BMP9-overexpressed spontaneous hypertension rat model as well as construction method and application thereof

Abstract

The invention discloses a spontaneous hypertension rat model for overexpressing BMP9 as well as a construction method and application thereof, creatively discovers that the spontaneous hypertension rat model can be successfully constructed by overexpressing BMP9 in a rat body for the first time, and provides an ideal experimental platform for research and development of new hypertension drugs. The invention lays a foundation for accelerating the research and development process of new hypertension drugs and improving the research and development efficiency, and has a good application prospect and important transformation significance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a spontaneously hypertensive rat model overexpressing BMP9, and a construction method and application thereof. Background Art

[0002] Hypertension is one of the most common cardiovascular diseases worldwide and is closely related to serious health problems such as heart disease, stroke, and kidney disease. As a complex multifactorial disease, the pathogenesis of hypertension involves the interaction of multiple systems such as genetics, nerves, endocrine system, and blood vessels. Although there are many antihypertensive drugs available, some patients still have unsatisfactory blood pressure control, and the pathogenesis of hypertension has not yet been fully elucidated. Therefore, in-depth research on the pathophysiological mechanisms of hypertension is crucial. Due to the many limitations of human research, such as ethical constraints and the difficulty in precisely controlling experimental conditions, animal models have become a key tool for in-depth exploration of the pathogenesis of hypertension.

[0003] The spontaneously hypertensive rat (SHR) model has become an ideal tool for studying hypertension and its complications due to its high similarity to human essential hypertension. Through the SHR model, relevant researchers can better understand the genetic, molecular and physiological mechanisms of hypertension, thereby promoting the development of new treatments. In addition, the SHR model plays a vital role in the development and evaluation of hypertension drugs. The development of new drugs for the treatment of hypertension requires rigorous pre-clinical trial evaluation to determine the effectiveness, safety and mechanism of action of the drugs. The SHR model can simulate the pathophysiological process of human hypertension, providing an ideal experimental platform for drug development, accelerating the development of new hypertension drugs and improving research and development efficiency.

[0004] The causes of spontaneous hypertension are very complex. Currently known common risk factors include high sodium intake, obesity and metabolic disorders. However, the single or combined effects of these risk factors can only induce stable hypertension in some animals and humans. Taking animal models as an example, the incidence of hypertension in rats induced by a long-term (usually 10-16 weeks) high-salt diet and high-fat, high-sugar diet is less than 50%, and after stopping the above diet, most of the hypertension can be significantly reduced or returned to normal levels. It can be seen that this simple dietary intervention method cannot effectively replicate the hypertension that occurs in humans. The hypertension model induced by pressor hormones such as glucocorticoids, mineralocorticoids and angiotensin can only simulate secondary hypertension in humans. Therefore, the development of a new SHR model has important practical significance and clinical needs. Summary of the Invention

[0005] In light of this, the present invention aims to provide a spontaneously hypertensive rat model overexpressing BMP9, as well as its construction method and application. This invention, for the first time, creatively discovered that overexpressing BMP9 in rats can successfully establish a spontaneously hypertensive rat model. This provides an ideal experimental platform for the research and development of new hypertension drugs, laying the foundation for accelerating the development of new hypertension drugs and improving their efficiency, and has promising application prospects.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0007] The first aspect of the present invention provides a method for constructing a spontaneously hypertensive rat model overexpressing BMP9.

[0008] Furthermore, the method comprises the following steps:

[0009] (1) Construction of a recombinant vector overexpressing BMP9;

[0010] (2) A spontaneously hypertensive rat model overexpressing BMP9 was prepared by microinjection.

[0011] Furthermore, in the recombinant vector overexpressing BMP9, the liver-specific ALB gene promoter drives the high expression of BMP9 specifically in rat liver.

[0012] In the present invention, BMP9 refers to human bone morphogenetic protein 9 (BMP9), whose corresponding Gene ID is 2658. BMP9 belongs to the transforming growth factor-β (TGF-β) superfamily. Currently, there are no studies or reports related to the establishment of spontaneously hypertensive rat models using BMP9.

[0013] In the present invention, the liver-specific ALB gene promoter refers to a DNA sequence that can regulate the initiation of albumin (ALB) gene expression and is liver-specific, that is, it mainly initiates gene transcription activity in liver cells, so that the regulated gene can be specifically expressed in the liver.

[0014] In some embodiments, the liver-specific ALB gene promoter comprises a core promoter element and a cis-acting element.

