A thrombocytosis thrombosis zebra fish model and a construction method and application thereof

Abstract

The application provides a thrombocytosis and thrombosis zebrafish model and a construction method and application thereof, and belongs to the technical field of biology. CALR del52 The transgenic zebrafish is obtained, and the transgenic zebrafish is added with a thrombus inducer of arachidonic acid to induce and construct; CALR del52 The nucleotide sequence is shown as SEQ ID NO. 1; the thrombocytosis and thrombosis disease zebrafish model provided by the application can be used for screening of anti-thrombus treatment drugs, has a short experimental cycle, a small dosage, and is favorable for high-throughput screening of trace anti-thrombus active natural products.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a thrombocytosis-induced thrombosis zebrafish model and a construction method and application thereof. BACKGROUND

[0002] Thrombosis, as a complex pathological process, seriously threatens human health, and is closely related to the occurrence and development of cardiovascular and cerebrovascular diseases, venous thromboembolism and other major diseases. In recent years, the incidence of thrombosis-related diseases has been increasing globally, which not only significantly increases the risk of disability and death of patients, but also brings heavy economic burden to public health systems and families. The existing anti-thrombotic drugs have obvious limitations: the narrow treatment window makes it difficult to balance the risk of bleeding; complex interactions with many commonly used drugs may reduce efficacy or cause adverse reactions; long-term use may also cause safety problems such as gastrointestinal mucosa damage and bone metabolism disorder, and there is an urgent need to develop new anti-thrombotic drugs.

[0003] In the screening research of anti-thrombotic active compounds, a suitable animal model is a key technical bottleneck for discovering lead compounds. The current commonly used cell models and mammalian models have their own limitations: cell models, although simple to operate, can quickly study platelet function and signaling pathways in vitro, but lack a complete physiological environment, making it difficult to simulate the complex factors such as hemodynamics and vascular endothelial interaction during thrombus formation in vivo; mammalian models represented by mice are closer to human physiological conditions, but have problems such as high feeding cost, long experimental period, and difficult technical operation, which limit the efficiency of early high-throughput screening.

[0004] Zebrafish, as an emerging model organism, has unique advantages in the field of thrombosis research. Zebrafish has a short breeding cycle and a large single ovulation capacity, which can quickly obtain a large number of experimental samples and significantly improve the screening efficiency of active compounds. In addition, the zebrafish genome has more than 70% homology with humans, and the molecular mechanisms and pathological processes of thrombosis are highly similar to humans. Active compounds screened using zebrafish models have higher clinical transformation potential, providing an efficient and reliable research tool for the development of new anti-thrombotic drugs.

[0005] In the field of constructing zebrafish thrombus model, the prior art has developed various modeling methods. The current commonly used zebrafish thrombus inducers include arachidonic acid (AA), ferric chloride, ponatinib and phenylhydrazine. Ferric chloride and phenylhydrazine start the blood coagulation mechanism by oxidative damage to vascular endothelium, and ponatinib activates platelets by causing vascular endothelial dysfunction, but these methods have differences with the physiological thrombus formation mechanism of the human body, or have problems such as complex operation and poor stability. In contrast, arachidonic acid can specifically activate platelets by metabolizing thromboxane A2 (TXA2), promote platelet adhesion, aggregation and vasoconstriction, and can quickly induce platelet thrombus formation, and has unique advantages in simulating the physiological thrombus formation mechanism of the human body, so it has been widely concerned.

[0006] However, there are still significant limitations in using arachidonic acid as a zebrafish thrombus modeling reagent. As a highly active polyunsaturated fatty acid, arachidonic acid is easily inactivated by factors such as oxidation and light, so high-purity (≥98% HPLC grade) products must be used, and strict light-avoiding and low-temperature storage measures must be taken. The current market price of research-grade arachidonic acid is high, and the unit price of ≥98% purity control product is as high as 6300 yuan / gram, which significantly increases the experimental cost. More importantly, the existing conventional modeling method has the problem of low thrombus formation efficiency, and usually needs to increase the concentration of arachidonic acid to 80-100 μM and prolong the treatment time to more than 1 hour to achieve acceptable modeling effect. This high-concentration and long-time treatment method not only greatly increases the reagent consumption, but also significantly prolongs the experimental period, resulting in a double increase in reagent cost and time cost, which seriously restricts the high-throughput anti-thrombotic drug screening research based on zebrafish model. Therefore, developing a low-cost, efficient and stable zebrafish thrombus model has become a key technical problem to be solved in the current anti-thrombotic drug research and development field.

[0007] At present, there is no report on constructing a stable genetically CALR del52 Overexpression of zebrafish model related reports. SUMMARY

[0008] In view of the shortcomings of the prior art, the present application provides a zebrafish model of thrombocytosis accompanied by thrombosis and a construction method and application thereof.

[0009] The technical scheme of the present application is as follows:

[0010] A zebrafish model of thrombocytosis accompanied by thrombosis is constructed by overexpressing CALR del52 A transgenic zebrafish is obtained, and a thrombus inducer arachidonic acid is added to the transgenic zebrafish to induce and construct;

[0011] CALR del52 The nucleotide sequence of the recombinant expression vector is shown as SEQ ID NO. 1.

[0012] According to the application, the thrombosis-inducing agent arachidonic acid (AA) is added to the transgenic zebrafish at 3-4 dpf to induce the construction.

[0013] According to the application, the thrombocytosis zebrafish model is constructed by introducing the recombinant expression vector Tol2-EF1a-h CALR del52 The recombinant expression vector -IRES2-mcherry-UTRsv40 is introduced into wild-type AB zebrafish to overexpress CALR del52 The zebrafish with red fluorescence is selected and bred to adult zebrafish, which is then crossed with the green fluorescence-labeled platelet transgenic zebrafish Tg (cd41 :eGFP) to obtain the thrombocytosis transgenic zebrafish expressing red and green fluorescence simultaneously Tg (cd41 :eGFP) eGFP; EF1 a:CALR del52 ）， In the above method for constructing the thrombocytosis zebrafish model, the recombinant expression vector Tol2-EF1a-h Tg (cd41 :eGFP; EF1 a:CALR del52 ） The thrombocytosis zebrafish model is constructed by treating the zebrafish with 20-80 μΜ thrombosis-inducing agent arachidonic acid for 20-60 min;

[0014] Tol2-EF1a-h CALR del52 The nucleotide sequence of the recombinant expression vector -IRES2-mcherry-UTRsv40 is shown as SEQ ID NO. 2.

