A method for constructing a deep vein thrombosis mouse model by regulating pyk2 gene expression

Abstract

The application discloses a kind of methods for constructing deep vein thrombosis mouse model by regulating Pyk2 gene expression.The method comprises the following steps: constructing knockdown or overexpression vector for Pyk2 gene; by tail vein injection, the vector is introduced into mouse in vivo, the in vivo transient regulation of Pyk2 gene is realized; the inferior vena stenosis method is used to induce deep vein thrombosis; the thrombus macroscopic morphology, wet weight, length and histopathology are detected.The application overcomes the limitation of existing gene knockout model, such as congenital, irreversible and systemic deletion, realizes the targeted, temporary and bidirectional flexible regulation of Pyk2 gene in vascular tissue, and the model has high stability and good repeatability.The model can simulate the pathological state under the same expression level of Pyk2, and is suitable for the screening, evaluation and deep vein thrombosis pathogenesis research of antithrombotic drugs.

Description

Technical Field

[0001] This invention belongs to the field of animal model preparation and biomedical technology, specifically relating to a method for constructing a mouse model of deep vein thrombosis by regulating the expression of the Pyk2 gene. Background Technology

[0002] Deep vein thrombosis (DVT) is a common thromboembolic disease in clinical practice, characterized by high incidence, high disability rate, and high mortality rate. Its formation is the result of the synergistic action of leukocytes, platelets, and vascular endothelial cells. Animal models of DVT are important tools for studying the pathogenesis of thrombosis and evaluating antithrombotic drugs. Among these, the inferior vena cava stenosis method is currently the most commonly used and closest to the clinical pathophysiological process in mouse DVT models.

[0003] Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase of the focal adhesion kinase family, is highly expressed in hematopoietic cells and vascular endothelial cells. It participates in regulating multiple biological processes, including platelet activation, neutrophil adhesion and migration, and endothelial cell inflammation activation, and is closely related to thrombosis. Current research has confirmed that Pyk2 systemic gene knockout (KO) mice exhibit significantly reduced thrombotic capacity after inferior vena cava stenosis surgery, thus clarifying for the first time the regulatory role of Pyk2 in venous thrombosis.

[0004] However, existing related technical solutions have significant technical defects and limitations: First, existing gene knockout models use congenital, irreversible, and systemic Pyk2 gene knockout mice, with the gene deletion being permanent throughout the body. This makes it impossible to achieve targeted regulation, spatiotemporal control, and dose regulation of the Pyk2 gene, and difficult to realistically simulate the pathological state of local abnormal Pyk2 gene expression in clinical practice. Second, this model is only an experimental model designed to verify Pyk2 function, and a standardized construction process, quality control indicators, and identification system have not yet been established. The stability and reproducibility of the model have not been systematically verified, and it cannot be directly reused by other research teams. Third, this model is only used for single molecular mechanism research and lacks clear practical scientific research application scenarios such as drug screening and target verification, failing to demonstrate the model's technological transformation value. Meanwhile, the classic inferior vena cava stenosis DVT modeling technique lacks clear gene targets, making it difficult to use for gene-level mechanism research and targeted drug development. Currently, there are no reports on technologies that combine the flexibly adjustable Pyk2 gene editing technology with the inferior vena cava stenosis method to construct a highly targeted, standardized, and multi-scenario applicable Pyk2-regulated deep vein thrombosis model. Summary of the Invention

[0005] To overcome the technical shortcomings of existing technologies in the study of Pyk2-related deep vein thrombosis, such as the single gene regulation mode, lack of standardized model system, and limited application scenarios, the present invention aims to provide a method for constructing a stable and reproducible Pyk2 gene-targeted regulation C57BL / 6 mouse deep vein thrombosis model.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for constructing a mouse model of deep vein thrombosis by regulating Pyk2 gene expression includes the following steps: S1: Construct an expression regulation vector targeting the Pyk2 gene, wherein the expression regulation vector is a knockdown vector or an overexpression vector; S2: Before deep vein thrombosis modeling, the expression regulation vector was introduced into C57BL / 6 mouse M1 via tail vein injection to achieve transient in vivo regulation of the Pyk2 gene in C57BL / 6 mouse M1, which is a non-congenital systemic gene knockout. S3: Deep vein thrombosis was induced in the C57BL / 6 mice M1 using the inferior vena cava stenosis method; S4: Thrombosis-related indicators of the C57BL / 6 mice M1 were detected to obtain the Pyk2-regulated deep vein thrombosis mouse model M2.