[0015] Among them, the core promoter elements include TATA box, CAAT box and other core promoter elements. These elements can bind to transcription factors to accurately determine the transcription start site, promote RNA polymerase II and related transcription factors to gather in the promoter region, and start the gene transcription process.

[0016] Cis-acting elements contain multiple cis-acting elements, such as hepatocyte nuclear factor (HNF) binding sites. Hepatocyte nuclear factors such as HNF-1 and HNF-3 can specifically bind to these sites, thereby activating the transcriptional activity of the ALB gene promoter and ensuring that the gene is efficiently expressed only in liver cells.

[0017] In the present invention, microinjection is a technique that uses a microscope to precisely inject trace amounts of substances into tiny structures such as cells, embryos, or tissues. The basic principle is as follows: Under high-power microscopic observation, using an extremely fine glass microneedle (typically with an outer diameter of approximately 0.5-10 microns) as an injection tool, a trace amount of substance (such as DNA, RNA, protein, or drug) is injected directly into a specific location of a target cell or embryo via manual or automated control. This method can overcome the limitations of cell membranes or tissue barriers, allowing the injected substance to directly enter the target, thereby achieving precise regulation of the genetic material and physiological functions of cells or embryos.

[0018] In some embodiments, the microinjection method is commonly used to prepare transgenic animal models. Specifically, vector DNA containing a target gene is injected into the pronucleus of a fertilized egg, allowing the target gene to integrate into the embryo's genome. After embryo transfer and development, a transgenic animal carrying the exogenous gene is obtained. In a specific embodiment of the present invention, the target gene or exogenous gene is a human BMP9 gene.

[0019] Furthermore, the sequence of the liver-specific ALB gene promoter is shown in SEQ ID NO: 2, and the sequence of BMP9 is shown in SEQ ID NO: 3.

[0020] Furthermore, the recombinant vector for overexpressing BMP9 is a plasmid pcDNA3.1-ALB-BMP9-WT, and its corresponding sequence is shown in SEQ ID NO: 1.

[0021] In the present invention, the recombinant vector is a common tool for carrying and delivering exogenous genes in genetic engineering. A recombinant vector refers to a new DNA molecule formed by artificially connecting an exogenous gene (target gene) to a carrier DNA. It is capable of autonomously replicating in a host cell and allowing the exogenous gene to be expressed in the host cell. The present invention does not particularly limit the specific type of recombinant vector for overexpressing BMP9, and those skilled in the art can prepare a recombinant vector for overexpressing BMP9 based on common knowledge in the art.

[0022] In some embodiments, the components of the recombinant vector include a replication origin, a multiple cloning site, a selection marker gene, a promoter, and a terminator, and the construction process generally includes: selection of the vector and the exogenous gene, enzyme digestion, ligation reaction, host cell transformation, screening, and identification.

[0023] In some embodiments, the vector includes a plasmid vector (one of the most commonly used recombinant vectors, usually a double-stranded circular DNA molecule, present in microbial cells such as bacteria. Plasmid vectors have the advantages of simple operation, easy transformation, and high copy number), a phage vector (a vector constructed based on phage, such as a λ phage vector. Phage vectors can accommodate larger exogenous DNA fragments) and a viral vector (including retroviral vectors, adenoviral vectors, lentiviral vectors, etc. Viral vectors have the advantages of high infection efficiency, the ability to integrate exogenous genes into the host cell genome or highly efficient expression).

[0024] In a specific embodiment of the present invention, the vector is a plasmid vector. Specifically, the plasmid vector is pcDNA3.1. pcDNA3.1 is essentially a circular double-stranded DNA molecule. This structure enables it to exist stably in host cells and replicate independently of the host genome. More specifically, the recombinant vector for overexpressing BMP9 is the plasmid pcDNA3.1-ALB-BMP9-WT, the corresponding sequence of which is shown in SEQ ID NO: 1.

[0025] Furthermore, the preparation of a spontaneously hypertensive rat model overexpressing BMP9 by microinjection comprises the following steps:

[0026] 1) Vasectomy of male rats: SD male rats were subjected to vasectomy to obtain vasectomized male rats;

[0027] 2) Superovulation: Inject hormones into SD female mice to induce superovulation and obtain fertilized eggs;

[0028] 3) Fertilized egg injection: injecting the recombinant vector overexpressing BMP9 into the fertilized egg using a microinjector to obtain an injected fertilized egg;

[0029] 4) Preparation of recipient mice: After mating with the ligated male mice, female SD mice with thrombosis were selected as recipient mice;

[0030] 5) Embryo transplantation: The injected fertilized eggs are transplanted into the ampulla of the oviduct of the recipient mice, and the recipient mice give birth to obtain a spontaneously hypertensive rat model overexpressing BMP9.

