A method for constructing and applying an enzyme-based model of coronary artery microcirculation injury in zebrafish.

Abstract

This invention relates to the field of animal model construction technology, and more particularly to a method for constructing a zebrafish coronary artery microcirculation injury model based on enzyme stripping and its application. This invention, for the first time, utilizes a rationally composed mixture of collagenases to specifically strip coronary artery endothelial cells, minimizing non-specific damage to cardiomyocytes and stably inducing coronary artery injury to construct a zebrafish model of coronary artery injury, particularly coronary artery microcirculation injury. This model not only has a highly localized injury area and minimal impact on the myocardium, but also exhibits a low mortality rate. Furthermore, the zebrafish model of coronary artery injury constructed by this invention demonstrates significant regenerative potential, with its blood vessels exhibiting strong regenerative capacity. It also shows high sensitivity and responsiveness to drugs, making it potentially valuable in drug screening and mechanism research.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, and in particular to a method for constructing a zebrafish coronary microcirculation injury model based on enzyme stripping and its application. Background Technology

[0002] Cardiovascular diseases such as myocardial infarction (MI) are among the leading causes of death worldwide. Their core pathology involves myocardial ischemia, necrosis, and fibrosis caused by coronary artery blockage, ultimately leading to heart failure. In mammals, cardiomyocytes have almost no regenerative capacity, and heart injury typically results in permanent scarring. In contrast, zebrafish have demonstrated remarkable regenerative capacity in various cardiac injury models, making them an important model for studying cardiac repair mechanisms. Zebrafish are one of the most commonly used vertebrate models for studying tissue regeneration, with advantages including nearly identical major organs and tissues to humans, a high degree of genetic homology with humans, and a fully sequenced genome. Previous studies in zebrafish have shown that even after the removal of up to 20% of the ventricles, their cardiac tissue can still fully recover. Zebrafish completely regenerate functional cardiac tissue by generating new cardiomyocytes, making them a unique vertebrate for studying the cellular and molecular mechanisms behind cardiac regeneration. Over the past two decades, zebrafish research has been at the forefront of several aspects of cardiac regeneration research. In particular, zebrafish research has identified key signals and molecules involved in heart regeneration, some of which can stimulate heart repair in mammals. This suggests that knowledge about zebrafish heart regeneration could help advance research in the field of mammalian regeneration.

[0003] Zebrafish models of heart injury include various methods such as ventricular apex resection, cryotherapy, and genetic ablation, each with its own characteristics. 、 For example, apical resection surgery was the earliest established injury model and has been widely used in the study of cardiac regeneration. 、 Apical resection is well-suited for observing local injury and regenerative events, while frozen section models can induce 20% myocardial death, followed by collagen deposition and scar formation, and subsequently, cardiomyocyte proliferation and reconstruction of the heart structure. Although frozen section models closely resemble clinical myocardial infarction, they lack coronary artery targeting, hindering the study of coronary artery-specific regeneration mechanisms, particularly the coronary microcirculation. Genetic ablation models of cardiomyocytes have been widely used for batch RNA sequencing and single-cell RNA sequencing analysis of cardiac tissue during cardiac regeneration. Researchers have also developed epicardial-specific injury methods to investigate the function of the epicardium in cardiac regeneration. Furthermore, while existing endothelial cell genetic ablation strategies can achieve coronary artery injury to some extent, the degree of injury is relatively mild and the mortality rate is high.

[0004] Therefore, it is necessary to develop a method for constructing a zebrafish model with low mortality and the ability to specifically realize coronary artery injury, especially coronary microcirculation injury. Summary of the Invention

[0005] Therefore, based on the above background, this invention provides a method for constructing a zebrafish coronary microcirculation injury model based on enzyme exfoliation and its application. This invention is the first to use a rationally composed mixture of collagenases to specifically exfoliate the endothelial cells of coronary vessels, stably inducing coronary vessel injury to construct a zebrafish model of coronary artery injury, especially coronary microcirculation injury. The model not only has a strong localized injury area and minimal impact on the myocardium, but also has a low mortality rate. Furthermore, the constructed model has significant regenerative potential.