[0015] The method for constructing the thrombocytosis zebrafish model comprises the following steps:

[0016] (1) constructing the recombinant expression vector Tol2-EF1a-h CALR del52 -IRES2-mcherry-UTRsv40 with the nucleotide sequence shown as SEQ ID NO. 2;

[0017] (2) introducing the recombinant expression vector Tol2-EF1a-h CALR del52 -IRES2-mcherry-UTRsv40 constructed in step (1) and Tol2 transposase mRNA into wild-type AB zebrafish embryos, and breeding, selecting the zebrafish with red fluorescence as F0;

[0018] (3) After raising the F0 generation zebrafish selected in step (2) to adult zebrafish, they were crossbred with green fluorescently labeled platelet transgenic zebrafish. Tg (cd41 :eGFP) The zebrafish were hybridized, raised, and selected for their red and green fluorescence as the F1 generation. They were then identified to confirm that the F1 generation zebrafish had successfully inherited the target gene.

[0019] (4) The F1 generation zebrafish selected in step (3) were raised to adulthood and then self-crossed. The zebrafish with red and green fluorescence were selected as the F2 generation. They were then identified to confirm that the F2 generation zebrafish had successfully inherited the target gene. The F2 generation zebrafish and their offspring were all overexpressing the gene. CALR del52 Genetically modified zebrafish Tg (cd41 :eGFP; EF1 a:CALR del52 ） ;

[0020] (5) The transgenic zebrafish obtained in step (4) Tg (cd41 :eGFP; EF1 a:CALR del52 ） Feed the fish to 3-4 days post-capillary gestation (dpf), then treat them with 20-80 μM arachidonic acid for 20-60 min to establish a zebrafish model of thrombocytosis accompanied by thrombosis.

[0021] According to a preferred embodiment of the present invention, in step (3) or step (4), the zebrafish genome is amplified by PCR using primers Primer-F and Primer-R, and the amplification product is sequenced and identified; the sequence of Primer-F is shown in SEQ ID NO.3, and the sequence of Primer-R is shown in SEQ ID NO.4.

[0022] According to a preferred embodiment of the present invention, in step (5), 20-60 μM arachidonic acid is added and the animal is treated for 20-40 min to construct a zebrafish model of thrombocytosis accompanied by thrombosis.

[0023] The application of the above-mentioned zebrafish model with thrombosis or the zebrafish model with thrombosis constructed by the above method in screening drugs for the prevention or treatment of thrombosis.

[0024] The beneficial effects of the present invention include at least the following:

[0025] 1. To address the issues of varying degrees of side effects from current antithrombotic drugs and the lack of suitable experimental animal models for screening complex and trace amounts of natural active products, this invention, for the first time, constructs a zebrafish model of thrombosis accompanied by thrombocytosis. Tg (cd41 :eGFP; EF1 a:CALR del52 ）, and to establish a new technology for evaluating the anti-thrombotic activity of compounds based on a zebrafish model, thereby providing a powerful tool for the development of new anti-thrombotic drugs and solving the problem of disconnection between the research of trace natural products and their active effects.

[0026] 2. The thrombocytosis-induced thrombosis zebrafish model provided by the present application uses platelet-specific green fluorescence expression to observe the aggregation of platelets in real time, and realizes the visual evaluation of thrombosis development. Since the zebrafish has a fast breeding speed, transparent embryos, a short experimental period and a small dosage, it is beneficial to the high-throughput screening of trace anti-thrombotic active natural products. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Tol2-EF1a-h CALR del52 -IRES2-mcherry-UTRsv40 recombinant expression vector map.

[0028] Figure 2 F1 generation CALR del52 Overexpression zebrafish model construction map;

[0029] In the figure: A is CALR del52 Overexpression zebrafish fluorescence microscopic observation map; B is an electrophoresis map of PCR amplification products; the first lane on the left is 2000 marker: from bottom to top, 100 bp, 250 bp, 500 bp, 750 bp, 1000 bp, 1500 bp, 2000 bp, the second lane is F1 generation genome, and the third lane is a no-template negative control; C is a sequencing result representative peak map of F1 generation; the left box is the EF1a partial promoter sequence, and the right box is CALR del52 partial sequence.

[0030] Figure 3 F2 generation CALR del52 Overexpression zebrafish model sequencing result representative peak map;

[0031] In the figure: the left box is the EF1a partial promoter sequence, and the right box is CALR del52 partial sequence.

[0032] Figure 4 Zebrafish whole-body circulating platelet number statistics map; compared with the same group Tg (cd41 :eGFP) , ** P <0.01.

[0033] Figure 5 Zebrafish tail platelet fluorescence area and fluorescence intensity statistics map;

[0034] Figure: A is the tail platelet aggregation of zebrafish, B is the tail platelet fluorescence area chart, C is the tail platelet fluorescence intensity chart; compared with the same group Tg (cd41 :eGFP) , ** P <0.01.

[0035] Figure 6 Figure: A is the tail platelet aggregation of zebrafish, B is the tail platelet fluorescence area chart, C is the tail platelet fluorescence intensity chart; compared with the same group

[0036] Figure: A is the tail platelet aggregation of zebrafish, B is the tail platelet fluorescence area chart, C is the tail platelet fluorescence intensity chart; compared with the same group Tg (cd41 :eGFP) , ** P <0.01, *** P <0.001.

[0037] Figure 7 Figure: A is the tail platelet aggregation of zebrafish, B is the tail platelet fluorescence area chart, C is the tail platelet fluorescence intensity chart; compared with the same group Tg (cd41 :eGFP; EF1 a:CALR del52 ） Figure: A is the tail platelet aggregation of zebrafish, B is the tail platelet fluorescence area chart, C is the tail platelet fluorescence intensity chart; compared with the same group Tg (cd41 :eGFP) , ** P <0.01, *** P <0.001.