[0031] In some embodiments, the hormones used in the superovulation process include, but are not limited to, pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). PMSG has follicle-stimulating hormone (FSH)- and luteinizing hormone (LH)-like activities, promoting the growth and development of ovarian follicles; hCG has LH-like effects, inducing ovulation. Those skilled in the art can perform the superovulation step using common knowledge in the art.

[0032] In some embodiments, superovulation can be performed using the following method: Female rats are superovulated to obtain more fertilized eggs. Pregnant mare serum gonadotropin (PMSG) is first injected to promote follicular growth and development. After a certain interval (usually 48-52 hours), human chorionic gonadotropin (hCG) is then injected to induce ovulation. The dose of injected hormones may vary between rats of different strains and ages, and those skilled in the art can make routine adjustments based on actual conditions.

[0033] In some embodiments, fertilized eggs can be obtained using the following method: At an appropriate time after hCG injection (generally 13-16 hours), female rats are sacrificed, the oviducts removed, and placed in a culture dish containing culture medium. Under a microscope, the oviducts are gently squeezed with forceps to release the fertilized eggs into the culture medium. Fertilized eggs with normal morphology and good development are selected for subsequent microinjection.

[0034] In some embodiments, the following methods can be used for microinjection: (1) Fixing the fertilized egg: Transfer the selected fertilized egg to a culture dish specially used for microinjection, which has been pre-dropped with a culture medium droplet and covered with mineral oil to prevent the culture medium from evaporating. Use an egg-holding needle to fix the fertilized egg under a microscope so that it is in a position convenient for injection. (2) Injecting the expression vector: Aspirate the constructed expression vector DNA solution into a microsyringe. Under a high-power microscope, pass the needle tip of the microsyringe through the zona pellucida and cell membrane of the fertilized egg, and inject an appropriate amount of the expression vector DNA solution into the male pronucleus of the fertilized egg. After the injection is completed, the fertilized egg is briefly cultured in the culture medium to observe its survival, and the surviving fertilized egg is selected for embryo transplantation.

[0035] In some embodiments, embryo transfer can be performed as follows: the injected fertilized egg is aspirated using a transfer tube. Under sterile conditions, the recipient female mouse is anesthetized and secured on an operating table. The abdominal cavity is opened and the fallopian tube is located. The transfer tube is inserted through the fimbria of the fallopian tube and the fertilized egg is slowly injected into the ampulla of the fallopian tube. After the procedure, the wound is sutured and disinfected, and the recipient mouse is returned to its home cage for appropriate care and feeding.

[0036] Furthermore, the hormones are PMSG and HCG hormones;

[0037] Optionally, the dosage of the recombinant vector overexpressing BMP9 is 1-10 ng / μL;

[0038] Optionally, the method further comprises performing genotype identification and / or disease phenotype identification on the obtained spontaneously hypertensive rat model overexpressing BMP9.

[0039] Furthermore, the primer sequences used for genotype identification are shown in SEQ ID NO: 4-5.

[0040] In some embodiments, the genotype identification refers to gene expression detection. When the pups born by the recipient female mice grow to a certain stage (determined according to experimental requirements), relevant tissues (such as tissues where the target gene is expected to be expressed) are collected, and the expression level of the target gene mRNA is detected by real-time quantitative PCR (qPCR). The level of the target gene expression product (protein) is detected by protein blotting (Western blot) to determine whether the target gene is overexpressed in the rat body.

[0041] In some embodiments, disease phenotype identification refers to the assessment of disease characteristics, which involves performing comprehensive physiological, biochemical, and pathological examinations on rats to assess whether they exhibit symptoms and pathological features associated with the target disease (spontaneous hypertension). For example, relevant biochemical indicators in the blood are measured, and pathological morphological changes in tissues and organs are observed. By comparing the rat model with normal rats, it is determined whether the constructed rat model meets the requirements of a disease model.

[0042] The second aspect of the present invention provides a recombinant vector for constructing a spontaneously hypertensive rat model overexpressing BMP9.

[0043] Furthermore, the recombinant vector is the recombinant vector for overexpressing BMP9 described in the first aspect of the present invention.