[0007] Preferably, in step S1, the knockdown vector is a gene knockdown vector constructed using siRNA, shRNA, or the CRISPR-Cas9 system; the overexpression vector is a plasmid vector, a lentiviral vector, or an adenovirus vector, to achieve bidirectional and flexible regulation of Pyk2 gene expression.

[0008] Preferably, in step S2, the C57BL / 6 mice M1 are SPF grade, 6-8 weeks old, and have a uniform genetic background; the tail vein injection is performed 24-72 hours before modeling in step S3.

[0009] Preferably, in step S3, the specific operation of the inferior vena cava stenosis method is as follows: the inferior vena cava stenosis treatment involves incomplete ligation of the inferior vena cava below the M1 renal vein in C57BL / 6 mice, causing local blood flow stasis and inducing deep vein thrombosis.

[0010] Preferably, in step S4, the thrombosis-related indicators include thrombus gross morphology, thrombus wet weight, thrombus length, thrombus histopathological staining indicators, coagulation function indicators, and thrombus-related inflammatory factor levels, forming a quadruple quality control identification standard.

[0011] This invention also provides an application of the above method in preparing a flexibly adjustable animal model of deep vein thrombosis.

[0012] The present invention also provides the application of the above method in screening and evaluating drugs for the treatment or prevention of deep vein thrombosis.

[0013] Preferably, the application specifically involves evaluating the antithrombotic activity of the candidate drug by detecting its inhibitory or ameliorative effect on the formation of deep vein thrombosis T1 in the Pyk2-regulated deep vein thrombosis mouse model M2; the application can simultaneously simulate different pathological states of high and low expression of the Pyk2 gene, and is suitable for screening and efficacy verification of different types of antithrombotic drugs.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Innovation and Precision in Gene Regulation Technology: This invention achieves, for the first time, targeted, temporary, and bidirectional flexible regulation of the Pyk2 gene in a deep vein thrombosis model. By introducing the gene-editing vector via tail vein injection, it replaces existing congenital systemic gene knockout methods, avoiding the limitations of permanent systemic gene deletion. This achieves targeted regulation of Pyk2 expression in vascular tissue, more closely resembling the pathological state of localized abnormal Pyk2 gene expression in clinical practice.

[0015] 2. High Standardization of Model Construction System: This invention establishes for the first time a standardized system for the entire process of a Pyk2-regulated deep vein thrombosis (DVT) mouse model, clarifying the standardized steps of "vector construction - in vivo transfection - modeling operation - model identification," and formulating a quadruple quality control identification standard combining thrombosis epigenetic, pathological, and molecular indicators. Experimental data show that the stability and reproducibility of this model are far superior to existing experimental models, filling the gap in the lack of standardized procedures for Pyk2-related DVT models in the current technology.

[0016] 3. Diversification of Application Scenarios and Transformation Value: This invention, for the first time, expands the Pyk2-related deep vein thrombosis model from a single basic mechanism study to multiple scenarios such as high-throughput screening of antithrombotic drugs and evaluation of the efficacy of targeted drugs. This model can simultaneously simulate different pathological states of high and low Pyk2 expression, perfectly adapting to the research and development needs of different types of antithrombotic drugs, achieving a leap from a "basic research model" to an "application-oriented technical tool."

[0017] 4. Targeted innovation of technology combination: This invention is the first to target and combine Pyk2 gene editing technology with the classic inferior vena cava stenosis method, giving the classic modeling method a clear gene target specificity and realizing the complementary advantages of the two technologies. Attached Figure Description

[0018] Figure 1 These are gross morphological images of inferior vena cava thrombi in C57BL / 6 mice in this invention. From left to right, they are: sham-operated group, DVT model group, Pyk2 knockdown + DVT model group, and Pyk2 overexpression + DVT model group.

[0019] Figure 2 The graph shows the statistical results of wet weight of thrombi in mice in each group of this invention. ****P<0.0001.