[0038] Figure 8 Figure: A is the tail platelet aggregation of zebrafish, B is the tail platelet fluorescence area chart, C is the tail platelet fluorescence intensity chart; compared with the same group

[0039] Figure: A is the tail platelet aggregation of zebrafish, B is the tail platelet fluorescence area chart, C is the tail platelet fluorescence intensity chart; compared with the same group Tg (cd41 :eGFP) control group, # P <0.05, ## P <0.01, ### P <0.001; AA represents arachidonic acid.

[0040] Figure 9 Figure: A is the tail platelet aggregation of zebrafish, B is the tail platelet fluorescence area chart, C is the tail platelet fluorescence intensity chart; compared with the same group

[0041] Figure: A is the tail platelet aggregation of zebrafish, B is the tail platelet fluorescence area chart, C is the tail platelet fluorescence intensity chart; compared with the same group & P <0.05,&&& P <0.001; vs. CALR del52 vs. AA group, # P <0.05, ## P <0.01; vs. CALR del52 vs. AA group, ** P <0.01, *** P <0.001. AA represents arachidonic acid, and ASA represents aspirin.

[0042] Figure 10 Figure 2 is a graph of blood flow velocity of caudal artery of zebrafish;

[0043] Figure 2 is a graph of blood flow velocity of caudal artery of zebrafish; &&& P <0.001; vs. CALR del52 vs. AA group, ### P <0.001; vs. CALR del52 vs. AA group, *** P <0.001; AA represents arachidonic acid, and ASA represents aspirin. DETAILED DESCRIPTION

[0044] The application will be further described in conjunction with specific examples, but the scope of the application is not limited thereto.

[0045] The contents not specified in the examples are carried out according to the conventional conditions; the reagents or instruments not specified by the manufacturer are all common commercially available products.

[0046] 1. Materials and instruments

[0047] 1.1 Experimental reagents

[0048] RNase-Free Deionized Water (Thermo Fisher Scientific, Cat# A57775), 50x TAE Buffer (Shanghai Generay Biotech Co., Ltd., Cat# B548101-0500), DL2000 Plus DNA Marker (Novizen Biotech Co., Ltd., Cat# MD101-01), Ultra GelRed (Novizen Biotech Co., Ltd., Cat# GR501-01), 2x Rapid Taq Master Mix (Novizen Biotech Co., Ltd., Cat# P222-01), Agarose (Yixing Shengao Biotech Co., Ltd., Cat# 10208ES60), Sodium Chloride (Shanghai Generay Biotech Co., Ltd., Cat# A610476-0001), FastDigest NotI (Thermo Fisher Scientific, Cat# FD0594), Potassium chloride (Shanghai Generay Biotech Co., Ltd., Cat# A610440-0500), Calcium chloride dihydrate (Shanghai Generay Biotech Co., Ltd., Cat# A610050-0500), Magnesium chloride hexahydrate (Shanghai Generay Biotech Co., Ltd., Cat# A601336-0500), Methylene Blue (Sigma-Aldrich Corporation, Cat# M9140), MS-222 (Sigma-Aldrich Corporation, Cat# A5040), 10x DNA Loading Buffer (Novizen Biotech Co., Ltd., Cat# P022-01), mMESSAGE mMACHINE™ SP6 Transcription Kit (Thermo Fisher Scientific, Cat# AM1340), One-step Cell Genotyping Kit (Nanjing Yaoshunyu Biotechnology Co., Ltd., Cat# K-101-100), YSY Buffer (Nanjing Yaoshunyu Biotechnology Co., Ltd., Cat# K-101-100), Axygen PCR Clean Kit (Axygen Biotech Co., Ltd., Cat# AP-PCR-50), Minerva SuperFusion Cloning Kit (Everbright inc, Cat# M2026S), pCS2-transposase plasmid (National Zebrafish Resource Center, Cat# CZP13), Nuclease-free Water (Thermo Fisher Scientific, Cat# AM9937), Lithium Chloride, Arachidonic acid (AA), Aspirin,ASA), phenylthiourea (1-Phenyl-2-thiourea, PTU).

[0049] 1.2 Experimental Apparatus

[0050] Zebrafish embryo microinjection instrument IM-300 (Neimo Corporation, Japan), Bori New XP gene amplification instrument TC-XP-G (Hangzhou Bori Technology Co., Ltd.), zebrafish rearing system (Beijing Aisheng Technology Co., Ltd.), horizontal electrophoresis system HE-120 (Shanghai Tianneng Technology Co., Ltd.), Tanon 1600 series multifunctional gel image analysis system (Shanghai Tianneng Life Science Co., Ltd.), Olympus inverted fluorescence microscope (Olympus Ltd., Japan, model SZX16), Ausen Nano-100 micro spectrophotometer (Hangzhou Ausen Instrument Co., Ltd., model Nano-100), low-temperature centrifuge (Eppendorf AG, Germany, model 5428000295), gradient PCR instrument (Thermal Cycler, model BIO-RAD C100), real-time PCR instrument (Roche, Switzerland, model LightCycler). 96) Micro-volume spectrophotometer (ThermoFisher Technology, USA, model NanoDrop™ OneC), ZebraBlood flowmeter v1.3.2 (ViewPoint, France).

[0051] 1.3 Experimental Animals

[0052] This experiment used wild-type AB strain zebrafish and transgenic zebrafish with green fluorescently labeled platelets. Tg (cd41 :eGFP) CALR Zebrafish can be purchased from the National Zebrafish Resource Center or from commercially available products. Male and female zebrafish should be raised separately under standard conditions of 14 hours of light / 10 hours of darkness at 28 ± 0.5℃, and fed with pelleted food at regular intervals. For egg collection, healthy, sexually mature zebrafish should be placed in a mating tank at a female-to-male ratio of 2:2 or 1:2. Fertilized eggs should be obtained between 10 and 11 am the following day. The embryos should be washed three times with zebrafish culture water, then disinfected with 0.1% methylene blue, and transferred to zebrafish culture water (5 mM NaCl, 0.33 mM CaCl2, 0.33 mM MgSO4•7H2O, and 0.17 mM KCl) and cultured under controlled light at 28 ± 0.5℃.