[0044] In a specific embodiment of the present invention, the recombinant vector for overexpressing BMP9 is plasmid pcDNA3.1-ALB-BMP9-WT, and its corresponding sequence is shown in SEQ ID NO:1.

[0045] The third aspect of the present invention provides a spontaneously hypertensive rat model constructed using the method described in the first aspect of the present invention.

[0046] A fourth aspect of the present invention provides any of the following applications:

[0047] (1) Use of the recombinant vector described in the second aspect of the present invention in constructing a spontaneously hypertensive rat model or preparing an agent for promoting vascular fibrosis;

[0048] (2) Application of the spontaneously hypertensive rat model described in the third aspect of the present invention in the development of new drugs for spontaneous hypertension, screening of antihypertensive drugs, evaluation of the efficacy of antihypertensive drugs, evaluation of the safety of antihypertensive drugs, or research on the pathogenesis of spontaneous hypertension.

[0049] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0050] This invention creatively discovered for the first time that overexpressing BMP9 in rats can successfully construct a spontaneously hypertensive rat model, providing an ideal experimental platform for the research and development of new hypertension drugs, laying the foundation for accelerating the research and development process of new hypertension drugs and improving research and development efficiency. It has good application prospects and important translational significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 : Expression of BMP9 in wild-type rats;

[0052] Figure 2 : Schematic diagram of pcDNA3.1-ALB-BMP9-WT transgenic plasmid;

[0053] Figure 3 : Reaction conditions and reagent ratios for genotyping rats using PCR;

[0054] Figure 4 : Representative gel images of genotyping of transgenic (Tg) rats, with bands indicating Tg-positive rats;

[0055] Figure 5 : RT-PCR identification of BMP9 expression in the livers of rats in the Tg-BMP9 group and the control group;

[0056] Figure 6 : Western-blot analysis of BMP9 expression in the livers of rats in the Tg-BMP9 group and the control group;

[0057] Figure 7 : The relative expression of plasma BMP9 in Tg and WT rats was detected by ELISA;

[0058] Figure 8 : The systolic blood pressure, diastolic blood pressure, and mean arterial pressure of Tg and WT rats at baseline were measured by tail-cuff method, n=10, *P<0.05, **P<0.01, ***P<0.001;

[0059] Figure 9: Spontaneous hypertension in 16-week-old BMP9-overexpressing rats. Implants were used to continuously monitor 24-hour changes in systolic blood pressure (A), diastolic blood pressure (B), and mean arterial pressure (C) in 16-week-old Tg and WT littermates; n = 8, *P < 0.05, **P < 0.01, ***P < 0.001;

[0060] Figure 10 : Biochemical indices of blood and urine in Tg and WT rats. (AE) Serum biochemical index test results, (FL) urine biochemical index test results. Data are expressed as mean ± SD, n = 4-9, ns indicates no statistical significance.

[0061] Figure 11 : Cardiac ultrasound indices of Tg and WT rats, (AH) Statistical graphs of cardiac ultrasound indices of rats, n=4-17, ns indicates no statistical significance, LVPWd: left ventricular posterior wall thickness at end-diastole; LVAWd: left ventricular anterior wall thickness at end-diastole; LVIDd: left ventricular internal diameter at end-diastole; LVPWs: left ventricular posterior wall thickness at end-systole; LVAWs: left ventricular anterior wall thickness at end-systole; LVIDs: left ventricular internal diameter at end-systole; EF%: ejection fraction; FS%: fractional shortening, *P<0.05, **P<0.01, ***P<0.001;

[0062] Figure 12 : Cardiac ultrasound parameters of Tg and WT rats, including: (A) ultrasound image of rat aorta, (B) statistical graph of aortic inner diameter and width, n=6, *P<0.05, **P<0.01, ***P<0.001;

[0063] Figure 13 Overexpression of BMP9 promotes vascular wall thickening. Representative HE staining images. Scale bars: 200 μm and 50 μm. Vascular structure statistics, n = 6, *P < 0.05, **P < 0.01, ***P < 0.001.

[0064] Figure 14 Overexpression of BMP9 promotes collagen deposition in vascular walls. (A) Representative Masson staining images, blue represents collagen fibers, scale bar is 50 μm. (B) Sirius red staining was used to detect the expression of type I and type III collagen in vascular tissue. (C) Statistical graph showing the percentage of vascular fibrosis area to total area, n=6. (D) RT-PCR detection of the expression of type I and type III collagen at the mRNA level in vascular tissue, n=6.