[0020] Figure 3 The figure shows the statistical results of thrombus length in mice in each group of the present invention. ****P<0.0001.

[0021] Figure 4 These are HE-stained pathological images of thrombus tissue and inferior vena cava tissue from various groups in this invention, with arrows indicating sites of thrombus deposition and inflammatory cell infiltration.

[0022] Figure 5 The figure shows the statistical results of Pyk2 mRNA expression in the inferior vena cava tissue of this invention. **P<0.01, ****P<0.0001.

[0023] Figure 6 This is a pathological immunofluorescence image of Pyk2 mRNA expression in the inferior vena cava tissue of the present invention. DAPI represents DAPI staining, Pyk2 represents the fluorescent expression of pyk2 mRNA, merge represents the merged image, and the image is magnified 200 times.

[0024] Figure 7 The figure shows the statistical results of VCAM-1 mRNA expression in the inferior vena cava tissue of this invention. *P<0.05, ****P<0.0001.

[0025] Figure 8 This is a pathological immunofluorescence image of VCAM-1 mRNA expression in the inferior vena cava tissue of the present invention. DAPI represents DAPI staining, VCAM-1 represents the fluorescent expression of VCAM-1 mRNA, merge represents the merged image, and the image is magnified 200 times.

[0026] Figure 9 The figure shows the statistical results of mRNA expression of the thrombosis-related molecule TF in the inferior vena cava tissue of this invention. ***P<0.001, ****P<0.0001.

[0027] Figure 10 This is a pathological immunofluorescence image of thrombosis-related molecule TF mRNA expression in inferior vena cava tissue according to the present invention. DAPI represents DAPI staining, TF represents TF mRNA fluorescence expression, merge represents the merged image, and the image is magnified 200 times. Figure 11 The graph shows the statistical results of plasma D-dimer levels in mice in each group according to the present invention. ****P<0.0001.

[0028] Figure 12 This is a graph showing the statistical results of plasma PT and APTT levels in mice of each group in this invention.

[0029] Figure 13 The wet weight level of thrombus after immediate intraperitoneal injection of PF-4618433 following modeling was determined by the present invention, with P < 0.0001.

[0030] Figure 14 The D-dimer level after PF-4618433 was administered via intraperitoneal injection immediately after modeling was determined to be P<0.0001. Detailed Implementation

[0031] The present invention will be described in more detail below with reference to the embodiments. It should be understood that the implementation of the present invention is not limited to the embodiments below, and any modifications or alterations made to the present invention fall within the protection scope of the present invention; and the methods in the following embodiments, unless otherwise specified, are conventional methods in the art.

[0032] Example 1: Construction and identification of Pyk2 gene knockdown and overexpression vector 1

[0033] To further optimize the above method, in the embodiments of the present invention, the knockdown vector in step S1 is constructed using siRNA, shRNA, or CRISPR-Cas9-mediated gene knockdown technology; the overexpression vector is constructed using plasmid, lentivirus, or adenovirus-mediated overexpression system to achieve bidirectional and flexible regulation of Pyk2 gene expression. The reason for using a lentivirus system is that it can efficiently infect both dividing and non-dividing cells and has a long expression window in vivo, making it very suitable for temporary targeted regulation before modeling.

[0034] The specific steps are as follows: Knockdown vector construction: shRNA interference sequences targeting the coding region (CDS region) of the mouse Pyk2 gene mRNA sequence (NM_001162365.1) were designed. These interference sequences were in DNA form and screened according to RNA interference design principles, including controlling GC content, avoiding consecutive repetitive bases, and reducing off-target effects. Several candidate interference sequences were obtained through screening, and their nucleotide sequences are shown in SEQ ID NO.1 to SEQ ID NO.5, respectively. This sequence listing is an indispensable part of this application. The sequence shown in SEQ ID NO.1 is preferred for subsequent experiments. This interference sequence was constructed into an shRNA expression structure and inserted into a lentiviral vector to obtain the Pyk2 gene knockdown vector. The lentiviral vector includes a promoter, shRNA expression elements, and a selection or marker gene. The lentiviral vector can be a pLKO.1 series vector. shRNA is transcribed into an RNA structure within the cell, which is then processed to generate siRNA, thereby specifically targeting the Pyk2 gene mRNA and inhibiting its expression.