[0053] 2. Experimental Methods

[0054] 2.1 Construction of a zebrafish platelet hyperplasia model

[0055] 2.1.1 Tol2-EF1a-h CALR del52Design and construction of the IRES2-mcherry-UTRsv40 recombinant expression vector

[0056] EF1a is a potent and broad-spectrum promoter capable of driving gene expression in various cell types and tissues. Highly conserved in mammals, fish, and other eukaryotes, it is commonly used to construct transgenic animals or cell lines to achieve stable and efficient expression of target genes. IRES2 is an internal ribosome entry site that allows ribosomes to directly bind to mRNA and initiate translation without relying on the 5' cap structure. Its main function is to achieve polycistronic expression, enabling multiple genes to be co-expressed under the regulation of the same promoter. mCherry is a red fluorescent protein with a relatively simple gene sequence, facilitating fusion expression with other genes. Through genetic engineering, the mCherry gene can be linked to the target gene to construct a fusion protein expression vector. After intracellular expression, mCherry acts as a fluorescent tag, visually indicating the location of the protein encoded by the target gene. UTRSV40 is a highly efficient and reliable gene expression regulatory element widely used in animal expression vectors, playing a crucial role in gene expression regulation. It is used to enhance gene expression, improve mRNA stability, and optimize vector design. The Tol2 vector, capable of expressing the mcherry reporter gene throughout zebrafish, was modified using molecular cloning methods, by inserting a gene between the EF1a promoter and the mcherry coding region. CALR The nucleotide sequence after removing 52 bp from exon 9 yields Tol2-EF1a-h. CALR del52 -IRES2-mcherry-UTRsv40 recombinant expression vector. The vector construction was commissioned to Nanjing Yaoshunyu Biotechnology Co., Ltd.

[0057] CALR del52 The nucleotide sequence is shown in SEQ ID NO.1.

[0058] Tol2-EF1a-h CALR del52 The nucleotide sequence of the -IRES2-mcherry-UTRsv40 recombinant expression vector is shown in SEQ ID NO.2.

[0059] 2.1.2 Synthesis of Tol2 transposase mRNA

[0060] The pCS2-transposase plasmid (purchased from the National Zebrafish Research Center) was linearized with Not1 restriction endonuclease and transcribed using the mMESSAGE mMACHINE™ SP6 (Thermo Fisher Scientific) transcription kit according to the manufacturer's instructions to obtain Tol2 transposase mRNA. Purification was then performed using lithium chloride precipitation, and the Tol2 transposase mRNA was resuspended in 20 μL of nuclease-free water. RNA concentration was determined, and the RNA was stored frozen at −70°C.

[0061] The specific operating steps are as follows:

[0062] 2.1.2.1 pCS2-transposase plasmid linearization by digestion

[0063] The pCS2-transposase plasmid was linearized using the Not1 restriction endonuclease. The experimental procedures are shown in Table 1. The reaction conditions were: 37℃ water bath for 2 h. 0.5 µL of the sample was taken for agarose gel electrophoresis (1%) to determine its integrity.

[0064] Table 1 Enzyme digestion reaction system

[0065]

[0066] 2.1.2.2 Purification of in vitro transcription template

[0067] The recovered products were collected using a nuclease-free PCR cleaning kit (Axygen). The procedure is as follows:

[0068] 1. Add 3 volumes of Buffer PCR-A to the linearized plasmid, mix well, transfer to a preparation tube, place the preparation tube in a 2 mL centrifuge tube (provided in the kit), centrifuge at 12,000×g for 1 min, and discard the filtrate.

[0069] 2. Place the preparation tube back into a 2 mL centrifuge tube, add 700 μL Buffer W2, centrifuge at 12,000×g for 1 min, and discard the filtrate.

[0070] 3. Place the preparation tube back into the 2 mL centrifuge tube, add 400 μL Buffer W2, centrifuge at 12,000×g for 1 min, and discard the filtrate.

[0071] 4. Place the preparation tube into a clean 1.5 mL centrifuge tube (provided in the kit), add 30 μL of Eluent to the center of the membrane in the preparation tube, and let it stand at room temperature for 1 min. Centrifuge at 12,000×g for 1 min and collect the eluent.

[0072] 2.1.2.3 In vitro transcription

[0073] mRNA was synthesized using the mMESSAGE mMACHINE™ SP6 Kit transcription reagent and SP6 transcriptase. The reaction system is shown in Table 2. The reaction conditions were: 37°C for 2 h. After in vitro transcription was completed, 1 µL of TURBO DNase I was added, and the mixture was incubated at 37°C for another 15 min to remove the DNA template.

[0074] Table 2 In vitro transcription reaction system

[0075]

[0076] 2.1.2.4 Purification of in vitro transcribed mRNA

[0077] 1. Add 50 μL of lithium chloride precipitate solution to the reaction product tube in 2.1.2.3 and store overnight at −80°C.

[0078] 2. Centrifuge at 12000 g for 10 min at 4℃.

[0079] 3. Carefully discard the supernatant and add 1000 µL of 75% ethanol (prepared with DEPC water). Vortex thoroughly to wash, and gently tap the bottom of the tube to suspend the precipitate.

[0080] 4. Centrifuge at 12000g for 10 min at 4℃, discard the supernatant, and be careful not to lose the RNA precipitate.

[0081] 5. Repeat steps 3-4.

[0082] 6. Dry in a fume hood for 1 min, add 20 µL of Nuclease-free water to dissolve the RNA. After complete dissolution, take 1 μL of the sample for gel electrophoresis analysis and spectrophotometer concentration determination. Store the remaining solution at −80℃.

[0083] 2.1.3 Zebrafish microinjection and embryo selection

[0084] Wild-type AB line zebrafish fertilized eggs were collected using standard methods, and Tol2-EF1a-h Tg (cd41 :eGFP) del52 The -IRES2-mcherry-UTRsv40 recombinant expression vector was mixed with Tol2 transposase mRNA at a final concentration of 200 ng / μL and microinjected into zebrafish fertilized eggs within 15 minutes of fertilization. The injection volume was 1 nL per embryo. The injected zebrafish were treated with phenylthiourea (PTU) to 48 hpf (Hours Post Fertilization) for fluorescence observation. The observation of red fluorescent spots in the zebrafish indicated that the transgenic expression system was functioning normally. Fluorescent zebrafish were selected and raised to adulthood (F0). The F0 cells were then compared with...CALR Embryos expressing both red and green fluorescence were selected at 48 hpf and cultured to adulthood (F1). F1 males and females were self-crossed to produce F2 generation, and zebrafish expressing red and green fluorescence (i.e., overexpression) were selected. Tg (cd41 :eGFP; EF1 a:CALR del52 Transgenic zebrafish with green fluorescently labeled platelets CALR del52 ） .

