Preparation method and application of aortic aneurysm stem cell fixed-point administration animal model
Through ultrasound-guided injection technology, a targeted drug delivery animal was established in aortic aneurysm stem cells, which solved the problem that drugs in the prior art are difficult to target aortic aneurysm, and achieved more effective aortic wall remodeling and drug screening.
Patent Information
- Application Number
- CN202510402971.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-01
- Publication Date
- 2025-06-20
AI Technical Summary
The prior art is difficult to target or enrich therapeutic drugs into aortic aneurysms through conventional routes of administration, resulting in the inadequate realization of therapeutic potential and the lack of animal models suitable for other routes of administration.
Ultrasound-guided injection technology was used to insert an ultrasound-guided injection device into the target area of the aortic aneurysm model animal, and perform targeted drug administration, establish an animal model for aortic aneurysm stem cells, and evaluate the aortic wall remodeling effect of the drug in combination with pathological analysis methods.
Through site-based drug delivery technology, the aortic wall can be reshape more effectively, prevent the occurrence of dissection aneurysms, and improve the therapeutic effect of drugs, providing a new idea and drug screening method.
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Figure CN120168171A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of animal model preparation and pathological analysis, and particularly relates to a method for preparing an animal model for targeted drug administration of aortic aneurysm, a pathological analysis method for evaluating the aortic wall remodeling effect of drugs using the animal model, and a method for screening drugs for preventing dissecting aneurysm and / or remodeling the aortic wall of aortic aneurysm. Background Art
[0002] Aortic aneurysm refers to the aneurysmal dilation of the aorta, exceeding 50% of the normal blood vessel diameter. When the blood flow breaks through the intima and tears the vessel wall into the middle layer, aortic dissecting aneurysm is formed. Although the incidence of aortic aneurysm is lower than that of common cardiovascular diseases such as hypertension and coronary heart disease, with the increase of age, the risk of the disease is also getting higher and higher. In recent years, the number of patients with aortic aneurysm has been increasing continuously. According to statistics, the incidence of aortic aneurysm shows an obvious upward trend with the increase of age, and the elderly over 60 years old are more likely to suffer from this disease. The early symptoms of aortic aneurysm are latent and the clinical manifestations are complex. Once dissection occurs, it is extremely easy to rupture, and the mortality rate after rupture is >80%, which is a very dangerous disease. Aortic aneurysm can be further divided into thoracic aortic aneurysm and abdominal aortic aneurysm according to the location of occurrence.
[0003] The main causes of aortic aneurysm mainly include hypertension, atherosclerosis, genetic diseases, infectious diseases (such as syphilis) and trauma, etc. At present, the treatment drugs for aortic aneurysm are mainly used to control blood pressure, heart rate, etc., in order to reduce the risk of aortic aneurysm rupture, and there is no drug that specifically improves the vascular pathological manifestations of aortic aneurysm. Once dissecting aneurysm occurs, in addition to using drugs to control blood pressure and heart rate, the main treatment methods include surgical treatments such as aortic replacement and endovascular repair.
[0004] At present, the treatment drugs for aortic aneurysm are often administered through conventional administration routes (for example, intravenously, orally). The disadvantage of these administration routes is that it is difficult to target or enrich the treatment drugs (especially cell preparations) into the aortic aneurysm to achieve directional treatment. Therefore, the existing administration routes do not fully exert the therapeutic potential of the drugs. However, in order to develop other potential administration routes for treatment drugs and then screen the treatment drugs suitable for these potential administration routes, first of all, a new aortic aneurysm drug administration model needs to be established to accurately evaluate the efficacy of the drugs under other administration routes. At present, there is a lack of animal models for other administration routes in this field.
[0005] In addition, the pathological analysis of disease models is also crucial for the evaluation of drug efficacy. There is also a need in this field for a pathological analysis and efficacy evaluation method for aortic aneurysm and suitable for specific administration routes. Summary of the Invention
[0006] To solve at least one of the above problems existing in the prior art, the present invention provides an animal model for targeted drug delivery of aortic aneurysm and a preparation method thereof, as well as a pathological analysis method and a drug screening method for evaluating the efficacy of drugs for treating aortic aneurysm by using the animal model for targeted drug delivery of aortic aneurysm.
