayscale threshold, it is determined that the myocardial cells are not damaged;

[0095] When the image grayscale value is greater than the image grayscale threshold, myocardial cell damage is determined.

[0096] In the embodiment of the present invention, the image grayscale threshold is 90, the captured image is an 8-bit grayscale image, and the image grayscale threshold ranges from 0 to 255. However, the above value is not limited thereto, and those skilled in the art may also adjust the value according to actual needs.

[0097] Specifically, under the condition of determining myocardial cell damage, the stability of the myocardial cell state is determined according to the comparison result of the oxidative stress index of the captured image and the preset oxidative stress index:

[0098] When the oxidative stress index is less than or equal to the preset oxidative stress index, it is determined that the state of the myocardial cells is unstable;

[0099] When the oxidative stress index is greater than the preset oxidative stress index, it is determined that the state of the myocardial cells is stable.

[0100] In the embodiment of the present invention, the preset oxidative stress index value is 0.82, but the above value is not limited thereto, and those skilled in the art may also adjust the value according to actual needs.

[0101] It is understandable that mitochondrial reactive oxygen species (ROS) are the core driving factor of myocardial cell damage. About 90% of cellular ROS come from abnormal mitochondrial metabolism. Excessive ROS can directly lead to myocardial cell apoptosis or necrosis. The oxidative stress index reflects the cellular redox balance by quantifying the ROS level.

[0102] During implementation, the oxidative stress index was the ratio of the grayscale mean of the control group minus the grayscale mean of the experimental group to the grayscale mean of the control group.

[0103] In the embodiments of the present invention, the present invention determines whether myocardial cells are damaged by comparing the image grayscale value with a preset image grayscale threshold, and determines the stability of the myocardial cell state by comparing the oxidative stress index with a preset oxidative stress index, taking into account both damage judgment and state warning, monitoring the oxidative stress level, optimizing the culture environment of myocardial cells, improving the repeatability and scientificity of the experiment, enhancing the detection accuracy, and quickly and accurately evaluating the degree of damage and stability of myocardial cells.

[0104] Specifically, in step S6, the process of conducting the hypoxia experiment is to inoculate the cardiomyocytes into the NEST confocal culture dish, perform the hypoxia experiment on the non-adherent cardiomyocytes using recombinant GDF-15, and perform the hypoxia experiment on the cardiomyocytes that have not undergone any treatment as a control group, and perform the hypoxia experiment on the non-adherent cardiomyocytes using recombinant GDF-15 at a concentration of 100 ng / ml.

[0105] Specifically, in step S6, the process of detecting the mitochondrial membrane potential of the cardiomyocytes includes:

[0106] Step S401, incubating the cardiomyocytes with JC-1 reagent at 37°C for 20 minutes;

[0107] Step S402, obtaining a fluorescence image after incubation using a fluorescence microscope;

[0108] Step S403: Analyze the captured fluorescence image to obtain mitochondrial membrane potential.

[0109] In the embodiment of the present invention, the JC-1 reagent is a cationic lipid fluorescent dye that can accumulate in mitochondria in a potential-dependent manner. The fluorescence characteristics of the JC-1 reagent can be changed according to changes in mitochondrial membrane potential.

[0110] See also Figure 4 As shown, it is a flow chart of determining the purity of cardiomyocytes according to an embodiment of the present invention.

[0111] Specifically, the embodiment of the present invention determines the purity of myocardial cells based on the comparison result of the fluorescence signal intensity of the fluorescence image and the preset fluorescence signal intensity;

[0112] When the fluorescence signal intensity is less than or equal to the preset fluorescence intensity, it is determined that the purity of the cardiomyocytes is unqualified;

[0113] When the fluorescence signal intensity is greater than a preset fluorescence intensity, it is determined that the purity of the cardiomyocytes is qualified.

[0114] In an embodiment of the present invention, the preset fluorescence signal intensity is 1000 AU. The preset fluorescence signal intensity is obtained by taking the average value of the fluorescence signal intensities of several historical cases in which non-cardiac cells are completely removed. However, the above value is not limited to this, and those skilled in the art can also adjust the value according to actual needs.

[0115] It is understandable that cardiomyocyte-specific markers are usually used in experiments, and their fluorescent antibodies only bind to target cardiomyocytes. The higher the purity of the cardiomyocytes, the greater the proportion of positively labeled cells per unit area, and the stronger the cumulative fluorescent signal.

