Application of CLCA1 as intervention target of thrombogenesis

Abstract

The invention provides application of CLCA1 as an intervention target spot of thrombus generation, and belongs to the field of biological medicine. It is found for the first time that CLCA1 can participate in thrombosis, and low expression of CLCA1 can reduce the blood flow velocity of blood vessels, promote erythrocyte aggregation in the blood vessels and up-regulate the expression level of thrombus-related genes Fga, f7 and PAI-1. The CLCA1, as an intervention target of thrombus occurrence, can be used for screening or preparing antithrombotic drugs, preparing products for evaluating thrombus occurrence risks and constructing animal thrombus models, and has a clinical application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to application of CLCA1 as an intervention target for thrombosis. BACKGROUND

[0002] Thrombus refers to a small piece formed on the surface of a blood vessel in the cardiovascular system at a peeling or repairing site, and is composed of insoluble fibrin, deposited platelets, accumulated white blood cells and red blood cells. The formation of thrombus is related to vascular injury, enhanced blood coagulation function, slow blood flow, genetic predisposition and other causes. Genetic factors such as antithrombin deficiency, congenital abnormal fibrinogenemia, thrombophilia gene mutation, etc. can all cause an increase in pro-coagulation components in the blood, inducing thrombosis.

[0003] Calcium-activated chloride channel regulator 1 (CLCA1) is a kind of secreted split and zinc-dependent metalloproteinase, which can activate calcium-dependent chloride ion current in mammalian cells and has the characteristics of zinc metalloproteinase. Current studies have shown that CLCA1 can play an important role in the pathogenesis of diseases related to high mucus secretion in the respiratory and digestive systems, such as bronchial asthma and ulcerative colitis, by exerting its metalloproteinase and anion channel activation effects. At the same time, due to its regulation of chloride ion and other anions in epithelial cells, it will also participate in the occurrence and development process of malignant tumors. However, there is no report on the correlation between CLCA1 and thrombosis. SUMMARY

[0004] In order to solve the problems existing in the prior art, the purpose of the present application is to provide the application of CLCA1 as an intervention target for thrombosis, including screening or preparing anti-thrombotic drugs, preparing products for evaluating the risk of thrombosis, and constructing an animal thrombosis model, which has a clinical application prospect.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0006] The present application provides the application of CLCA1 as an intervention target for thrombosis in screening or preparing anti-thrombotic drugs.

[0007] Preferably, the low expression of CLCA1 reduces the blood flow speed of the blood vessel.

[0008] Preferably, the low expression of CLCA1 promotes the aggregation of red blood cells in the blood vessel.

[0009] Preferably, the low expression of CLCA1 up-regulates the expression level of thrombus-related genes; the thrombus-related genes include Fga, f7 and PAI-1.

[0010] The present application also provides the application of a CLCA1 expression promoter in the preparation of a drug for preventing and / or treating thrombosis.

[0011] Preferably, the medicine further comprises a pharmaceutically acceptable carrier.

[0012] The present application also provides the use of CLCA1 in the preparation of a product for evaluating the risk of thrombosis.

[0013] Preferably, the product comprises an antibody, a medicine and a kit.

[0014] The use of a CLCA1 expression inhibitor in the construction of an animal thrombosis model.

[0015] Preferably, the CLCA1 expression inhibitor comprises a CLCA1 gene-specific siRNA.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] The present application first discovers that CLCA1 can participate in thrombosis, low expression of CLCA1 can reduce the blood flow rate of a blood vessel, promote the aggregation of red blood cells in the blood vessel, and up-regulate the expression level of thrombosis-related genes Fga, f7 and PAI-1. CLCA1 as an intervention target of thrombosis can be used for screening or preparing an anti-thrombosis medicine, preparing a product for evaluating the risk of thrombosis, and constructing an animal thrombosis model, and has a clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 : Silencing efficiency of the CLCA1 gene;

[0019] Figure 2 : Effect of CLCA1 gene silencing on blood flow rate;

[0020] Figure 3 : Effect of CLCA1 gene silencing on the aggregation of zebrafish red blood cells;

[0021] Figure 4 : Effect of CLCA1 gene silencing on thrombosis-related genes Fga, f7 and PAI-1. DETAILED DESCRIPTION