[0007] In the first aspect, a preparation method of an animal model for targeted drug delivery of aortic aneurysm (hereinafter sometimes simply referred to as "the targeted drug delivery model of the present invention") is provided, including the following steps: 1) Establish an animal model of aortic aneurysm; 2) Determine the target area for targeted drug delivery of the aortic aneurysm in the aortic aneurysm model animal obtained through step 1); 3) Insert an ultrasound-guided injection device into the adventitia of the aortic aneurysm in the target area of the aortic aneurysm model animal under the guidance of an ultrasound imaging system to obtain an animal model for targeted drug delivery of aortic aneurysm.
[0008] In one embodiment, the aortic aneurysm may be an aortic aneurysm caused by hypertension, atherosclerosis, or genetic diseases. In a preferred embodiment, the aortic aneurysm may be a thoracic aortic aneurysm. In a specific embodiment, the aortic aneurysm may be an aortic dissection aneurysm. In a preferred embodiment, the aortic aneurysm may be a thoracic aortic aneurysm with a risk of developing dissection aneurysm. Preferably, the aortic aneurysm is a thoracic aortic aneurysm caused by Marfan syndrome.
[0009] Therefore, in a preferred embodiment, the aortic aneurysm animal model is a thoracic aortic aneurysm model with a risk of developing dissection aneurysm. Preferably, the thoracic aortic aneurysm model can be constructed using FBN1 gene mutant mice. FBN1 gene mutant mice refer to the mutation of the mouse fibrillin-1 gene (FBN1), which causes a point mutation in the amino acid of fibrillin-1. Specifically, cysteine (C) at position 1041 can be mutated to glycine (G). The FBN1 gene mutant mice are named Fbn1 C1041G / + . FBN1 is the earliest identified pathogenic gene for thoracic aortic aneurysm. FBN1 mutation can cause a connective tissue disease called Marfan syndrome, and about 75% of patients have thoracic aortic aneurysm and dissection. The lifespan of FBN1 gene heterozygous mutant mice is normal. The aortic structure is normal within 2 months after birth, and then progressive lesions occur in the middle layer of the blood vessels. This model is used as an animal model of Marfan syndrome for the study of thoracic aortic aneurysm and dissection.
[0010] In one embodiment, the target area for targeted drug delivery determined in step 2) is the adventitia area at the junction of the anterior wall of the innominate artery and the aortic arch.
[0011] In one embodiment, in the ultrasound-guided injection step of step 3), the ultrasound-guided injection device is inserted into the adventitia at the junction of the anterior wall of the innominate artery and the aortic arch. The innominate artery (brachiocephalic trunk) is the largest first branch of the aortic arch, and the junction of its anterior wall and the aortic arch is a common involved area of aortic dissection (AD). The dissection here may extend to the ascending aorta, aortic arch or descending aorta, affecting the blood supply of the brachiocephalic vessels. Once ruptured, it may lead to massive hemorrhage, directly threatening the patient's life.
[0012] Clinically, Stanford type A aortic dissection (referred to as type A dissection) is the most dangerous aortic disease, accounting for 66% of the cases, involving the ascending aorta or aortic arch (such as the origin of the innominate artery), belonging to the high-risk type, which is a difficult point in clinical treatment and cannot be completely cured.
[0013] In a specific embodiment, the injection needle insertion direction of the ultrasound-guided injection device is at an angle of 45° with the chest wall of the animal. At such an angle, the injection needle insertion direction is consistent with the probe direction, which can reduce errors and accurately reach the adventitia of the ascending aorta at the centripetal end of the opening of the brachiocephalic trunk branch of the ascending aorta after the injection needle passes through the chest wall.
[0014] In a second aspect, a method for preparing an animal model for targeted drug delivery of aortic aneurysm stem cells is provided, including the following steps: 1) Establish an animal model of aortic aneurysm; 2) Determine the target area for targeted drug delivery of the aortic aneurysm in the aortic aneurysm model animal obtained through step 1); 3) Under the guidance of an ultrasound imaging system, insert the ultrasound-guided injection device into the adventitia of the aortic aneurysm in the target area of the aortic aneurysm model animal, and inject vascular stem cells into the target area through the ultrasound-guided injection device, thereby obtaining an animal model for targeted drug delivery of aortic aneurysm stem cells.