[0065] Figure 15 : Representative EVG staining images, blue represents collagen fibers, scale bars are 1 mm and 100 μm; BMP9 overexpression in rats increases the disruption of the elastic lamina in the vascular media;

[0066] Figure 16 : Oxidative stress in vascular tissue of rats overexpressing BMP9. RT-PCR detection of the relative expression of oxidase isoforms in vascular tissue of four groups of rats, n = 6. Data are expressed as mean ± SD. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 compared with the WT group. Representative DHE staining images. Scale bar, 100 μm.

[0067] Figure 17 : BMP9 overexpression in rats promotes aortic atherosclerosis;

[0068] Figure 18 : BMP9 overexpression promotes vascular dysfunction in rats. Concentration-response curves of phenylephrine (PE)-induced vasoconstriction in Tg and WT rats, and concentration-response curves of endothelium-dependent ACH- and endothelium-independent SNP-induced vasodilation in Tg and WT rats. DETAILED DESCRIPTION

[0069] The present invention will be further described below with reference to specific embodiments. The following specific embodiments are intended only to illustrate the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the claims and their equivalents.

[0070] The reagents, raw materials, and experimental consumables used in the present invention are readily available to those of ordinary skill in the art and, unless otherwise specified, can be obtained commercially. Experimental methods for which specific conditions are not specified in the present invention are generally performed under conventional conditions or as recommended by the manufacturer. In particular, the following examples are intended only to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are intended only to illustrate the present invention and should not, and will not, limit the present invention described in detail in the claims.

[0071] Example 1 Construction and identification of BMP9 overexpressing transgenic rats

[0072] 1. Construction of vector

[0073] We detected the expression of BMP9 in wild rats and found that BMP9 was highly expressed in the liver tissue of wild rats and was slightly expressed in the heart and lung (e.g. Figure 1 As shown). Therefore, the liver-specific ALB gene promoter sequence was constructed to drive the high expression of human BMP9 gene in rat liver. The plasmid containing human BMP9 was named pcDNA3.1-ALB-BMP9-WT. The plasmid was independently constructed in the laboratory. The schematic diagram of the transgenic plasmid is shown in Figure 2 .

[0074] The sequence of the plasmid pcDNA3.1-ALB-BMP9-WT is as follows (SEQ ID NO: 1):

[0075]

[0076] The liver-specific ALB gene promoter sequence is shown below (SEQ ID NO: 2):

[0077]

[0078] The sequence corresponding to the human BMP9 gene is shown below (SEQ ID NO: 3):

[0079]

[0080] 2. Microinjection

[0081] (1) Vasectomy of male rats: SD male rats were vasectomized at 5 weeks of age.

[0082] (2) Superovulation: Ten 3-4 week old SD mice were injected with PMSG and HCG hormones for superovulation.

[0083] (3) Fertilized egg injection: About 100 fertilized eggs were injected (the plasmid DNA was injected into the fertilized eggs using a microinjection instrument), and the amount of the plasmid DNA used was 5 ng / μL.

[0084] (4) Preparation of recipient mice: 8-week-old SD female mice were mated with ligated male mice and the female mice with ligature were selected.

[0085] (5) Embryo transplantation: The injected fertilized egg is transplanted into the ampulla of the oviduct of the recipient mouse.

[0086] 3. Genotype identification

[0087] (1) Tail cutting and numbering: For rats 7-10 days old, the tail tip is cut and numbered.

[0088] (2) Genomic DNA extraction: Rat genomic DNA was extracted using the Transgen genomic DNA extraction kit (EE101-12).

[0089] (3) PCR detection: Synthesize PCR genotyping primers, the primer sequences are as follows:

[0090] R-BMP9-WT-F: 5'-TCCAGATGGCAAACATACGC-3' (SEQ ID NO: 4);

[0091] R-BMP9-WT-R: 5'-TCCACCCTTGTCTTGTCCTG-3' (SEQ ID NO: 5).

[0092] Use Transgen kit 2× PCR SuperMix (+dye), according to Figure 3 The rat genotype was identified using the indicated reagent ratios and reaction conditions.

[0093] 4. Agarose gel electrophoresis

[0094] After the PCR amplification was completed according to the set reaction conditions, 1% agarose gel electrophoresis was performed. Only rats carrying the human BMP9 fragment could amplify a 552bp positive band, while negative rats in the same litter had no band ( Figure 4 ). In the figure, 1, 2, 3, and 4 are positive rats, and 5, 6, and 7 are negative rats.