[0116] Specifically, under the condition that the cardiomyocyte state is determined to be stable and the cardiomyocyte purity is unqualified, the osmotic pump rate is adjusted according to the comparison result of the relative difference between the oxidative stress index and the preset oxidative stress index and the preset relative difference;

[0117] When the relative difference is less than or equal to the preset relative difference, it is determined to increase the reduced osmotic pump rate to a corresponding value using a first preset increase adjustment coefficient of 1.02;

[0118] When the relative difference is greater than the preset relative difference, it is determined to increase the reduced osmotic pump rate to a corresponding value using a second preset increase adjustment coefficient of 1.05;

[0119] The relative difference is the relative difference between the oxidative stress index and the preset oxidative stress index.

[0120] In the embodiment of the present invention, the preset relative difference value is 50, but the above value is not limited thereto, and those skilled in the art may also adjust the value according to actual needs.

[0121] In an embodiment of the present invention, the increased osmotic pump rate is the product of the decreased osmotic pump rate and a preset increase adjustment coefficient, and the preset increase adjustment coefficient includes a first preset increase adjustment coefficient, which is 1.02, and a second preset increase adjustment coefficient, which is 1.05.

[0122] Specifically, the present invention determines that the state of myocardial cells is stable and the purity of myocardial cells is unqualified, adjusts the osmotic pump rate, and intelligently links the osmotic pump rate with the oxidative stress state to accurately maintain cell homeostasis, improve cell purification efficiency and survival rate, inhibit the proliferation of contaminating cells and enhance the survival advantage of target cells, optimize the cellular environment, ensure the purity of myocardial cells and the reliability of experimental results, improve the construction of high-purity myocardial cell models, and provide a more reliable basis for the study of myocardial ischemia-reperfusion injury, thereby improving the accuracy and efficiency of the model.

[0123] Specifically, under the condition that the cardiomyocyte state is determined to be unstable and the cardiomyocyte purity is qualified, the osmotic pump rate is optimized according to the comparison result of the ratio of the oxidative stress index to the preset oxidative stress index and the ratio threshold;

[0124] When the ratio is less than or equal to the ratio threshold, it is determined to reduce the preset rate adjustment coefficient to a corresponding value using the first preset optimization adjustment coefficient of 0.99;

[0125] When the ratio is greater than the ratio threshold, it is determined to reduce the preset rate adjustment coefficient to a corresponding value using a second preset optimization adjustment coefficient of 0.97;

[0126] The ratio is the ratio of the oxidative stress index to the preset oxidative stress index.

[0127] In the embodiment of the present invention, the ratio threshold is set to 1.10, but the above value is not limited thereto, and those skilled in the art may also adjust the value according to actual needs.

[0128] In an embodiment of the present invention, the reduced preset rate adjustment coefficient is the product of the preset rate adjustment coefficient and the preset optimization adjustment coefficient. The preset optimization adjustment coefficient includes a first preset optimization adjustment coefficient, whose value is 0.99 and a second preset optimization adjustment coefficient, whose value is 0.97. The preset rate adjustment coefficient includes a first preset rate adjustment coefficient, whose value is 0.96 and a second preset rate adjustment coefficient, whose value is 0.93.

[0129] Specifically, the present invention determines that the state of myocardial cells is unstable and the purity of myocardial cells is qualified, adopts a progressive adjustment strategy, reduces the osmotic pump rate in stages, and realizes a gradual decrease in the osmotic pump rate. This not only prevents the imbalance of the cellular environment caused by a sudden drop in the rate, but also specifically alleviates oxidative stress, gradually reduces oxidative damage, promotes the recovery of mitochondrial function, reduces the impact of sudden changes in drug concentration on cellular metabolism, slowly releases and regulates the balance of cellular metabolic pressure, maintains the purity advantage, and improves the dynamic stability of the in vitro culture system.

[0130] Specifically, under the condition that the cardiomyocyte state is determined to be unstable and the cardiomyocyte purity is unqualified, the pre-plating time or pre-plating temperature is adjusted according to the comparison result of the difference between the fluorescence signal intensity and the preset fluorescence signal intensity and the preset difference;

[0131] When the difference is less than or equal to the preset difference, it is determined to increase the pre-plating time to a corresponding value using a preset pre-plating time adjustment coefficient of 1.02;

[0132] When the difference is greater than the preset difference, it is determined to increase the pre-plating temperature to a corresponding value using a preset pre-plating temperature adjustment coefficient of 1.05;

[0133] The difference is the difference between the fluorescence signal intensity and the preset fluorescence signal intensity, that is, the preset fluorescence signal intensity is subtracted from the fluorescence signal intensity.

[0134] In the embodiment of the present invention, the preset difference value is 70, but the above value is not limited thereto, and those skilled in the art may also adjust the value according to actual needs.

[0135] In the embodiment of the present invention, the increased pre-plating time is the product of the pre-plating time and the preset pre-plating time adjustment coefficient of 1.02; the increased pre-plating temperature is the product of the pre-plating temperature and the preset pre-plating temperature adjustment coefficient of 1.05.