[0015] In one embodiment, the vascular stem cells may be Sca-1⁺ vascular stem cells. Sca-1⁺ vascular stem cells (stem cell antigen-1 positive cells) are a type of vascular wall stem cells with differentiation potential, mainly present in the vascular wall, and have the potential to differentiate into endothelial cells and smooth muscle cells under injury stress. Due to the limitation of the administration route, it is currently not known whether vascular stem cells have the effect of remodeling the aortic wall in aortic aneurysm through administration methods other than intravenous administration, so as to achieve the effect of treating aortic aneurysm.
[0016] It should be noted that the various embodiments in the first aspect above are also applicable to the various embodiments in the second aspect.
[0017] In a third aspect, a pathological analysis method for in vivo evaluating the effect of a drug on aortic wall remodeling in aortic aneurysm (hereinafter sometimes simply referred to as "the pathological analysis method of the present invention") is provided, including the following steps: 1) Obtain an aortic aneurysm targeted drug delivery model animal through the preparation method of the above-mentioned targeted drug delivery model, and inject the drug to be tested into the target area of the aortic aneurysm model animal through an ultrasound-guided injection device; 2) Sacrifice the animal at a specified sampling time point to obtain an aortic sample; 3) Transparently process the aortic tissue sample, and perform fluorescence imaging on the transparently processed aortic tissue sample through a light sheet microscope to obtain a fluorescence image; 4) Evaluate the effect of the drug to be tested on aortic wall remodeling in aortic aneurysm by using the change in aortic diameter measured by ultrasound before and after injecting the candidate drug.
[0018] In one embodiment, the drug to be tested may be a potential therapeutic drug for aortic aneurysm with an aortic wall remodeling effect, including the vascular stem cells (e.g., Sca-1⁺ vascular stem cells) as described above, or non-stem cell drugs, such as non-stem cell cell preparations, chemical drugs (synthetic drugs, natural drug extracts), biological drugs (protein-based, polypeptide-based, nucleic acid-based, antibody-based, blood products, etc.), traditional Chinese medicines, etc.
[0019] In one embodiment, step 2) further includes: after obtaining the aortic tissue sample, measuring and recording the part with the largest outer diameter of the aorta.
[0020] Tissue clearing is a method of applying water-soluble organic solvents or hydrophilic reagents to transparently process fixed tissues, applying a high refractive index medium to match the tissue refractive index, reducing light scattering, making the tissue optically transparent, and thereby increasing the imaging depth and image contrast. It is used to observe the expression and localization of target proteins in relatively large tissue samples. There are mainly three tissue clearing methods: oil-based, hydrogel-based, and water-based. CUBIC solution belongs to a highly hydrated degreasing transparent reagent. Since the fluorescent protein molecules in tissues carry hydrophilic groups, compared with oil-based solvents, hydrophilic solvents are more conducive to the preservation of fluorescent protein signals and are more suitable for the clearing of tissue samples with inherent fluorescence.
[0021] In one embodiment, step 3) includes the following steps: Place the obtained aortic sample in 4% paraformaldehyde fixative for fixation, soak the fixed aortic sample in CUBIC-L solution for degreasing, perform immunostaining on the decolorized aortic sample, and soak the immunostained aortic sample in CUBIC-RA solution again for transparentization treatment.
[0022] Preferably, the aortic tissue sample is incubated in the above clearing reagent (i.e., CUBIC-L solution and CUBIC-RA solution) at room temperature in a shaker until it becomes transparent, and more preferably, incubated at 37°C. For the aortic tissue of mice, preferably, the soaking time of the aortic tissue sample in the CUBIC-L solution is 2 days, and the soaking time in the CUBIC-RA solution is 2 days.