[0095] 5. Identification of BMP9 expression in liver and plasma of BMP9-overexpressing rats

[0096] We further verified the expression levels of BMP9 mRNA and protein in the liver of overexpressed rats by RT-PCR and western-blot, and found that the expression levels of BMP9 in the liver of overexpressed rats were significantly higher than those in the control group (e.g. Figure 5-6 As shown). At the same time, we collected plasma from two groups of rats and detected the expression levels of BMP9 in the plasma of the two groups of rats by ELISA. The ELISA method is as follows: The circulating BMP9 concentration was determined using an ELISA kit (R&D Systems, USA; DY3209). 100 μL of plasma sample was used to test the BMP9 concentration. 100 μL of specific biotin-conjugated anti-human BMP9 was added to each well and incubated at 25°C for 2 hours. Wash three times. Horseradish peroxidase-coupled streptavidin was used as a substrate, and the colorimetric reaction was performed for 20 minutes. The calibration curve was a calibrator constructed by plotting the absorbance value at 450 nm against the known BMP9 concentration, and the concentration of each sample was determined using this curve. The results showed that the plasma BMP9 level in rats overexpressing BMP9 was significantly higher than that in the healthy control group (such as Figure 7 shown).

[0097] The above results confirmed that the BMP9 overexpression transgenic rat model was successfully constructed.

[0098] Example 2 BMP9 promotes blood pressure elevation in rats

[0099] This experiment used the tail cuff method and telemetry method to measure blood pressure. The tail cuff method uses a non-invasive tail artery blood pressure monitor (BP-98A; Softron, Japan) to detect rat blood pressure. To ensure accurate measurements, rats are placed in a blood pressure monitoring device for training when they are 6-8 weeks old. After two weeks of training, formal measurements are taken. The heating device is adjusted to 38°C, and the exposed rat tail is placed in the sensor. After the rat stabilizes, the measurement begins. The instrument automatically records systolic blood pressure, diastolic blood pressure, mean arterial pressure, and heart rate values. Each measurement records 5 data points, and the average value is obtained after removing the maximum and minimum values.

[0100] In this experiment, the tail-cuff method was used to continuously measure the blood pressure changes of the BMP9 overexpressing transgenic rats (Tg-BMP9 rats) constructed in Example 1 and the control group rats from 8 weeks to 16 weeks of age. Figure 8The results showed that the blood pressure of BMP9-overexpressing rats was significantly higher than that of the control group, and a stable hypertensive phenotype appeared at 16 weeks of age. Using an electronic sphygmomanometer, it was found that the systolic, mean arterial, and diastolic blood pressures of 16-week-old BMP9-overexpressing rats were significantly higher than those of their WT littermates (SBP: 119.3±11.0 vs 171.7±8.9 mmHg, P<0.001; MBP: 100.7±7.8 vs 149.1±9.2 mmHg, P<0.001; DBP: 90.80±8.5 vs 136.8±11.0 mmHg, P<0.001).

[0101] Telemetry was used to measure the 24-hour blood pressure changes in 16-week-old Tg-BMP9 rats and control group rats: Before telemetry, the implant must first be buried in the rat's body. During the operation, the rat was first anesthetized with isoflurane, the left common carotid artery was gently separated, and the catheter connected to the implant was inserted into the carotid artery and advanced until the tip was just inside the thoracic aorta. The implant was then buried subcutaneously in the abdomen. All operations were performed under sterile conditions. After the rat recovered for one week after surgery, the cage was placed on a flat plate. After the instrument was connected, the rat's blood pressure could be detected and the rat could move freely. Blood pressure was continuously recorded using a receiver platform (DSI), and data was acquired using the Dataquest system. Blood pressure continued to be monitored for 24 hours. The results are as follows. Figure 9 As shown, the results showed that the diurnal systolic blood pressure, diastolic blood pressure, and mean arterial pressure of BMP9-overexpressing rats were stably higher than those of the control rats, confirming that Tg-BMP9 rats developed spontaneous hypertension.