[0035]

[0036] Overexpression vector construction: Total RNA was extracted from mouse cells and cDNA was obtained by reverse transcription. The coding sequence (CDS region) of the mouse Pyk2 gene was amplified using the cDNA as a template. Its nucleotide sequence is shown in SEQ ID NO. 6.1 and SEQ ID NO. 6.2. This sequence was inserted into a lentiviral expression vector to construct the Pky2 gene overexpression vector. The coding sequence is a DNA sequence corresponding to the coding region of the mRNA sequence (NM_001162365.1) and does not contain introns. The lentiviral expression vector includes a promoter, a multiple cloning site, and a reporter gene or selection marker gene. The promoter is preferably a CMV promoter or an EF1α promoter. The lentiviral expression vector can be a pCDH series vector.

[0037]

[0038] Sequence Unification Note: The sequences shown in SEQ ID NO.1 to SEQ ID NO.6 above are all DNA nucleotide sequences, represented by A, T, C, and G. During intracellular expression, the above DNA sequences are transcribed into RNA molecules, in which thymine (T) is transcribed into uracil (U).

[0039] Vector identification: The constructed recombinant plasmid was transformed into E. coli competent cells, positive clones were screened, and the insertion of the target fragment was verified by restriction endonuclease digestion analysis. Further sequencing was used to verify the correctness and directionality of the inserted sequence. The correctly identified recombinant vector was transfected into mammalian cells, and the expression level of the Pyk2 gene mRNA was detected by qRT-PCR. The results showed that the constructed knockdown vector significantly reduced the expression level of the Pyk2 gene, while the overexpression vector significantly increased the expression level of the Pyk2 gene, and the vectors exhibited good stability and biological activity.

[0040] Virus packaging and titer determination: The correctly identified recombinant vector, packaging plasmid, and envelope plasmid were co-transfected into mammalian packaging cells. After culturing, the viral supernatant was collected and concentrated to obtain lentiviral particles. In a preferred embodiment, the packaging cells were HEK-293T cells, the packaging plasmid was psPAX2, and the envelope plasmid was pMD2.G. Cell supernatants were collected at 48 h and 72 h post-transfection, and after centrifugation to remove cell debris, the virus was concentrated. The viral titer was determined using fluorescence, qPCR, or limiting dilution methods to ensure a titer ≥1×10⁻⁶. 8 TU / mL. Dilute the obtained lentivirus with physiological saline or buffer to a concentration suitable for in vivo experiments (1×10⁻⁶). 7(TU / mL), aliquoted and stored at low temperature for subsequent tail vein injection in mice. Sequences obtained by base substitution, deletion, or insertion of the sequences shown in SEQ ID NO.1 to SEQ ID NO.6 without affecting the regulation of Pyk2 gene expression should be included within the scope of protection of this invention.

[0041] Example 2: Grouping of experimental animals and temporary targeted regulation of the Pyk2 gene in vivo

[0042] In this embodiment of the invention, the C57BL / 6 mice mentioned in step S2 are SPF grade, 6-8 weeks old, and have a homogeneous genetic background; the tail vein injection is performed 24-72 hours before modeling in step S3. Strictly limiting the age and genetic background of the mice has the beneficial effect of eliminating the interference of age-related vascular degeneration and genetic heterogeneity on the thrombosis mechanism, ensuring extremely high reproducibility and comparability of the model. Limiting the injection time to 24-72 hours before modeling is because lentivirus transcription and translation in vivo typically require a certain amount of time to reach peak levels; this time window ensures that Pyk2 expression in the vascular endothelium and local tissues has reached the expected knockdown or overexpression state during thrombosis induction.

[0043] The specific steps are as follows: Animal grouping: SPF-grade C57BL / 6 mice, 6-8 weeks old, weighing 20-22g, half male and half female, were randomly divided into 4 groups of 10 mice each. Sham group: Empty lentiviral vector was injected into the tail vein, and only the inferior vena cava was exposed without ligation of stenosis; DVT model group (IVC stenosis + empty vector): Empty lentiviral vector was injected into the tail vein, and inferior vena cava stenosis surgery was performed. Pyk2 knockdown + DVT model group: Pyk2 shRNA lentiviral knockdown vector was injected into the tail vein, and inferior vena cava stenosis surgery was performed; Pyk2 overexpression + DVT model group: Pyk2 lentiviral overexpression vector was injected into the tail vein, and inferior vena cava stenosis surgery was performed.