[0023] In a preferred embodiment, the immunostaining includes immunostaining of the vascular intima. In a specific embodiment, the vascular intima staining includes visualizing the vascular intima using CD31 as a marker, for example, by an anti-CD31 antibody, such as visualizing the vascular intima by immunofluorescence staining.
[0024] In one embodiment, in step 4), the change in the aortic diameter measured by ultrasound refers to the change in the diameter size of the aortic blood vessel measured by an ultrasound imaging system. Preferably, the ultrasound measurement is performed at the ascending aorta and aortic arch of the aorta. Further preferably, in step 4), it also includes using the change in the elastic index measured by ultrasound to evaluate the remodeling effect of the test drug on the aortic wall of the aortic aneurysm. Specifically, the elastic index is the Global Radial Strain, which is used to reflect the elastic deformation ability of the blood vessel.
[0025] Therefore, in a preferred embodiment, the pathological analysis method of the present invention further includes step 5): observing the number and distribution of Sca-1⁺ vascular stem cells in the aortic wall in the fluorescence image to evaluate the potential of the test drug for aortic wall remodeling.
[0026] After applying the drug, by observing the number and distribution of Sca-1⁺ vascular stem cells in the blood vessel wall, it can be understood whether the drug has a regulatory effect on the self-renewal and differentiation ability of Sca-1⁺ vascular stem cells, which helps to evaluate the potential of the drug for aortic wall remodeling.
[0027] In a fourth aspect, a method for screening drugs for preventing aortic dissection and / or remodeling the aortic wall of an aortic aneurysm (hereinafter sometimes simply referred to as "the drug screening method of the present invention") is provided, including the following steps: 1) Obtaining an aortic aneurysm targeted drug administration model animal through the preparation method of the above targeted drug administration model, and injecting the candidate drug into the target area of the aortic aneurysm model animal through an ultrasound-guided injection device; 2) Repeating the operation of step 1) using Sca-1 + vascular stem cells; 3) At a specified time point, with Sca-1 +Aortic aneurysm animals treated with vascular stem cells are used as a positive control group, and it is evaluated whether the candidate drug can be used as a drug for preventing dissecting aneurysm and / or remodeling the aortic wall of aortic aneurysm by comparing with the positive control group.
[0028] In a specific embodiment, the method further includes: measuring the diameter size of the aortic blood vessel by an ultrasonic imaging system and obtaining ultrasonic images before injecting the candidate drug and Sca-1 + vascular stem cells and at specified time points. Preferably, the aorta measured by ultrasound is the ascending aorta and the aortic arch.
[0029] In step 3), the evaluation further includes evaluating the long-term therapeutic effect of the candidate drug on aortic aneurysm and / or the preventive effect on dissecting aneurysm through the change in the aortic diameter measured by ultrasound. Preferably, the evaluation further includes evaluating the long-term therapeutic effect of the candidate drug on aortic aneurysm and / or the preventive effect on dissecting aneurysm through the change in the elastic index measured by ultrasound.
[0030] In a preferred embodiment, the long-term therapeutic effect refers to the remodeling effect of the aortic wall of aortic aneurysm (based on the change in the aortic diameter measured by ultrasound, or based on both the change in the aortic diameter and the change in the elastic index) at least 4 weeks after treatment, preferably 4 to 8 weeks. The preventive effect on dissecting aneurysm means the ability to prevent the occurrence of aortic aneurysm dissection in the aortic aneurysm model animals. In this case, the specified time point is at least 4 weeks after injecting the candidate drug or Sca-1 + vascular stem cells. If the long-term therapeutic effect of the candidate drug on aortic aneurysm or the preventive effect on the occurrence of dissecting aneurysm is close to or exceeds that of Sca-1 + vascular stem cells (that is, there is no statistical difference between the two, or the candidate drug is statistically significantly superior to Sca-1 + vascular stem cells), then the candidate drug can be used as a drug for preventing dissecting aneurysm and / or remodeling the aortic wall of aortic aneurysm.
[0031] Advantages of the present invention: 1. The present invention prepared an animal model for targeted administration of stem cells for aortic aneurysm by combining an ultrasound-guided method, and found that compared with the mouse tail injection route (i.e., the intravenous administration route), administering Sca-1 + vascular stem cells through targeted administration of stem cells for aortic aneurysm can more effectively remodel the aortic wall and prevent the occurrence of dissecting aneurysm, demonstrating the advantages of targeted administration of stem cells for aortic aneurysm over the traditional intravenous administration method.