[0102] Example 3: Exploring the mechanism of BMP9 in increasing blood pressure

[0103] 1. Blood and urine biochemical results of the two groups of rats

[0104] In order to explore the mechanism of BMP9 in increasing blood pressure and to rule out the possibility of secondary hypertension, we collected 24-hour urine and serum from two groups of rats. The rats included in the experimental study were placed in metabolic cages, and urine was collected for 24 hours. After centrifugation, the urine was collected into EP tubes and stored at -80°C. When the rats were taken, the eyeballs were removed to collect blood. The beard around the eyes was first cut off, and the eyeballs were removed with tweezers. The blood was collected into EP tubes. After being placed at room temperature for half an hour, the tubes were centrifuged at 5500rpm for 5 minutes. The serum was transferred to a new EP tube and stored at -80°C for later use. After the blood and urine of all mice were collected, they were analyzed by a fully automatic biochemical analyzer. The results are as follows: Figure 10 Results showed no significant differences in serum and urine levels of sodium, potassium, chloride, uric acid, urea nitrogen, creatinine, creatinine clearance, or urine total protein between Tg-BMP9 and WT rats (P>0.05). This suggests that BMP9 transgenic rats may not have experienced early kidney damage, and therefore, the resulting blood pressure increase may have other mechanisms.

[0105] 2. Ultrasound test results of the two groups of rats

[0106] Ultrasound was used to examine the cardiac function and aortic diameter of 16-week-old Tg and WT rats. Figure 11 and Figure 12 Results showed that compared with the WT group, there were no significant differences in left ventricular anterior wall (LVAW), left ventricular posterior wall (LVPW), ejection fraction (EF%), and fractional shortening (FS%) in Tg rats during systole and diastole (P>0.05). The ascending aorta diameter in Tg rats was reduced (P<0.05, n=6). This suggests that overexpression of BMP9 has no significant effect on cardiac structure and function in rats, but may play an important role in vascular structure and function.

[0107] 3. BMP9 overexpression promotes vascular remodeling

[0108] Vascular remodeling is a major contributor to elevated blood pressure. To investigate the role of BMP9 in elevating blood pressure, we investigated the effects of BMP9 overexpression on vascular remodeling. Inflammation, oxidative stress, and vascular fibrosis are key pathological processes of vascular remodeling. This study further examined the effects of BMP9 overexpression on vascular fibrosis, inflammation, and oxidative stress.

[0109] At the tissue level, we used HE pathological staining and measured the parameters of the thoracic aorta of BMP9-overexpressing rats and negative littermates at 8, 16, and 24 weeks. Figure 13 The results showed that BMP9-overexpressing transgenic rats had thickened vascular walls, a smaller lumen diameter, increased media thickness, and a significantly increased ratio of media to lumen diameter, indicating that BMP9 mediates pathological vascular remodeling.

[0110] Vascular fibrosis is one of the important pathological processes of vascular remodeling, which is mainly manifested by collagen deposition. We used Masson staining to stain aortic sections and found that compared with the WT group, the vascular tissue of rats with BMP9 overexpression showed a significant increase in blue fibrotic tissue, and the blood vessels showed obvious fibrosis ( Figure 14 Sirius red staining revealed increased vascular collagen expression. Detection of vascular collagen type I and type III expression at the mRNA level revealed that BMP9 overexpression in rats significantly increased both type I and type III collagen expression ( Figure 14 The above results confirmed that BMP9 overexpression promotes vascular fibrosis.

[0111] The aorta of the two groups of rats was stained with EVG to detect elastic fibers, which were colored as black thin lines. Figure 15As shown in the results, under both low- and high-power microscopes, the elastic fibers in the thoracic aorta of the WT group were arranged in an orderly and regular pattern, with small spacing between elastic fibers and a dense and intact elastic fiber layer throughout the media. In contrast, in the BMP9 overexpression group, the entire media layer was significantly thickened, with increased spacing between elastic fibers and coarsened elastic fiber lines. Furthermore, we counted elastic lamina ruptures under each high-power microscope field and found an increased number of lamina ruptures in the BMP9 overexpression group. BMP9-promoted elastic fiber rupture indicates severe structural damage to the vascular media.

[0112] The production of reactive oxygen species (ROS) mediated by the NADPH oxidase NOX family plays a key role in vascular changes in hypertension. Excessive vascular superoxide production can cause increased vascular tension and dysfunction, ultimately leading to hypertension. We used DHE staining to detect the expression of ROS in vascular tissues of the two groups of rats. The results are as follows: Figure 16 The results showed that ROS levels in Tg rats' blood vessels increased significantly. Furthermore, BMP9 overexpression was found to significantly increase the mRNA expression of various NADPH oxidase isoforms in aortic tissue, suggesting that BMP9 overexpression promotes vascular oxidative stress in rats.