[0085] 2.1.3.1 Extraction of F1 generation transgenic zebrafish genome

[0086] Five F1 embryos were collected to prepare genomic DNA templates. The genomic DNA templates were prepared using a one-step cell genotyping kit from Nanjing Yaoshunyu Biotechnology Co., Ltd. The reaction conditions were: 65℃ for 30 min, 95℃ for 5 min, 16℃ for 1 min, and 4℃.

[0087] 2.1.3.2 PCR detection of F1 generation transgenic zebrafish genome

[0088] h was determined by PCR amplification. CALR del52 Whether it integrates into the F1 generation zebrafish genome. PCR amplification system: 20 μL 2×Mastermix (Vazyme), 14 μL ultrapure water, 2 μL forward and reverse (Primer-F and Primer-R) primers (10 μM), and 2 μL genomic DNA template prepared in 2.1.3.1. PCR reaction conditions: 95℃ for 3 min, 34× (95℃ for 15 s, 56℃ for 15 s, 72℃ for 15 s), 72℃ for 5 min, 4℃. Amplification primer sequences are shown in Table 3.

[0089] Table 3 Primer sequences for PCR amplification

[0090]

[0091] 2.1.3.3 Extraction of F2 generation transgenic zebrafish genome

[0092] Five F2 generation zebrafish fertilized eggs were collected using standard methods. Genomic DNA templates were prepared using a one-step cell genotyping kit produced by Nanjing Yaoshunyu Biotechnology Co., Ltd. The specific reaction conditions were: 65℃ for 30 min, 95℃ for 5 min, 16℃ for 1 min, and 4℃.

[0093] 2.1.3.4 PCR detection of F2 generation transgenic zebrafish genome

[0094] Detection of h via PCR amplificationTg (cd41 :eGFP) del52 Whether it was successfully integrated into the F2 generation zebrafish genome. PCR amplification reaction system (40 μL): 20 μL 2×Mastermix (Vazyme), 14 μL ultrapure water, 2 μL forward and reverse (Primer-F and Primer-R) primers (10 μM), and 2 μL genomic DNA template prepared in 2.1.3.3. The amplified PCR products were sent to GE Sanger for sequencing. The amplification primer sequences are shown in Table 3.

[0095] 2.1.4 Research on Disease Progression in Transgenic Zebrafish

[0096] use Tg Zebrafish were used as a blank control to construct a zebrafish model with increased platelets. (cd41 :eGFP; EF1 a:CALR Tg (cd41 :eGFP) del52 ） Phenotypic observation was conducted. Starting from 3 dpf (Day Post Fertilization), 10 juvenile fish were randomly selected and placed under a Zeiss fluorescence microscope and blood flow analyzer. For 5 consecutive days, the number of circulating platelets, the area of ​​platelet aggregation in the tail and fluorescence intensity, changes in tail arterial blood flow velocity, and changes in the content of thrombosis-related factors were detected.

[0097] 2.1.4.1 Experimental Grouping

[0098] Experimental setup: blank control group Tg and the transgenic zebrafish model group with primary thrombocytosis (cd41 :eGFP; EF1 a:CALR Tg (cd41 :eGFP; EF1 a:CALR del52 ） Zebrafish with a dpf of 3-7 were selected under a microscope and transferred into a 24-well plate, with 10 zebrafish per well and 3 duplicate wells.

[0099] 2.1.4.2 Circulating platelet count detection

[0100] Select Tg (cd41 :eGFP) del52 ） and Tg (cd41 :eGFP; EF1 a:CALR Zebrafish strains were used, and the experimental groups are described in section 2.1.4.1. Ten juvenile zebrafish were randomly selected from each group and washed three times with zebrafish culture water. The number of circulating platelets flowing through the tail of the zebrafish within 15 seconds was recorded under an Olympus inverted fluorescence microscope, and statistical analysis was performed.

[0101] 2.1.4.3 Detection of platelet aggregation area and fluorescence intensity at the tail end

[0102] use Tg (cd41 :eGFP) del52 ）and Tg (cd41 :eGFP; EF1 a:CALR Zebrafish strains were used, and the experimental groups are described in section 2.1.4.1. Ten juvenile zebrafish were randomly selected from each group, and images of the zebrafish were acquired under an Olympus inverted fluorescence microscope. The platelet aggregation area and fluorescence intensity in the tail of the zebrafish were measured and calculated using Image-Pro Plus software, and statistical analysis was performed.

[0103] 2.1.4.4 Detection of blood flow velocity in the tail artery

[0104] Select Tg (cd41 :eGFP) del52 ） and Tg (cd41 :eGFP) Zebrafish strains were used, and the experimental groups are described in section 2.1.4.1. Ten juvenile zebrafish were randomly selected from each group, and the blood flow in the caudal artery of the zebrafish was recorded for 15 seconds using a blood flow meter. The blood flow velocity in the caudal artery of each group was statistically analyzed using ZebraBlood software.

[0105] 2.1.4.5 Detection of thrombosis-related factor levels

[0106] collect Tg (cd41 :eGFP; EF1 a:CALR Blank control group and Tg (cd41 :eGFP; EF1 a:CALR del52 ） Zebrafish in the thrombocytosis model group were washed three times with phosphate-buffered saline (PBSS). Following the manufacturer's instructions, PBSS (0.01 M, pH 7.4) was added, and the tissue homogenate was obtained by sonication. According to the manufacturer's instructions, thromboxane A2 (TXA2), von Willebrand factor (vWF), tissue factor (TF), and D-dimer, all closely related to thrombosis, were detected using an enzyme-linked immunosorbent assay (ELISA) kit. Sixty zebrafish were used in each group, and each experiment was performed three times.