[0032] 2. The present invention combines ultrasound-guided technology and tissue clearing technology to form a pathological analysis method for evaluating the aortic wall remodeling effect of drugs using an animal model, and evaluates the therapeutic effect of drugs from two aspects: the physical properties of blood vessels and the remodeling of blood vessels by vascular stem cells, providing a relevant research animal model and research method for the evaluation and screening of drugs for the treatment of aortic aneurysms.
[0033] 3. On the basis of the pathological analysis method of the present invention, a method for screening drugs for preventing dissecting aneurysm and / or remodeling the aortic wall of aortic aneurysm is further formed, thereby providing new ideas and drug screening methods for the clinical treatment of aortic aneurysm. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 The figure illustrates some of the experimental equipment and operations used in the examples, where Figure 1 A shows a color Doppler ultrasound imaging system integrating an ultrasound-guided injection device and a Vevo rail system, Figure 1 B shows the syringe control device of the ultrasound-guided injection device, Figure 1 C shows the optimal positioning of the ultrasound-guided needle injection of a mouse on the animal platform.
[0035] Figure 2 is a schematic diagram showing the ultrasound-guided injection operation in Example 3, where Figure 2 A and Figure 2 B are long-axis views showing two-dimensional ultrasound images of the aortic arch, Figure 2 A shows that the injection needle (with the tip facing down) is aligned with the probe direction and inserted into the chest wall at a 45° angle, and injected into the adventitial region at the junction of the innominate artery and the anterior wall of the aortic arch. The yellow arrow indicates the position of the needle, and the red dotted line indicates the boundary of the injected artery; Figure 2 B shows that when the stem cells are slowly injected, the cells are delivered to the adventitial region near the junction of the innominate artery and the anterior wall of the aortic arch; Figure 2 C is a schematic diagram of the aortic injection site, where a represents the ascending aorta, b represents the aortic arch, and c represents the descending aorta.
[0036] Figure 3 is a light sheet microscopy image of the thoracic aorta, where Figure 3 A is an image of the thoracic aorta obtained by light sheet microscopy 4 weeks after ultrasound-guided injection of mCherry-labeled Sca1⁺ stem cells into the aorta of Fbn1C 1041G / + mice, Figure 3 B is a longitudinal view along the Figure 3 yellow dotted line in Figure 3 A, Figure 3 C is an enlarged view of the area within the yellow dotted box in Figure 3In A-3C, the punctate red fluorescence represents Sca-1⁺ stem cells, and the yellow arrows indicate the migration of stem cells to the media or intima of blood vessels.
[0037] Figure 4 The ultrasonic images of the stem cell tail vein injection group and the stem cell fixed-point aortic injection group are shown. Fbn1C 1041G / + The dilation conditions of the two groups of mice at the measurement positions of the ascending aorta and aortic arch of mice (I is the measurement site of the ascending aorta, II is the measurement site of the aortic arch) are shown. Both groups were measured at baseline (day 0) and 4 weeks after injection.
[0038] Figure 5 The statistical chart showing the changes in the aortic lumen measured by ultrasound of the stem cell tail vein injection group and the stem cell fixed-point aortic injection group is shown. Among them Figure 5 A shows that compared with the stem cell tail vein injection group (n = 5), the diameter of the ascending aorta lumen in the stem cell fixed-point aortic injection group (n = 6) was significantly reduced (P < 0.05, independent samples t-test); Figure 5 B shows that compared with the stem cell tail vein injection group (n = 5), the diameter of the aortic arch lumen in the stem cell fixed-point aortic injection group (n = 6) showed a decreasing trend.
[0039] Figure 6 The analysis chart showing the change trend of the Global Radial Strain values measured by ultrasound before and one month after injection in the stem cell tail vein injection group and the stem cell fixed-point aortic injection group, and the comparison result of the difference between the ultrasound strain values one month after injection and one month before injection. Specific implementation manners
[0040] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention, and they should not be construed as limitations to the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for the purpose of description and cannot be construed as indicating or implying relative importance.