[0022] The application provides application of CLCA1 as an intervention target of thrombosis in screening or preparation of an anti-thrombus drug. The amino acid sequence of the CLCA1 is MDWRTVFVVLWMLLPYPSTAI KLDGGGYVDITIAIGAKVKQDDTLIDKIKEMVTDGSFYLYHALDKKVYLKDATILVPSHWSCKSCSKARTESFEKAKIKIDHAKLMEPRTKLYGECGKGGEYIHFTPDFLLNDSAIQMYGPRGKVFLHEWAHLRWGVYDEYNEEKPFYLSNGRVEYTRCTTNIEGQIFEVNGGSPQSCRINPETFLPSSDCKFFPNKDQNTDSSVMFSPSLEAVTTFCRETEHNYEAETQQNIICNNKATWTVIFEDSVDKDALSFLPPLPSTPSPPIFKLLQRKKRAVCLILDVSGSMATESRILRMRQAATHLLRNYVEEQASVGIVKFSTAASIVSSLTIIESDATRDHLINLLPETPGGSTNMCNGLRLGLQVLSEDDMDAIGDEIIFLTDGQATDDVTLCIPDAINSGAIIHTIALSDSAHNALQEMADKTGGIFFYSKDDFTSNQLMDAFASLTLSTGDHSNEPVQLESVGTRTSDWFNGTVSVDQTVGNKTSFVIIYERIFPAVYIQSPSGIVYTEAQMNHDNRLKTVTLKVAETAEPGDWAYSIKTSTDQAFTITVTSQASHEDVPPIIVKTRMNQQVSDGTKPMTVFAEVSQNYRPVINVEVWATLEPATGPIQSLQLLDNGAGADVMANDGIYSRYFTKMVNGRCSLKVRAKNQDGKARFAVQKRSGAPYVPGYVVDGVVELNPPNPSVSDEPIEVGSFTRTATGESFEVTLTGSTPPNFPPSKVTDLGAKILEEAVLLSWTAPGEDLDQGRAKSYEIRWSFDLDMLRQDFSNGHVVDTATVSPQEAGSVEQHAFNLSLPIQNGTTLFFAVQTLDEHDAKSDTSNIASASMILPNPKPPGISNPGLNLTVLVSSLCAVTIVICIIAGVTTWAVRRRRPALRI, as shown in SEQ ID No. 1.

[0023] The present application firstly discovers that the expression amount of CLCA1 is related to the thrombus phenotype, and the low expression of CLCA1 (CLCA1 silencing) can reduce the blood flow rate of the blood vessel and promote the aggregation of red blood cells in the blood vessel.

[0024] Fga is a gene encoding fibrinogen A chain, and fibrinogen is one of the main coagulation factors in blood, which participates in the blood clotting process. f7 is a gene encoding coagulation factor VII (prothrombin C), and factor VII is part of the extrinsic coagulation pathway, which can be activated by tissue factor (TF) to start the coagulation cascade. PAI-1 gene (SERPINE1), i.e. complete plasminogen activator inhibitor 1, is a member of the serpin superfamily of serine protease inhibitors. The low expression of CLCA1 (CLCA1 silencing) in the present application can up-regulate the expression levels of thrombus-related genes Fga, f7 and PAI-1, and promote the formation of thrombus.

[0025] The present application also provides the use of a CLCA1 expression promoter in the preparation of a medicine for preventing and / or treating thrombus. The present application uses CLCA1 as a target for anti-thrombus drugs to prepare a medicine for preventing and / or treating thrombus, and the active ingredient of the anti-thrombus drug is a CLCA1 expression promoter. By including a promoter for the expression of CLCA1, the expression amount of CLCA1 is promoted, and the blood flow rate of the blood vessel is too low, the aggregation of red blood cells is too excessive, and the expression level of thrombus-related genes is too high, so as to achieve the control of thrombus. The thrombus in the present application is preferably a venous thrombus.

[0026] Preferably, the anti-thrombus drug in the present application further comprises a pharmaceutically acceptable carrier, and a pharmaceutically acceptable excipient is added to the anti-thrombus drug to ensure the preparation of the anti-thrombus drug and the clinical application of the prepared anti-thrombus drug. Further preferably, the excipient in the present application is selected from at least one of diluents, wetting agents, binders, lubricants, coloring agents, and coating agents.

[0027] Preferably, the dosage form of the anti-thrombus drug in the present application is selected from at least one of tablets, capsules, granules, pills, injections, suspensions, dispersions, and syrups. However, the dosage form of the anti-thrombus drug in the present application is not limited to this, and other dosage forms that can be achieved are within the protection scope of the present application.