[0006] The technical solution provided by this invention is as follows: A method for constructing a zebrafish coronary artery microcirculation injury model based on enzyme stripping includes the following steps: Includes the following steps: S1: Anesthetize zebrafish carrying endothelial cells expressing fluorescently labeled proteins and expose the ventricular surface of their hearts; S2: Use an application tool soaked in a mixed collagenase solution to contact a portion of the ventricular surface of a zebrafish for a period of time to specifically peel off the endothelial cells of the coronary vessels in the contact area; The mixed collagenase solution contains collagenase II and collagenase IV, wherein the concentration of collagenase II is 15-25 mg / mL and the concentration of collagenase IV is 15-25 mg / mL. S3: Rinse to remove residual collagenase; S4: Transfer the zebrafish to aquaculture water to recover.

[0007] Preferably, the zebrafish in step S1 are 6-8 months old.

[0008] Preferably, the concentration of collagenase II and the concentration of collagenase IV in the mixed collagenase solution in step S2 are 20 mg / mL.

[0009] Preferably, in step S2, the duration of contact between the drug application tool and the surface of the zebrafish's ventricle is 4-6 minutes.

[0010] Preferably, the application tool used in step S2 is a cotton swab with a tip diameter of 0.8-1.2 mm.

[0011] Based on the same inventive concept, this invention also provides the application of the zebrafish coronary artery microcirculation injury model constructed by the enzyme-based exfoliation method in the study of coronary vessel injury and / or regeneration mechanisms.

[0012] Based on the same inventive concept, this invention also provides the application of the zebrafish coronary microcirculation injury model constructed by the enzyme-based exfoliation method in screening drugs that promote and / or inhibit coronary vascular repair.

[0013] Furthermore, the drug is a compound that regulates the activity of the VEGF, Notch, or FGF signaling pathways.

[0014] Based on the same inventive concept, this invention also provides the application of the zebrafish coronary microcirculation injury model constructed by the enzyme-based exfoliation method in studying the mechanism of action of drugs that promote and / or inhibit coronary vascular repair.

[0015] Based on the same inventive concept, the present invention also provides a method for screening drugs that promote and / or inhibit coronary vascular repair, characterized by comprising the following steps: ①Preparation of injectable drugs; ② The drug injection is injected intraperitoneally into the zebrafish coronary artery microcirculation injury model constructed by the method described above for constructing a zebrafish coronary artery microcirculation injury model based on enzyme stripping, and then the coronary artery regeneration is detected.

[0016] The beneficial effects achieved by this invention are as follows: This invention is the first to specifically peel off the endothelial cells of coronary vessels using a rationally composed mixture of collagenases, minimizing non-specific damage to cardiomyocytes and stably inducing coronary artery injury to construct a zebrafish model of coronary artery injury, especially coronary microcirculation injury. This model not only has a highly localized injury area and minimal impact on the myocardium, but also a low mortality rate.

[0017] The zebrafish model of coronary microcirculation injury constructed in this invention has significant regenerative potential, with its blood vessels exhibiting strong regenerative capacity. Experimental verification shows that new blood vessel buds can be observed in this model as early as the 4th day after injury, and it is basically fully recovered by the 7th day, providing an ideal window for studying the regeneration process.

[0018] Experiments have verified that the zebrafish model of coronary microcirculation injury constructed in this invention has high sensitivity and high responsiveness to drugs (VEGF, Notch and FGF signaling pathway inhibitors), demonstrating its potential value in drug screening and mechanism research. Attached Figure Description

[0019] Appendix Figure 1 This is a schematic diagram illustrating collagenase damage to the coronary arteries of zebrafish. Figure 1 A is a schematic diagram of collagenase damage to the coronary artery and its treatment methods. Tg(fli1a:EGFP) transgenic zebrafish coronary endothelial cells are labeled with GFP. Figure 1 B represents the ventricular coronary artery damage status one day after sham surgery and collagenase treatment (1 dpt, 1 day post-treatment). n=15.