[0041] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.
[0042] Example 1 Establishment of a thoracic aortic aneurysm model Measure using a high-resolution small animal ultrasound imaging system (Vevo 2100 Imaging System, FUJIFILM VisualSonics Inc., USA) Fbn1 C1041G / + the diameter of the aortic vessel of mice (purchased from Model Animal Research Center, https: / / www.modelorg.com / portal / article / index / id / 10625 / post_type / 3.html). Then Fbn1 C1041G / + Anesthetize the mice with isoflurane and fix them in the supine position on a special operating table for mice. Place the snout of the mice inside the anesthesia mask and adjust the amount of isoflurane to 1.5% - 2.0%. Fix the mice in the supine position and perform transthoracic echocardiography. Detect the diameter of the thoracic aortic blood vessel of the mice by ultrasound and capture clear and stable ultrasound images.
[0043] Example 2 Sca-1 + Preparation of vascular stem cells After sacrificing 8 - 10-week-old C57BL / 6J mice (purchased from Vital River Laboratories) by anesthesia, isolate the adventitial tissue of the aorta under a microscope, cut it into pieces about 0.5 mm in size, disperse and spread them on the bottom of a culture flask coated with gelatin. After 3 hours, add DMEM / F12 medium containing 10% FBS, streptomycin / penicillin 100 U / mL, and LIF (10 ng / ml). Keep cell culture in an incubator at 37°C and 5% CO2, and change the medium every 3 days. When reaching 80 - 100% confluence, screen through Sca-1 + immunomagnetic beads (Miltenyi Biotec, catalog number 130 - 123 - 124) to screen and collect Sca-1 + cells, continue to culture them and perform lentiviral transfection of mCherry on them, and amplify and culture the cells after successful transfection and screening.
[0044] Example 3 Preparation of a model for targeted drug delivery of aortic aneurysm stem cells under ultrasound guidance Apply an ultra-high-resolution small animal color Doppler ultrasound imaging system, Vevo orbital system, and ultrasound-guided injection needle device (Vevo 2100 Imaging System, FUJIFILM VisualSonics Inc., USA), use an MS-400 probe with a central frequency of 40 MHz for the probe. Use a BD 29G micro-injection needle. Fbn1 C1041G / +The mice were anesthetized with isoflurane and fixed in the supine position on a special operating table for mice. The nasal and oral parts of the mice were placed inside the anesthesia mask, and the amount of isoflurane was adjusted to 1.5% - 2.0%. The chest wall was depilated, and the probe was placed on the right side of the sternum to display the long-axis section of the aortic arch. A syringe containing 25 μl of the suspension of Sca-1 vascular wall stem cells expressing mCherry prepared in Example 2 was fixed on the ultrasound-guided injection needle device. The direction of the injection needle was the same as that of the probe and was at a 45° angle to the chest wall. Observe the screen, slowly guide the injection needle to make it enter the field of view, adjust the injection needle to enter the focal plane, and make the tip of the injection needle overlap with the guiding needle path marking line. When the injection needle approaches the skin edge, quickly move the injection needle to pass through the chest wall to the adventitial part of the ascending aorta at the centripetal end of the branch opening of the brachiocephalic trunk of the ascending aorta, push the syringe to inject the suspension into the target area. Slowly withdraw the injection needle, and perform routine disinfection on the puncture injection site (see 6 and + ). Figure 1 and Figure 2 ).