[0107] 2.2 Construction of a zebrafish model of thrombocytosis accompanied by thrombosis

[0108] 2.2.1 Determination of Arachidonic Acid Treatment Concentration and Treatment Time

[0109] Select 3-4 dpf Tg (cd41 :eGFP) del52 ） and Tg (cd41 :eGFP; EF1 a:CALR Transgenic zebrafish, with a blank control group (cd41 / CALR) set up in the experiment. del52 -Control), Arachidonic acid (AA) treatment group (cd41 / CALR) del52(+different concentrations of AA), 10 zebrafish per well, with 3 replicates. The blank control group was treated with zebrafish culture water, while the arachidonic acid treatment groups were treated with different concentrations of arachidonic acid solution. All groups were incubated at 28 ± 0.5℃ in the dark for different durations. After arachidonic acid treatment, 15 juvenile zebrafish were randomly selected from each group, washed three times with zebrafish culture water, and the blood flow in the caudal artery was recorded for 15 seconds using a blood flow meter. The blood flow velocity in the caudal artery of each group was statistically analyzed using ZebraBlood software.

[0110] 2.3 Applicability Validation of Antithrombotic Drug Screening in a Zebrafish Model with Thrombosis and Thrombosis

[0111] 2.3.1 Experimental Grouping

[0112] Zebrafish with a 3-4 dpf growth rate were selected under a microscope and transferred into 24-well plates. Two blank control groups (cd41, CALR) were set up for the experiment. del52 ), thrombosis model group (CALR) del52 +AA), positive drug aspirin (ASA) group (CALR) del52 (+AA+ASA), 10 strips per well, with 3 replicates. The blank control group and thrombosis model group were treated with zebrafish culture water, while the positive control group was treated with 125 μM aspirin solution. Zebrafish in all groups were incubated at 28 ± 0.5℃ for 6 h. The solution was then aspirated. The blank control group was treated with zebrafish culture water, while the other groups were treated with arachidonic acid (AA) solution at a final concentration of 20 μM and kept in the dark for 20 min.

[0113] 2.3.2 Circulating platelet count detection

[0114] Select Tg (cd41 :eGFP) del52 ） and Tg (cd41 :eGFP; EF1 a:CALR For transgenic zebrafish, the experimental grouping and drug treatment are described in 2.3.1. The experimental procedure is the same as in 2.1.4.2.

[0115] 2.3.3 Detection of platelet aggregation area and fluorescence intensity at the tail end

[0116] use Tg (cd41 :eGFP) del52 ） and Tg (cd41 :eGFP; EF1 a:CALR For transgenic zebrafish, the experimental grouping and drug treatment are described in 2.3.1. The experimental procedure is the same as in 2.1.4.3.

[0117] 2.3.4 Detection of blood flow velocity in the tail artery

[0118] Select Tg (cd41 :eGFP) del52 ）and CALR For zebrafish strains, experimental grouping and drug treatment are described in 2.3.1. Experimental procedures are the same as in 2.1.4.4.

[0119] 3. Experimental Results

[0120] 3.1 Construction of a zebrafish platelet hyperplasia model

[0121] 3.1.1 Tol2-EF1a-h CALR del52 Construction results of the -IRES2-mcherry-UTRsv40 recombinant expression vector

[0122] Tol2-EF1a-h Figure 1 del52 The map of the -IRES2-mcherry-UTRsv40 recombinant expression vector is shown below. Tg (cd41 :eGFP; EF1 a:CALR As shown, its sequence is SEQ ID NO.2.

[0123] 3.1.2 Tg (cd41 :eGFP; EF1 a:CALR del52 ） Zebrafish screening and identification results

[0124] 3.1.2.1 CALR del52 ） F1 generation screening and identification

[0125] Choose those with red fluorescence Tg (cd41 :eGFP) Genetically mutated zebrafish were raised to adulthood (F0), and then F0 was compared with... Figure 2 Hybridization was performed, and zebrafish embryos expressing both red and green fluorescence were selected at 48 hpf (F1). CALR As shown in Figure A, the development of the organism to 48 hours was observed under an inverted fluorescence microscope. Figure 2 In F1 generation zebrafish with gene mutation, red and green fluorescent spots were found in the heart area, indicating that the transgenic expression system can function normally and that platelets are specifically labeled with green fluorescent protein. CALR B is CALR52 Electrophoresis image of PCR amplification products from F1 generation of mutant zebrafish, showing the PCR amplification covering part of the EF1a promoter sequence and part of the EF1a promoter sequence. Figure 2 Sequence. The results showed that the mutant gene was successfully transferred into the zebrafish genome. CALR The C-value indicates that Tol2-EF1a-h can be detected by sequencing the F1 generation. Tg (cd41 :eGFP; EF1 a:CALR del52 The key elements of the -IRES2-mcherry-UTRsv40 recombinant expression vector demonstrate that the mutated gene is heritable.

[0126] 3.1.2.2 Figure 3del52 ） F2 generation screening and identification

[0127] Transgenic F1 zebrafish exhibiting red and green fluorescence were raised to adulthood and then self-crossed to obtain the F2 generation. The genome sequence of the F2 generation zebrafish was then determined. CALR As shown, Tol2-EF1a-h can be detected by sequencing the F2 generation. Tg (cd41 :eGFP; EF1 a: del52 The key elements of the -IRES2-mcherry-UTRsv40 recombinant expression vector indicate stable inheritance. CALR Tg (cd41 :eGFP; EF1 a:CALR del52 ） The transgenic zebrafish has been successfully constructed.

[0128] 3.1.3 Transgenic Zebrafish Figure 4 del52 ） Disease progression research results

[0129] 3.1.3.1 Changes in circulating platelet count

[0130] Platelet count and platelet aggregation function are important indicators for assessing platelet function and activation status, and help to determine the risk of thrombosis. Tg (cd41 :eGFP; EF1 a:CALR visible, Tg del52 ） Transgenic zebrafish and control group (cd41 :eGFP) Figure 5 Compared to zebrafish, the number of circulating platelets increased at 3 dpf, 4 dpf, 5 dpf and 6 dpf, with a significant difference at 5 dpf.