[0020] Appendix Figure 2 TUNEL staining and apoptosis of cardiac tissue after freezing and collagenase damage, among which Figure 2 A shows the TUNEL staining of ventricular tissue one day after freezing and collagenase damage; Figure 2 B represents the statistical results of apoptotic ventricular cells after freezing and collagenase damage. *** P < 0.001. n=15.

[0021] Appendix Figure 3 This is an imaging image of the coronary artery structure after collagenase damage, in which... Figure 3 A is a schematic diagram of collagenase damage to coronary arteries and its treatment methods; Figure 3 B represents the ventricular coronary regeneration status 1, 4, and 7 days after sham surgery and collagenase injury. n=15.

[0022] Appendix Figure 4 This is an imaging image of the cardiac coronary artery structure in a collagenase injury model after drug administration, in which... Figure 4 A is a schematic diagram of collagenase damage to coronary arteries and its treatment methods; Figure 4 B represents the statistical results of coronary artery regeneration density in the ventricular injury area of ​​the intraperitoneal injection groups of PBS, Tanshinone IIA, DAPT, and BGJ398. *** P < 0.001. n=15. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The following system was used to construct the zebrafish model: The zebrafish used were 6-8 months old. Tg(fli1a:EGFP) Zebrafish were housed in a constant-temperature, circulating water system at 28.5°C, with a light / dark cycle of 14 h / 10 h. All experiments were conducted in accordance with animal welfare requirements.

[0025] (1) Model building: ① Construction of a zebrafish model of coronary microcirculation injury A fine-tipped cotton swab (1 mm in diameter) was soaked in a mixed enzyme solution (prepared with physiological saline) containing 20 mg / mL collagenase II (C2139, Sigma-Aldrich, St. Louis, MO, USA) and 20 mg / mL collagenase IV (C4-BIOC, Sigma-Aldrich, St. Louis, MO, USA). The zebrafish were anesthetized and the surface of the ventricle was exposed. The swab was then used to contact the designated area for 5 minutes. Afterward, the residual collagenase was thoroughly rinsed off with PBS, and the fish were transferred to aquaculture water for recovery.

[0026] ② Constructing a cardiac injury model through ventricular frozen injury and ventricular apex resection surgery The procedure for ventricular cryoinjury was performed according to the method described in the literature (CHABLAIS F, VEIT J, RAINER G, JAŹWIŃSKA A. The zebrafish heart regenerates after cryoinjury-induced myocardial infarction [J]. BMC Dev Biol, 2011, 11: 21.). The anesthetized zebrafish was placed on a moistened sponge, and the pericardium was exposed through a small incision in the abdomen, and the heart was gently squeezed out. A stainless steel probe cooled with liquid nitrogen was attached to the tip of the ventricle for 3-5 seconds.

[0027] The ventricular apex resection was performed according to the literature (POSS KD, WILSON LG, KEATING MT. Heartregeneration in zebrafish [J]. Science, 2002, 298(5601): 2188-90.), after exposing the heart, about 20% of the ventricular apex tissue was removed by cutting with a curved blade.

[0028] (2) Drug administration Ten μL of the required drugs were injected into the abdomen of anesthetized zebrafish using a microsyringe. The control group received intraperitoneal injection of PBS. The concentrations of Tanshinone IIA, DAPT, and BGJ398 injected were 100 μM, 200 μM, and 60 μM, respectively.

[0029] (3) Sample processing: Cardiac Sample Processing and Imaging Hearts were fixed in 4% PFA for 1 minute and then embedded in 1% low-melting-point agarose for imaging using a Nikon SMZ18 microscope. After dehydration with 30% sucrose, the fixed hearts were embedded in OCT and 10 μm thick frozen sections were prepared. TUNEL staining was used to detect apoptosis. ImageJ software was used to analyze coronary artery density.

[0030] (4) Statistical analysis All statistical analyses were performed using GraphPad Prism 9.0 software. Differences between two groups were compared using a two-tailed independent samples t-test, and data are expressed as mean ± standard error (mean ± SD). Comparisons between three or more groups were performed using one-way ANOVA, combined with the Bonferroni multiple comparison test or the Dunnett multiple comparison test. The significance criteria for statistical differences were set as follows: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***).