[0045] Example 4 Transparentization and Imaging of Aortic Tissue Four weeks after injecting Sca-1 + vascular wall stem cells according to the steps of Example 3, the mice were sacrificed, and the aortic tissue was removed after perfusion, fixed in 4% paraformaldehyde for 4 hours, and washed with PBS 1 h × 3 times. Subsequently, the blood vessels were placed in CUBIC-L solution (10 w / v% N-butyl diethanolamine (MACKLIN, N802391) and 10 w / v% Triton X-100 (Sigma-Aldrich, T9284) dissolved in ddH2O), and transparentized at 37°C on a vertical shaker for 2 days. Washed with PBS at room temperature 2 h × 3 times, incubated with the primary antibody (CD31 (R&D, AF3628, 1:50)) at 4°C on a vertical shaker for 24 h, and washed with PBS at room temperature 1 h × 3 times. Incubated with the secondary antibody donkey anti-goat (Invitrogen, A11055, 1:800) at room temperature for 3 - 4 h, washed with PBS 1 h × 3 times, and then transferred to CUBIC-RA solution (45 w / v% Antipyrine (MACKLIN, A800873) and 30 w / v% Nicotinamide (MACKLIN, N814605) dissolved in an appropriate amount of ddH2O. After mixing and dissolving, add 0.5 w / v% N-butyl diethanolamine (MACKLIN, N802391) and mix well) for transparentization for more than 2 days. After transparentization, it was embedded in agarose gel, fixed on the holder of the light sheet microscope, and 3D scanned (light sheet microscope (Bruker Luxendo, MuVi-SPIM)) for photography. Figure 3It is a light sheet imaging map 4 weeks after injecting mCherry-labeled Sca-1⁺ stem cells, as Figure 3 shown in A-3C, the Sca-1⁺ stem cells migrate to the middle or inner layer of blood vessels.
[0046] Example 5 Sca-1 + Comparison between the mouse tail injection model of vascular wall stem cells and the ultrasound-guided stem cell targeted drug delivery model Sca-1 + Vascular wall stem cells are administered by ultrasound-guided stem cell targeted drug delivery as described in Example 3. A suspension of vascular wall stem cells containing 1×10 6 expressing mCherry prepared in Example 2 diluted in 100 μl of DMEM + is injected into the mouse tail as a control. Four weeks after injection of the two groups of mice, as described in Example 1, the diameter of the mouse aorta is measured using a high-resolution small animal ultrasound imaging system (Vevo 2100 Imaging System, FUJIFILM VisualSonics Inc., USA) Fbn1 C1041G / + At two sites of the aorta, the lumen diameter (at the maximum diameter) measurement images are shown, Fbn1 C1041G / + and the statistical results of the dilation changes at two sites in the two groups 4 weeks after injection are shown. The results show that at the ascending aorta and aortic arch, the therapeutic effect of aortic stem cell targeted drug delivery on aortic dilation (reduction in the degree of dilation) is better than mouse tail injection. Given the greater risk of dissecting aortic aneurysm at these two sites, it indicates that stem cell targeted drug delivery is more beneficial for preventing thoracic dissecting aortic aneurysm compared to the mouse tail injection route of administration. Figure 4 In the ultrasound two-dimensional speckle tracking image, it can be seen that the vascular wall contains many evenly distributed acoustic speckles, and these acoustic speckles move synchronously with the tissue. The 2D-ST technique is used to identify these speckles in the ultrasound image and track the movement of the speckles, thereby obtaining the movement parameters of the tissue as elastic indices (for example, the overall radial strain value) (see, Xu Qing, Liu Lina, Song Yanting, et al. Methodological study on analyzing the stiffness of mouse carotid artery by ultrasound two-dimensional speckle tracking technique [J]. Journal of Capital Medical University, 2018, 39(06): 883-888). Figure 5
[0047] Figure 6 The figure shows the analysis of the changing trend of the Global Radial Strain values measured by ultrasound before injection and one month after injection in the group of rats administered with stem cells via the tail and the group of rats administered with stem cells at the fixed point of the aorta. It also shows the comparison result of the difference between the ultrasound strain values one month after injection and those one month before injection. The data shows that the tissue strain values in the group administered with stem cells at the fixed point showed an increasing trend after injection (n = 3), suggesting that ultrasound-guided fixed-point injection of stem cells may have a beneficial effect on the recovery of tissue elasticity. The direction of the change provides a reference basis for further exploring the mechanism of action of ultrasound-guided fixed-point injection of stem cells.