[0131] 3.1.3.2 Changes in platelet aggregation area and fluorescence intensity at the tail end

[0132] Tg (cd41 :eGFP; EF1 a:CALR visible, Tg (cd41 :eGFP) del52 ） Transgenic zebrafish and control group Figure 6 Compared to zebrafish, the area of ​​platelet aggregation in the tail increased and the fluorescence intensity increased at 3 dpf and 4 dpf. As the number of days increased, the platelets aggregated in the tail gradually entered the systemic circulation after 4 dpf, and there were fewer platelets aggregated in the tail of zebrafish at 5 dpf and beyond.

[0133] 3.1.3.3 Changes in blood flow velocity in the tail artery

[0134] Tg (cd41 :eGFP; EF1 a:CALR It can be seen that 3 dpf and 4 dpf Tg del52 ）Transgenic zebrafish and control group ​ (cd41 :eGFP) Compared to zebrafish, the blood flow velocity in the caudal artery was significantly slower, but no spontaneous thrombus formation occurred; the caudal artery blood flow velocity recovered at 5-7 days post-flood (dpf), suggesting... CALR del52 The blood circulation disorder in overexpressing transgenic zebrafish was self-healed to a certain extent, meaning that the blood flow velocity returned to normal.

[0135] 3.1.3.4 Changes in the levels of thrombosis-related factors

[0136] Increased platelet count leads to increased blood viscosity, microcirculatory disturbances, and the release of procoagulant substances such as thromboxane A2 (TXA2). These substances can activate other platelets, promoting platelet aggregation and increasing the probability of thrombus formation. Vonoblastic hemophilia factor (vWF) is an important plasma component that plays a hemostatic role by binding to platelets and collagen fibers to form thrombi. Tissue factor (TF) is the receptor for coagulation factor VII, responsible for initiating the extrinsic coagulation cascade and promoting fibrin clot formation. D-dimer is produced after the degradation of cross-linked fibrin, indicating intravascular coagulation and serving as a marker of overall activation of the coagulation and fibrinolytic systems. Therefore, this study examined transgenic zebrafish at 3-7 dpf and 14 dpf. Tg (cd41 :eGFP; EF1 a:CALR del52 ） Changes in the levels of TXA2, vWF, TF, and D-dimer in the body. Results showed Tg (cd41 :eGFP; EF1 a:CALR del52 ） The levels of thrombosis-related factors in transgenic zebrafish were significantly higher than those in other species. Tg (cd41 :eGFP) Blank control group ( Figure 7 ).

[0137] The above disease progression research results show that, compared with the control group Tg (cd41 :eGFP) Compared to zebrafish, Tg (cd41 : eGFP; EF1 a:CALR del52 ） Transgenic zebrafish showed an increase in circulating platelet count at 3-6 days post-flop (dpf), with a significant difference at 5 dpf. At 3 and 4 dpf, the area and fluorescence intensity of platelet aggregation in the tail increased; after 4 dpf, these tail-aggregated platelets gradually entered systemic circulation, with fewer at 5 dpf and beyond. At 3 and 4 dpf, the blood flow velocity in the tail artery significantly decreased without spontaneous thrombosis; the blood flow velocity recovered at 5-7 dpf, suggesting... Tg (cd41 :eGFP; EF1 a:CALR del52 ） Zebrafish exhibited spontaneous remission. Furthermore, this study examined the expression levels of thrombosis-related factors in zebrafish. It was found that... Tg (cd41 :eGFP; EF1 a:CALR del52） The levels of thrombosis-related factors in zebrafish were significantly higher than those in other fish. Tg (cd41 :eGFP) In summary, this study selected patients with a 3-4 day post-thrombotic risk (dpf) of higher risk. Tg (cd41 :eGFP; EF1 a:CALR del52 ） Construction of a zebrafish model of thrombocytosis accompanied by thrombosis.

[0138] 3.2 Construction of a zebrafish model of thrombocytosis accompanied by thrombosis

[0139] 3.2.1 Effects of different concentrations of arachidonic acid treatment for different durations on blood flow velocity in zebrafish

[0140] In this study, the zebrafish thrombocytosis model showed a significant decrease in blood flow velocity, but no spontaneous thrombosis was observed. Thrombosis is a complex pathological process, triggered not only by intrinsic factors such as coagulation system abnormalities, platelet activation, and endothelial injury, but also by exogenous factors such as chemical substances, hemodynamic changes, and inflammatory stimulation. Therefore, this study used exogenous arachidonic acid (AA) to treat transgenic zebrafish with thrombocytosis to construct a thrombosis model. Different concentrations of arachidonic acid were used to treat 3-4 dpf transgenic zebrafish. Tg (cd41 :eGFP; EF1 a:CALR del52 ） Different times. Figure 8 visible, Tg (cd41 :eGFP) The blood flow velocity in the tail artery of zebrafish after treatment with 20 μM arachidonic acid for 20 min was compared with... Tg (cd41 :eGFP) There was no significant difference compared to the control group. Under the same treatment concentration and treatment time conditions, CALR del52 Overexpression of zebrafish Tg (cd41 :eGFP; EF1 a:CALR del52 ） The blood flow velocity in the tail artery was significantly lower than that in other arteries. Tg (cd41 :eGFP) In the control group, local blood flow obstruction and thrombus formation were observed. In the treatment groups with higher concentrations (40 μM, 60 μM, 80 μM) and longer treatment times (40 min, 60 min), Tg (cd41 :eGFP; EF1 a:CALR del52 ） The blood flow velocity in the tail artery was significantly lower than that in other arteries. Tg (cd41 :eGFP) Control group. Therefore, treatment with 20-80 μM arachidonic acid for 20-60 min was used as the control group for transgenic zebrafish in this study. Tg (cd41 :eGFP; EF1 a:CALR del52 ） A method for thrombosis modeling was used to construct a zebrafish model of thrombosis accompanied by thrombocytosis. Previous literature often used arachidonic acid at concentrations of 80 μM or higher to treat non-thrombotic platelets. CALR del52A thrombosis model was induced in zebrafish by overexpression for more than 1 hour. In this study... CALR del52 Overexpression of arachidonic acid, which is required for thrombosis in zebrafish, requires lower concentrations and shorter treatment times, enabling the screening of antithrombotic compounds at lower cost and higher efficiency.