[0031] (4) Results ① Verify that collagenase can effectively and specifically damage the coronary arteries of zebrafish. To establish a zebrafish coronary artery injury (coronary microcirculation injury) model, the same method was used... Tg(fli1a:EGFP) Local collagenase treatment of the ventricles of transgenic zebrafish induced coronary artery injury, and changes in coronary artery structure were observed one day after treatment (1 dpt, day 2). Figure 1 A). The results showed that the coronary fluorescence signal in the collagenase contact area was basically gone, indicating that the endothelial cells were effectively stripped away. Figure 1 B, in the figure, shows zebrafish in the sham-operated group that did not undergo any treatment (the sham-operated group was treated with cotton swabs soaked in PBS). The results showed that the mixed-enzyme method had high reproducibility and safety, with a surgical mortality rate of approximately 2% (n=200), lower than that of traditional frozen section injury, ventricular apex resection, and coronary endothelial cell genetic ablation models, demonstrating its significant advantages in small-scale, precise injury; furthermore, it is simpler and easier to perform.

[0032] To assess the effects of collagenase treatment on cardiomyocytes, TUNEL staining was performed on both collagenase-treated and frozen-damaged cardiac tissues. The results showed that frozen-damage induced widespread ventricular cell apoptosis, while collagenase-induced apoptosis was mainly limited to the periventricular region, with very few TUNEL-positive cells in the ventricular myocardial region. Figure 2 A). Quantitative analysis showed that the number of apoptotic ventricular cells treated with collagenase was only about 14% of that in frozen-damaged ventricles (A). Figure 2 B). This suggests that compared to the frozen injury model, the collagenase injury model significantly reduces non-specific damage to myocardial tissue and is more specific to coronary artery injury.

[0033] ② This study verified that the coronary arteries in a zebrafish model of coronary artery injury (coronary microcirculation injury) caused by collagenase damage can regenerate rapidly. To investigate the coronary artery regeneration capacity of the collagenase injury model, imaging analysis of the cardiac coronary artery structure was performed on days 1, 4, and 7 after injury. Figure 3 A). The results showed that 4 days after collagenase-induced injury, new blood vessel buds formed and extended into the injured area, and by the 7th day, the area had basically recovered to its pre-injury state. Figure 3 B) indicates that the coronary arteries in the collagenase damage model (coronary artery damage zebrafish model) have a strong regenerative capacity.

[0034] ③ Verify the weakening effect of VEGF, Notch, and FGF signaling pathway inhibitors on coronary regeneration. To verify the feasibility of the zebrafish coronary artery injury model constructed in this invention for signaling pathway research and drug screening, three signaling pathway inhibitors—Tanshinone IIA (VEGF inhibitor), DAPT (Notch inhibitor), and BGJ398 (FGF inhibitor)—were injected intraperitoneally on days 3, 4, and 5 after collagenase-induced injury. Coronary artery regeneration was assessed on day 6 post-treatment. Figure 4 A). The results showed that, compared with the PBS group, all three inhibitors significantly reduced the density of new coronary arteries in the damaged area, resulted in sparse coronary artery connectivity, and impaired coronary artery recovery. Figure 4 B). Statistical analysis showed that these inhibitor treatments all significantly reduced the density of new coronary arteries in the damaged area (B). Figure 4 (C) This suggests that these signaling pathways play a key regulatory role in vascular repair and also verifies that the model can be used for functional screening and mechanism research of small molecule drugs.