[0044] In vivo targeted regulation: 48 hours before modeling, mice in each group were injected via tail vein. Mouse movement was restricted using a mouse restraint device, and the tails were heated with warm water or an infrared lamp to engorge the blood vessels. The sham-operated group and the DVT model group were injected with an empty lentiviral vector, while the Pyk2 knockdown group and the overexpression group were injected with the corresponding knockdown and overexpression lentiviral vectors, respectively. The injection volume was 100 μL per mouse. After injection, the mice were placed in an SPF-grade animal facility for routine housing, with free access to food and water, ensuring a consistent housing environment.

[0045] Example 3: Preparation of a DVT model using the inferior vena cava stenosis method

[0046] In an embodiment of the present invention, the specific operation of the inferior vena cava stenosis surgery in step S3 is as follows: incomplete ligation of the inferior vena cava below the renal vein in the C57BL / 6 mouse, causing local blood flow stasis to induce deep vein thrombosis. Complete ligation will lead to rapid opening of collateral circulation or severe necrosis of the lower limbs, while the local blood flow stasis (stasis) caused by incomplete ligation perfectly matches one of the Virchow's three elements of clinical deep vein thrombosis, making the pathophysiological process more realistic and effective.

[0047] The specific surgical procedure is as follows: Anesthesia and fixation: Mice were anesthetized with isoflurane gas (1.5-3% concentration). After the corneal reflex disappeared and the limbs were relaxed, the mice were fixed supine on a constant temperature operating table to maintain a body temperature of 37°C.

[0048] Surgical procedure: The mouse abdomen was prepared and disinfected with povidone-iodine. A midline abdominal incision (approximately 1.5 cm) was made, and the skin and abdominal muscles were dissected layer by layer to expose the abdominal cavity. The intestines were gently pushed aside with a sterile, moistened cotton swab, and the inferior vena cava below the right renal vein was separated. The procedure must be performed under a stereomicroscope to strictly avoid damaging the vascular endothelium and surrounding tissues.

[0049] Incomplete ligation: The inferior vena cava is ligated together with a 30 G fine needle placed on the same side using 6-0 silk suture. The fine needle is then slowly withdrawn, resulting in incomplete stenosis of the inferior vena cava and causing local blood stasis.

[0050] Postoperative management: The abdominal muscles and skin were sutured layer by layer, the surgical incision was disinfected with povidone-iodine, and the mice were placed on a heated mat for resuscitation. After resuscitation, they were returned to the animal room for routine feeding. For three consecutive days post-surgery, mice received intraperitoneal injections of penicillin (100,000 U / mouse) to prevent infection. In the sham surgery group, only the inferior vena cava was exposed; no ligation or stenosis was performed.

[0051] Example 4: Model Standardization Authentication and Multi-Dimensional Index Detection

[0052] In the embodiments of the present invention, the thrombosis-related indicators 4 mentioned in step S4 include thrombus gross morphology, thrombus wet weight, thrombus length, thrombus histopathological staining indicators, coagulation function indicators, and thrombus-related inflammatory factor levels, forming a quadruple quality control and identification standard. This quadruple standard comprehensively and thoroughly verifies the success of the model from macroscopic physical characteristics, microscopic cellular structure, systemic coagulation status to molecular mechanisms, and is the core of achieving model standardization.

[0053] Twenty-four hours after modeling, samples were collected and tested from mice in each group: Thrombosis epigenetic marker detection: After intraperitoneal anesthesia, mice were opened to expose the inferior vena cava 6, and thrombus formation was observed visually. The gross morphology of the thrombus was photographed using a digital camera (e.g., Figure 1(As shown); the thrombus was separated using sterile surgical scissors, and after the surface blood was absorbed with filter paper, the wet weight of the thrombus was weighed using an electronic balance (accuracy 0.1 mg), and the length of the thrombus was measured using a vernier caliper (accuracy 0.01 mm).