[0141] 3.3 Applicability Validation of Aspirin (ASA), an Antithrombotic Therapy Drug, in a Zebrafish Model of Thrombosis-Related Thrombotic Disease with Thrombosis

[0142] To verify the applicability of the established zebrafish model of thrombocytosis accompanied by thrombosis for drug screening, aspirin, an internationally recognized antithrombotic drug, was selected to test its antithrombotic effect on the model. The core mechanism of aspirin's antithrombotic effect is the inhibition of platelet aggregation. This is achieved by irreversibly inhibiting cyclooxygenase (COX)-1 and (COX)-2, preventing the production of TXA2 from arachidonic acid, thereby blocking TXA2-induced platelet aggregation and exerting an antithrombotic effect. In this study, aspirin was used to treat juvenile zebrafish in the thrombocytosis-associated disease model. The antithrombotic activity of aspirin was assessed by detecting indicators such as circulating platelet count, tail platelet aggregation area and fluorescence intensity, and tail artery blood flow velocity to verify whether this model is suitable for antithrombotic drug screening.

[0143] 3.3.1 Effects of aspirin on circulating platelet count, tail platelet aggregation area, and fluorescence intensity

[0144] Arachidonic acid causes thrombosis by inducing platelet aggregation. Figure 9 The results showed that, compared with the blank control group, platelet aggregation occurred in the tail of zebrafish in the thrombosis model group, the number of circulating platelets was significantly reduced, and the area of ​​aggregated platelets and fluorescence intensity in the tail were significantly increased. Compared with the thrombosis model group, the number of circulating platelets in zebrafish in the positive drug aspirin group was significantly increased, and the area of ​​aggregated platelets and fluorescence intensity in the tail were significantly reduced.

[0145] 3.3.2 Effect of Aspirin on Blood Flow Velocity in the Caudal Artery

[0146] An increased platelet count increases blood viscosity, leading to slower blood flow. Slower blood flow makes it easier for formed elements in the blood to deposit on the blood vessel walls, causing platelets to adhere to vascular endothelial cells and aggregate, thereby promoting thrombus formation. Figure 10 It can be seen that, compared with the blank control group, CALR del52 +AA group: The blood flow velocity in the caudal artery of zebrafish was significantly slowed, and thrombi formed locally in the arteries and veins, indicating that the thrombosis model was successfully established. CALR del52 The caudal artery flow velocity in the +AA+ASA group of zebrafish was lower than that in the CALR group. del52The significant increase in AA levels indicates that aspirin has antithrombotic efficacy in a zebrafish model of thrombocytosis accompanied by thrombosis.

[0147] This study investigated the overexpression of [a specific gene] in zebrafish somatic cells. CALR del52 By combining low-concentration arachidonic acid (RAA) as a thrombotic inducer with short-term induction, a zebrafish model of thrombosis accompanied by thrombosis was successfully constructed. Tg (cd41 :eGFP; EF1 a: CALR del52 ） Aspirin is a commonly used antithrombotic drug in clinical practice. In a zebrafish model of thrombocytosis accompanied by thrombosis, aspirin treatment significantly increased the number of circulating platelets, significantly reduced the platelet aggregation area and fluorescence intensity in the tail, significantly restored the circulatory disturbances caused by arachidonic acid, and significantly increased the blood flow velocity in the tail artery. The antithrombotic therapeutic effect of aspirin was significant and highly consistent with its efficacy in human clinical treatment, verifying that the zebrafish model of thrombocytosis accompanied by thrombosis provided by this invention can be used for screening antithrombotic drugs. The zebrafish model of thrombocytosis accompanied by thrombosis provided by this invention has a short experimental cycle and requires small dosage, which is beneficial for high-throughput screening of trace amounts of antithrombotic active natural products.

Claims

1. A method for constructing a thrombophilia zebrafish model, characterized in that, Comprising the following steps: (1) Constructing a Tol2-EF1a-h CALR del52 -IRES2-mcherry-UTRsv40 recombinant expression vector; (2) Through microinjection, the recombinant expression vector Tol2-EF1a-h CALR del52 IRES2-mcherry-UTRsv40 and Tol2 transposase mRNA into wild type AB line zebrafish embryos, feeding, and selecting red fluorescent zebrafish as F0 generation; (3) After the F0 generation zebra fish selected in step (2) is bred to adult zebra fish, the adult zebra fish is crossed with the green fluorescent marker platelet transgenic zebra fish Tg (cd41:eGFP) Hybridization, breeding, and selection of red and green fluorescent zebra fish as F1 generation, identification, and determination of successful inheritance of target genes in F1 generation zebra fish; (4) The F1 generation zebra fish selected in step (3) is bred to adult zebra fish, and then self-crossed, and the zebra fish with red and green fluorescence is selected as F2 generation, identified, and determined that the F2 generation zebra fish successfully inherit the target gene, and the F2 generation zebra fish and its offspring are all over-expressed CALR del52 transgenic zebra fish Tg (cd41:eGFP; EF1a:CALR del52 ） ; (5) the transgenic zebrafish obtained in step (4) is fed until 3-4 dpf, 20-80 μM arachidonic acid is added for 20-60 min, and a thrombocytosis with thrombosis zebrafish model is constructed. Tg (cd41:eGFP; EF1a:CALR del52 ） The transgenic zebrafish obtained in step (4) is fed until 3-4 dpf, 20-80 μM arachidonic acid is added for 20-60 min, and a thrombocytosis with thrombosis zebrafish model is constructed.

2. The construction method of claim 1, wherein, In step (3) or step (4), the zebrafish genome is amplified by PCR using primers Primer-F and Primer-R, and the amplified product is sequenced and identified; the sequence of Primer-F is shown in SEQ ID NO. 3, and the sequence of Primer-R is shown in SEQ ID NO.

4.

3. The construction method of claim 1, wherein, In step (5), 20-60 μM arachidonic acid is added for 20-40 min to construct a thrombocytosis with thrombosis zebrafish model.

4. Use of the thrombocytosis with thrombosis zebrafish model constructed by the method of any one of claims 1-3 in screening drugs for preventing or treating thrombosis.

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