[0113] 4. BMP9 overexpression promotes vascular dysfunction

[0114] Oxidative stress and inflammation are key mechanisms of large and small blood vessel dysfunction and remodeling, which mediate the occurrence of hypertension. Among them, large vessel dysfunction is mainly manifested as increased vascular stiffness. Pulse wave velocity (PWV) refers to the speed of pulse propagation along the walls of large arteries generated by each beat of the heart pumping blood. It is one of the indicators commonly used in clinical practice to evaluate vascular stiffness. The higher the PWV, the lower the elasticity of the arterial wall. The results of our study are as follows Figure 17 As shown, the results showed that in BMP9-overexpressing rats, aortic PWV was significantly increased, indicating damage to large blood vessel function.

[0115] We further conducted aortic and mesenteric vascular function experiments to observe the differences in the function of large and small blood vessels. Figure 18 As shown, vascular contractility measurements showed increased phenylephrine-dependent contractile tension in the aorta and mesenteric arteries of Tg rats. Vasodilation measurements showed decreased responsiveness to both the endothelium-dependent relaxant ACH and the endothelium-independent relaxant SNP in the aorta and mesenteric arteries of Tg rats. These results suggest that BMP9 overexpression exacerbates vascular dysfunction.

[0116] The above results demonstrate that the Tg-BMP9 rats constructed using the method described in Example 1 of the present invention are a spontaneously hypertensive rat model, rather than a secondary hypertensive rat model.

Claims

1. A method for constructing a spontaneously hypertensive rat model overexpressing BMP9, characterized in that: The method comprises the following steps: (1) Construction of a recombinant vector overexpressing BMP9; (2) A spontaneously hypertensive rat model overexpressing BMP9 was prepared by microinjection.

2. The method according to claim 1, characterized in that In the recombinant vector overexpressing BMP9, the liver-specific ALB gene promoter drives the high expression of BMP9 specifically in the rat liver.

3. The method according to claim 2, characterized in that The sequence of the liver-specific ALB gene promoter is shown in SEQ ID NO: 2, and the sequence of BMP9 is shown in SEQ ID NO:

3.

4. The method according to claim 1, wherein The recombinant vector for overexpressing BMP9 is plasmid pcDNA3.1-ALB-BMP9-WT, and its corresponding sequence is shown in SEQ ID NO:

1.

5. The method according to claim 1, wherein The microinjection method for preparing a spontaneously hypertensive rat model overexpressing BMP9 comprises the following steps: 1) Vasectomy of male rats: SD male rats were subjected to vasectomy to obtain vasectomized male rats; 2) Superovulation: Inject hormones into SD female mice to induce superovulation and obtain fertilized eggs; 3) Fertilized egg injection: injecting the recombinant vector overexpressing BMP9 into the fertilized egg using a microinjector to obtain an injected fertilized egg; 4) Preparation of recipient mice: After mating with the ligated male mice, female SD mice with thrombosis were selected as recipient mice; 5) Embryo transplantation: The injected fertilized eggs are transplanted into the ampulla of the oviduct of the recipient mice, and the recipient mice give birth to obtain a spontaneously hypertensive rat model overexpressing BMP9.

6. The method according to claim 5, characterized in that The hormones are PMSG and HCG hormones; Optionally, the dosage of the recombinant vector overexpressing BMP9 is 1-10 ng / μL; Optionally, the method further comprises performing genotype identification and / or disease phenotype identification on the obtained spontaneously hypertensive rat model overexpressing BMP9.

7. The method according to claim 6, characterized in that The primer sequences used for genotype identification are shown in SEQ ID NO: 4-5.

8. A recombinant vector for constructing a spontaneously hypertensive rat model overexpressing BMP9, characterized in that: The recombinant vector is the recombinant vector for overexpressing BMP9 according to any one of claims 1 to 4.

9. A spontaneously hypertensive rat model constructed using the method according to any one of claims 1 to 7.

10. Any of the following applications: (1) Use of the recombinant vector according to claim 8 in constructing a spontaneously hypertensive rat model or preparing an agent for promoting vascular fibrosis; (2) Use of the spontaneously hypertensive rat model described in claim 9 in the development of new drugs for spontaneous hypertension, screening of antihypertensive drugs, evaluation of the efficacy of antihypertensive drugs, evaluation of the safety of antihypertensive drugs, or research on the pathogenesis of spontaneous hypertension.

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