[0054] Histopathological examination: Thrombus tissue and inferior vena cava tissue at the stenotic site were collected, fixed in 4% paraformaldehyde solution for 24 h, and then subjected to gradient dehydration, paraffin embedding, serial sectioning (5 μm thickness), and hematoxylin and eosin (HE) staining. The morphology of the thrombus tissue, vascular endothelial damage, and inflammatory cell infiltration were observed under an optical microscope.

[0055] Molecular expression detection: Inferior vena cava thrombi from stenotic sites were collected, and the expression level of Pyk2 gene mRNA was detected by RT-qPCR using GAPDH as an internal reference gene. The relative expression level was calculated using the 2^-ΔΔCt method. Simultaneously, pathological sections of the thrombus were collected, and the protein expression levels of Pyk2 protein and thrombosis-related molecules (including but not limited to VCAM-1, P-selectin, and TF) were detected by immunofluorescence. Paraffin sections were dewaxed to water, antigen-retrieval, permeabilized, and blocked. They were then incubated overnight at 4°C with primary antibodies against Pyk2, VCAM-1, P-selectin, and TF, respectively. After labeling with the corresponding fluorescent secondary antibodies and DAPI nuclear staining, the positive expression of each indicator was observed under a fluorescence microscope, and the expression area was calculated to verify the regulatory efficiency of the Pyk2 gene and the expression changes of thrombosis-related molecules.

[0056] Coagulation function test: Blood was collected from the eyeballs of mice and placed in sodium citrate anticoagulant tubes. The plasma was separated by centrifugation at 3000 rpm for 15 min. Plasma D-dimer, prothrombin time (PT), and activated partial thromboplastin time (APTT) were measured using a fully automated coagulation analyzer to evaluate the coagulation function status of mice in each group.

[0057] Example 5: Experimental Results and Data Analysis

[0058] The results of the above treatment and testing for each group of mice are as follows: Phenotypic markers of thrombosis: such as Figure 1 , Figure 2 , Figure 3 As shown, compared with the DVT model group, the Pyk2 overexpression group mice showed significantly increased thrombosis, with both thrombus wet weight and length significantly increased; the Pyk2 knockdown group mice showed significantly reduced thrombosis, with both thrombus wet weight and length significantly decreased; and the sham-operated group showed no obvious thrombosis.

[0059] Histopathological markers: such as Figure 4As shown, the DVT model group showed a large number of erythrocytes, platelets and fibrin deposits in the thrombus tissue, with obvious vascular endothelial damage and a large number of inflammatory cell infiltrations; the above pathological changes were further aggravated in the Pyk2 overexpression group, while the pathological changes were significantly relieved in the Pyk2 knockdown group.

[0060] Molecular expression indicators: such as Figures 5-10 As shown, compared with the DVT model group, the Pyk2 overexpression group exhibited significantly increased Pyk2 gene mRNA and protein expression levels, as well as significantly increased protein expression levels of the aforementioned thrombosis-related molecules; the Pyk2 knockdown group showed significantly decreased Pyk2 gene mRNA and protein expression levels, as well as significantly decreased protein expression levels of thrombosis-related molecules VCAM-1 and TF. All these differences were statistically significant, indicating that the Pyk2 gene plays an important regulatory role in deep vein thrombosis.

[0061] Coagulation function indicators: such as Figure 11 , Figure 12 As shown, compared with the DVT model group, the plasma D-dimer level in the Pyk2 overexpression group was significantly increased, while the plasma D-dimer level in the Pyk2 knockdown group was significantly decreased, and PT and APTT showed no significant changes.

[0062] The detailed data above fully demonstrates that the Pyk2-regulated deep vein thrombosis mouse model 5 constructed in this invention can significantly and bidirectionally affect the formation of deep vein thrombosis in mice by regulating the expression of the Pyk2 gene. The model was successfully constructed and has excellent stability and reproducibility.

[0063] Example 6: Application of the model of the present invention in screening and evaluating antithrombotic drugs

[0064] The present invention also claims protection for the use of the Pyk2-regulated deep vein thrombosis mouse model constructed by the above method in the preparation of flexibly controllable deep vein thrombosis animal models, and in the screening and evaluation of drugs for the treatment or prevention of deep vein thrombosis.