[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for preparing an aortic aneurysm fixed-point drug delivery animal model, comprising the following steps: 1) Establish an aortic aneurysm animal model; 2) determining the target area of aortic aneurysm targeted drug delivery in the aortic aneurysm model animal obtained in step 1); 3) Under the guidance of the ultrasound imaging system, an ultrasound-guided injection device is inserted into the aortic aneurysm adventitia of the target area of the aortic aneurysm model animal to obtain an aortic aneurysm targeted drug delivery animal model.
2. The aortic aneurysm fixed-point drug delivery animal model according to claim 1, characterized in that: The aortic aneurysm is caused by hypertension, atherosclerosis, aortic media degeneration, genetic disease, or congenital aortic malformation.
3. The aortic aneurysm fixed-point drug delivery animal model according to claim 1, characterized in that: The target area for the targeted drug delivery determined in step 2) is the adventitia area at the junction of the innominate artery and the aortic arch.
4. The aortic aneurysm fixed-point drug delivery animal model according to claim 3, characterized in that: In the ultrasound-guided injection step of step 3), the ultrasound-guided injection device is inserted into the adventitia at the junction of the anterior wall of the innominate artery and the aortic arch.
5. The aortic aneurysm fixed-point drug delivery animal model according to claim 1, characterized in that: The injection needle of the ultrasound-guided injection device was inserted at a 45° angle to the animal's chest wall.
6. A pathological analysis method for evaluating the effect of a drug on aortic aneurysm and aortic wall remodeling using an animal model, comprising the following steps: 1) obtaining an aortic aneurysm fixed-point drug delivery model animal by the method for preparing an aortic aneurysm fixed-point drug delivery animal model according to any one of claims 1 to 5, and injecting the drug to be tested into the target area of the aortic aneurysm model animal through an ultrasound-guided injection device; 2) Animals were sacrificed at designated sampling time points to obtain aortic samples; 3) performing a transparent treatment on the aortic tissue sample, and performing fluorescence imaging on the transparent aortic tissue sample by light sheet microscopy to obtain a fluorescence image; 4) Using ultrasound to measure the change in aortic diameter before and after injection of the candidate drug to evaluate the remodeling effect of the candidate drug on the aortic wall of the aortic aneurysm.
7. The pathological analysis method according to claim 6, characterized in that: The method also includes step 5): observing the number and distribution of Sca-1⁺ vascular stem cells in the aortic vascular wall in the fluorescent image obtained in step 3) to evaluate the potential of the test drug to remodel the aortic wall.
8. A method for screening a drug for preventing dissecting aneurysm and / or remodeling the aortic wall of aortic aneurysm, comprising the following steps: 1) obtaining an aortic aneurysm targeted drug delivery model animal by the method for preparing an aortic aneurysm targeted drug delivery animal model according to any one of claims 1 to 5, and injecting the candidate drug into the target area of the aortic aneurysm model animal by an ultrasound-guided injection device; 2) Using Sca-1 + Repeat step 1) for vascular stem cells; 3) At the designated time point, the Sca-1 + The aortic aneurysm animals treated with vascular stem cells serve as a positive control group, and whether the candidate drug can be used as a drug for preventing dissecting aneurysms and / or remodeling the aortic wall of aortic aneurysms is evaluated by comparison with the positive control group.
9. The method for screening drugs for preventing dissecting aneurysms and / or remodeling aortic walls of aortic aneurysms according to claim 8, characterized in that: The method further comprises: injecting the candidate drug and Sca-1 + Before and at indicated time points in the vascular stem cell implantation procedure, ultrasound measurements of aortic vessel lumen diameter dimensions were obtained.
10. The method for screening drugs for preventing dissecting aneurysms and / or remodeling aortic walls of aortic aneurysms according to claim 8 or 9, characterized in that: The evaluation also includes evaluating the long-term therapeutic effect of the candidate drug on aortic aneurysm and / or the preventive effect of dissecting aneurysm by measuring the change of aortic diameter by ultrasound. If the long-term therapeutic effect of the candidate drug on aortic aneurysm or the effect of preventing dissecting aneurysm is close to or exceeds that of Sca-1 + If the candidate drug contains vascular stem cells, the candidate drug can be used as a drug for preventing dissecting aneurysms and / or remodeling the aortic wall of aortic aneurysms.