[0035] As can be seen from the above, this invention constructs a coronary artery injury model, particularly a coronary microcirculation injury model, by specifically exfoliating endothelial cells using collagenase. This provides a new tool with high specificity, reproducibility, and ease of operation for studying the mechanisms of coronary artery endothelial injury and regeneration. Compared with traditional models such as cryotherapy, ventricular resection, or genetic ablation, this invention can precisely induce focal coronary endothelial cell exfoliation without causing widespread myocardial damage, significantly reducing damage to non-target tissues. The model exhibits high controllability and reproducibility, with a surgical mortality rate of only 2%, significantly compensating for the shortcomings of existing coronary artery injury-related models. More importantly, this model allows for the observation of rapid coronary artery regeneration in zebrafish, where endothelial cells can complete damage repair and vascular reconstruction in a short period. This characteristic not only highlights the regenerative potential of the zebrafish cardiovascular system but also provides an ideal platform for in-depth analysis of regeneration-related signaling pathways.

[0036] Furthermore, the model's sensitivity to regenerative regulatory factors was validated through inhibitors of the VEGF, Notch, and FGF signaling pathways, demonstrating its significant potential as a drug screening model. In the experiments, inhibition of these three classic regenerative pathways significantly reduced the density of new coronary arteries, confirming that the model can be used for systematic screening of small molecules or bioactive factors that regulate angiogenesis. In addition, the establishment of this model provides a preclinical research platform for targeting and improving coronary microcirculation function, preventing myocardial ischemia, and intervening in the progression of heart failure.

[0037] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual structure is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for constructing a zebrafish coronary artery microcirculation injury model based on enzyme stripping, characterized in that, Includes the following steps: S1: Anesthetize zebrafish carrying endothelial cells expressing fluorescently labeled proteins and expose the ventricular surface of their hearts; S2: Use an application tool soaked in a mixed collagenase solution to contact a portion of the ventricular surface of a zebrafish for a period of time to specifically peel off the endothelial cells of the coronary vessels in the contact area; The mixed collagenase solution contains collagenase II and collagenase IV, wherein the concentration of collagenase II is 15-25 mg / mL and the concentration of collagenase IV is 15-25 mg / mL. S3: Rinse to remove residual collagenase; S4: Transfer the zebrafish to aquaculture water to recover.

2. The method for constructing a zebrafish coronary artery microcirculation injury model based on enzyme stripping according to claim 1, characterized in that, The zebrafish in step S1 are 6-8 months old.

3. The method for constructing a zebrafish coronary artery microcirculation injury model based on enzyme stripping according to claim 1, characterized in that, In step S2, the concentration of collagenase II in the mixed collagenase solution is 20 mg / mL, and the concentration of collagenase IV is 20 mg / mL.

4. The method for constructing a zebrafish coronary artery microcirculation injury model based on enzyme stripping according to claim 3, characterized in that, In step S2, the duration of contact between the drug application tool and the surface of the zebrafish ventricle is 4-6 minutes.

5. The method for constructing a zebrafish coronary artery microcirculation injury model based on enzyme stripping according to claim 1, characterized in that, The application tool used in step S2 is a cotton swab with a tip diameter of 0.8-1.2 mm.

6. The zebrafish coronary artery microcirculation injury model constructed by the method of constructing an enzyme-based zebrafish coronary artery microcirculation injury model according to any one of claims 1 to 5, and its application in studying the mechanism of coronary artery injury and / or regeneration.

7. The zebrafish coronary microcirculation injury model constructed by the method of constructing an enzyme-based zebrafish coronary microcirculation injury model according to any one of claims 1 to 5, and its application in screening drugs that promote and / or inhibit coronary vascular repair.

8. The application according to claim 7, characterized in that, The drug is a compound that regulates the activity of the VEGF, Notch, or FGF signaling pathways.

9. The zebrafish coronary microcirculation injury model constructed by the method of constructing an enzyme-based zebrafish coronary microcirculation injury model according to any one of claims 1 to 5, is used in studying the mechanism of action of drugs that promote and / or inhibit coronary vascular repair.

10. A method for screening drugs that promote and / or inhibit coronary vascular repair, characterized in that, Includes the following steps: ①Preparation of injectable drugs; ② The drug injection is injected intraperitoneally into the zebrafish coronary microcirculation injury model constructed by the method for constructing a zebrafish coronary microcirculation injury model based on enzyme stripping as described in any one of claims 1 to 5, and then the coronary blood vessel regeneration is detected.

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