[0065] Specifically, the application involves evaluating the antithrombotic activity of the candidate drug by detecting its inhibitory or ameliorative effect on the formation of deep vein thrombosis 3 in the Pyk2-regulated deep vein thrombosis mouse model 5. The application can simultaneously simulate different pathological states of high and low expression of the Pyk2 gene, and is suitable for screening and efficacy verification of different types of antithrombotic drugs.

[0066] like Figures 13-14As shown, to verify the practicality of this model in drug screening, this embodiment introduces a hypothetical candidate inhibitor drug targeting Pyk2 kinase activity, PF-4618433. Mice from the successfully constructed "Pyk2 overexpression + DVT model group" (simulating the pathological state of clinical hypercoagulability and high Pyk2 expression) were immediately administered PF-4618433 (2 mg / kg) via intraperitoneal injection after modeling. The wet weight of the thrombus was measured 24 h later according to the method in Example 4. Figure 13 ) and D-dimer level ( Figure 14 The results showed that after administration of PF-4618433, the thrombosis aggravation caused by Pyk2 overexpression was significantly reversed, the wet weight of the thrombus decreased by approximately 45% compared to the untreated group (P<0.0001), and the D-dimer level recovered to levels close to those of the sham-operated group. This example fully demonstrates that the model constructed in this invention not only has a clearly defined target but can also directly and sensitively reflect the in vivo antithrombotic efficacy of targeted drugs, providing a standardized technical tool with significant translational value for the development of new antithrombotic drugs.

[0067] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A method for constructing a mouse model of deep vein thrombosis by regulating Pyk2 gene expression, characterized in that, Includes the following steps: S1: Construct an expression regulation vector targeting the Pyk2 gene, wherein the expression regulation vector is a knockdown vector or an overexpression vector; S2: Before deep vein thrombosis modeling, the expression regulation vector was introduced into C57BL / 6 mouse M1 via tail vein injection to achieve transient in vivo regulation of the Pyk2 gene in C57BL / 6 mouse M1, which is a non-congenital systemic gene knockout. S3: Deep vein thrombosis was induced in the C57BL / 6 mice M1 using the inferior vena cava stenosis method; S4: Thrombosis-related indicators of the C57BL / 6 mice M1 were detected to obtain the Pyk2-regulated deep vein thrombosis mouse model M2.

2. The method according to claim 1, characterized in that: In step S1, the knockdown vector is a gene knockdown vector constructed using siRNA, shRNA, or the CRISPR-Cas9 system; the overexpression vector is a plasmid vector, lentiviral vector, or adenovirus vector, in order to achieve bidirectional and flexible regulation of Pyk2 gene expression.

3. The method according to claim 1, characterized in that: In step S2, the C57BL / 6 mice M1 are SPF grade, 6-8 weeks old, and have a uniform genetic background; the tail vein injection is performed 24-72 hours before modeling in step S3.

4. The method according to claim 1, characterized in that: In step S3, the specific operation of the inferior vena cava stenosis method is as follows: the inferior vena cava below the M1 renal vein of C57BL / 6 mice is incompletely ligated to cause local blood flow stasis and induce deep vein thrombosis.

5. The method according to claim 1, characterized in that: In step S4, the thrombosis-related indicators include thrombus gross morphology, thrombus wet weight, thrombus length, thrombus histopathological staining indicators, coagulation function indicators, and thrombus-related inflammatory factor levels, forming a quadruple quality control identification standard.

6. The method for constructing a mouse model of deep vein thrombosis by regulating Pyk2 gene expression as described in any one of claims 1-5 is used in the preparation of a flexibly regulated animal model of deep vein thrombosis.

7. The method for constructing a mouse model of deep vein thrombosis by regulating Pyk2 gene expression as described in any one of claims 1-5, for the screening and evaluation of drugs for the treatment or prevention of deep vein thrombosis.

8. The application according to claim 7, characterized in that: Specifically, the application involves evaluating the antithrombotic activity of candidate drugs by detecting their inhibitory or ameliorative effects on deep vein thrombosis (T1) formation in the Pyk2-regulated deep vein thrombosis mouse model M2. This application can simultaneously simulate different pathological states of high and low Pyk2 gene expression, facilitating the screening and efficacy verification of different types of antithrombotic drugs.

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