[0136] Specifically, the present invention determines that the state of cardiomyocytes is unstable and the purity of cardiomyocytes is unqualified, adjusts the pre-plating time or pre-plating temperature, thereby enhancing the adhesion selectivity of target cells, improving the mildness and specific expression of the enzymatic hydrolysis reaction, and fine-tuning the cell attachment conditions to improve the purity and state in a targeted manner, thereby improving the attachment efficiency of cardiomyocytes, reducing the impact of mechanical damage on cell integrity, improving the repairability and subsequent culture plasticity of unqualified cardiomyocytes, and improving the accuracy and reliability of modeling.

[0137] It is understandable that the adjustment of the pre-plating time and pre-plating temperature will result in corresponding adjustment of the physiological saline group.

[0138] It is understood that all data are expressed as mean ± standard error of the mean (SEM). Statistical comparisons between two groups were performed using unpaired two-tailed t-tests. For comparisons of multiple groups, one-way analysis of variance (ANOVA) was used, followed by Fisher's least significant difference (LSD) post hoc test. Differences were considered statistically significant when P value < 0.05. All statistical analyses were performed using GraphPad Prism software.

[0139] Example 1:

[0140] In step S3, the process of detecting the AMPK signaling pathway expressed by myocardial tissue proteins in the myocardial tissue samples includes:

[0141] Step S201, using a BCA protein detection kit to determine the protein concentration;

[0142] Step S202, using 8% to 12.5% SDS-PAGE to separate equal amounts of proteins, and transferring the separated proteins to a nitrocellulose membrane;

[0143] Step S203, blocking the cell membrane with TBS containing 0.1% Tween 20 and 5% skim milk at room temperature for 1 hour;

[0144] Step S204, after incubation with the primary antibody at 4°C overnight, wash three times with TBST, each wash lasting 5 minutes;

[0145] Step S205, protein detection using horseradish peroxidase-coupled secondary antibody;

[0146] Step S206: obtaining the phosphorylation status value of the AMPK signaling pathway expressed by myocardial tissue proteins.

[0147] Table 1 AMPK phosphorylation level and osmotic pump rate adjustment

[0148]

[0149] As can be seen in the table, GDF-15 activates the AMPK pathway, and the pump speed is dynamically adjusted to avoid over-activation and maintain the protective effect.

[0150] Example 2:

[0151] In step S5, the process of detecting the ROS level of the cardiomyocytes includes:

[0152] Step S301, diluting DCFH-DA and Hoechst 33342 in serum-free culture medium;

[0153] Step S302, incubate the diluted DCFH-DA, Hoechst33342 and cardiomyocytes in the dark at 37°C for 15 minutes;

[0154] Step S303, obtaining a captured image after incubation using a fluorescence microscope;

[0155] Step S304 : Analyze the captured image using image analysis software to analyze the ROS level in the cardiomyocytes.

[0156] In the embodiment of the present invention, the DCFH-DA is a fluorescent probe for detecting intracellular reactive oxygen species (ROS), and the Hoechst 33342 is a blue fluorescent dye that can penetrate cell membranes.

[0157] Table 2: ROS levels and cardiomyocyte status assessment

[0158]

[0159] As shown in the table, the normal saline group was judged to be unstable because the gray value was >90 and the oxidation index was ≤0.82, and the conditions needed to be optimized. After the pump speed was adjusted, the gray value of the GDF-15 group decreased, and the oxidation index increased to >0.82, indicating a stable state.

[0160] Thus far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art may make equivalent changes or substitutions to the relevant technical features, and the technical solutions after such changes or substitutions will fall within the scope of protection of the present invention.

[0161] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A modeling method based on GDF-15 defects, characterized in that: include: Several GDF-15 knockout mice were ligated at the left anterior descending coronary artery, and myocardial ischemia-reperfusion models were established using recombinant GDF-15 and normal saline, respectively. taking a number of myocardial tissue samples from the perfused mouse heart, and processing the myocardial tissue samples under pre-plating temperature and pre-plating time conditions to obtain a number of myocardial cells; detecting the AMPK signaling pathway of myocardial tissue protein expression in several of the myocardial tissue samples; determining whether the activity of the AMPK signaling pathway is inhibited based on the phosphorylation state value of the AMPK signaling pathway, and determining the osmotic pump rate according to the ratio of the phosphorylation state value to a preset phosphorylation state value; or, detecting the ROS level of the cardiomyocytes to obtain a captured image, determining the cardiomyocyte state based on an image grayscale value of the captured image, and determining the stability of the cardiomyocyte state according to an oxidative stress index of the captured image; taking at least one cardiomyocyte to perform a hypoxia experiment, and detecting the mitochondrial membrane potential of the cardiomyocyte to obtain a fluorescence image; The purity of the cardiomyocytes is determined based on the fluorescence signal intensity of the fluorescence image, and the osmotic pump rate is adjusted according to the relative difference or ratio between the oxidative stress index and the preset oxidative stress index, or the pre-plating time or pre-plating temperature is adjusted according to the difference between the fluorescence signal intensity and the preset fluorescence signal intensity.

2. The modeling method based on GDF-15 defect according to claim 1, characterized in that: The process of determining the myocardial tissue protein expression of the myocardial tissue sample includes: The protein concentration was determined using the BCA protein assay kit; Equal amounts of protein were separated using 8% to 12.5% SDS-PAGE and transferred to a nitrocellulose membrane; The cell membrane was blocked with TBS containing 0.1% Tween20 and 5% skim milk for 1 h at room temperature; After incubation with primary antibodies overnight at 4°C, the membranes were washed three times with TBST, each wash lasting 5 minutes; Protein detection was performed using horseradish peroxidase-conjugated secondary antibodies; The phosphorylation status values of the AMPK signaling pathway expressed in myocardial tissue proteins were obtained.

3. The modeling method based on GDF-15 defect according to claim 2, characterized in that: Based on the comparison result that the phosphorylation state value of the AMPK signaling pathway is greater than the preset phosphorylation state value, it is determined that the activity of the AMPK signaling pathway is not inhibited, and based on the comparison result of the ratio of the preset phosphorylation state value to the phosphorylation state value and the preset ratio, it is determined that the osmotic pump rate is reduced by the first preset rate adjustment coefficient or the second preset rate adjustment coefficient.

4. The modeling method based on GDF-15 defect according to claim 1, characterized in that: The process of detecting the mitochondrial membrane potential of the cardiomyocytes includes: The cardiomyocytes were incubated with JC-1 reagent at 37° C. for 20 minutes; Fluorescence images after incubation were obtained using a fluorescence microscope; The captured fluorescence images were analyzed to obtain the mitochondrial membrane potential.

5. The modeling method based on GDF-15 defect according to claim 4, characterized in that: Myocardial cell damage is determined based on the comparison result that the image grayscale value of the captured image is greater than the image grayscale threshold, the myocardial cell state is determined to be stable based on the comparison result that the oxidative stress index of the captured image is less than or equal to the preset oxidative stress index, and the myocardial cell purity is determined to be unqualified based on the comparison result that the fluorescence signal intensity of the fluorescence image is less than or equal to the preset fluorescence intensity.

6. The GDF-15 defect-based modeling method according to claim 5, characterized in that: Under the condition that the myocardial cell state is stable and the myocardial cell purity is unqualified, the osmotic pump rate after increasing or decreasing with the first preset increase adjustment coefficient or the second preset increase adjustment coefficient is determined based on the comparison result of the relative difference between the oxidative stress index and the preset oxidative stress index and the preset relative difference.

7. The GDF-15 defect-based modeling method according to claim 6, characterized in that: The cardiomyocyte state is determined to be unstable based on the comparison result that the oxidative stress index of the captured image is greater than the preset oxidative stress index, and the cardiomyocyte purity is determined to be qualified based on the comparison result that the fluorescence signal intensity of the fluorescence image is greater than the preset fluorescence intensity.

8. The GDF-15 defect-based modeling method according to claim 7, characterized in that: Under the condition that the state of the myocardial cells is determined to be unstable and the purity of the myocardial cells is qualified, based on the comparison result of the ratio of the oxidative stress index to the preset oxidative stress index and the ratio threshold, it is determined to reduce the preset rate adjustment coefficient by the first preset optimization adjustment coefficient or the second preset optimization adjustment coefficient.

9. The GDF-15 defect-based modeling method according to claim 8, characterized in that: The cardiomyocyte state is determined to be unstable based on the comparison result that the oxidative stress index of the captured image is greater than the preset oxidative stress index, and the cardiomyocyte purity is determined to be unqualified based on the comparison result that the fluorescence signal intensity of the fluorescence image is less than or equal to the preset fluorescence intensity.

10. The GDF-15 defect-based modeling method according to claim 9, characterized in that: Under the condition that the state of the myocardial cells is determined to be unstable and the purity of the myocardial cells is unqualified, based on the comparison result of the difference between the fluorescence signal intensity and the preset fluorescence signal intensity and the preset difference, it is determined to increase the pre-plating time by the preset pre-plating time adjustment coefficient or to increase the pre-plating temperature by the preset pre-plating temperature adjustment coefficient.

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