[0028] The application also provides the use of CLCA1 in the preparation of a product for evaluating the risk of thrombosis. Preferably, the product comprises an antibody and a kit. The application preferably evaluates the risk of thrombosis by detecting the expression amount of CLCA1 protein or the transcription amount of CLCA1 mRNA. The lower the expression amount of CLCA1 protein or the transcription amount of CLCA1 mRNA, the higher the risk of thrombosis. As an alternative embodiment, the antibody of the application is an antibody for detecting the expression of CLCA1 protein, which can be an antibody ab180851 from Abeam; and the kit of the application can be a kit for detecting the gene expression amount of CLCA1 by qPCR.

[0029] The application also provides the use of a CLCA1 expression inhibitor in the construction of an animal thrombosis model. As an alternative embodiment, the CLCA1 expression inhibitor of the application comprises a CLCA1 gene-specific siRNA, the sense strand of the siRNA being CUCCAGACUUUCUCUUAAATT, as shown in SEQ ID No. 2; and the antisense strand of the siRNA being UUUAAGAGAAAGUCUGGAGTT, as shown in SEQ ID No. 3. The animal of the application is preferably a zebrafish.

[0030] In the following examples, unless otherwise specified, the methods are conventional. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0031] In the application, the Axygen RNA extraction kit (AP-MN-MS-RNA-250G) is purchased from Axygen; the Bio-Rad reverse transcription kit (1708891) is purchased from Bio-Rad; and the Bio-Rad qPCR detection kit (172-5121) is purchased from Bio-Rad.

[0032] Fish liquid: 3.6g sodium bicarbonate, 10g sea salt, 15mL methyl blue solution.

[0033] O-anisidine staining solution: weigh 30mg 3-3′ dimethoxy benzidine, 41mg anhydrous sodium acetate, add 323μL 30% hydrogen peroxide, 20mL anhydrous ethanol, after dissolution, add ultrapure water to 50mL, avoid light, until the staining solution turns dark red and can be used for staining.

[0034] In the application, the primer sequences used are shown in Table 1. The data are statistically analyzed using GraphPad Prism 8.0. *, P<0.05, **, P<0.01, ***, P<0.001.

[0035] Table 1 Primer sequences of each gene

[0036]

[0037] Example 1

[0038]

[0039] Zebrafish breeding: Zebrafish were maintained in water at 28±0.5℃ with a 14:10 h light:dark cycle and were mated after four months of age. After spawning, fertilized embryos were collected and cultured in fish water, which was changed daily and dead embryos were removed by pipetting. Embryos of zebrafish with red blood cell fluorescence labeling at half an hour after fertilization were used for microinjection. Zebrafish (Tg(gata1:DsRed)) were purchased from the National Zebrafish Resource Center, ID number CZ67: http: / / www.zfish.cn / .

[0040] The above synthetic siRNA and nonsense siRNA were respectively diluted with DEPC water to obtain a 50 μM stock solution. 1 μL of the stock solution was taken and microinjected into Tg(gata1:DsRed) zebrafish embryos (about 200 embryos per microliter). The experimental group (labeled as Clca1 SiRNA) was injected with CLCA1 gene-specific siRNA, and the control group (labeled as Control) was injected with nonsense siRNA.

[0041] Microinjection steps are as follows:

[0042] Step 1: Preparation of microinjection samples

[0043] The injection sample needs to be prepared in advance, 1 μL for 200 embryos.

[0044] Step 2: Preparation of microinjection needles

[0045] Before the microinjection experiment, the injection needle needs to be drawn using a needle puller.

[0046] Step 3: Preparation of injection plates

[0047] 1.5 g of agarose powder was dissolved in 100 mL of deionized water, and the mixture was heated in a microwave oven until dissolved. 15 mL of the agarose solution was poured into a 90 mm culture dish. After it cooled slightly and could be touched by hand but had not yet begun to solidify, the plate preparation mold was slowly inverted onto the surface of the agarose solution. After it completely solidified, the mold was pulled out from one side of the short edge, and the microinjection plate was obtained.

[0048] Step 4: Embryo preparation

[0049] The parent fish were mated the night before, and the fertilized eggs were collected the next morning. The collected fertilized eggs were injected at the 1-cell stage.

[0050] Step 5: Microinjection of zebrafish embryos

[0051] Turn on the microinjector. First, open the main valve of the nitrogen tank and adjust the pressure to between 0 and 0.5 MPa. Then, turn on the microinjector switch and adjust the injection pressure value on the main control panel to between 15 and 20 psi, the pulse time to between 40 and 50 ms, and the pressure value on the negative pressure panel to approximately 0.5 psi to prevent backflow and sample leakage during injection. During the experiment, fine-tune the pressure and pulse time according to the size of the ruptured needle until the appropriate injection pressure and pulse time are achieved. After rupture, load the sample into the needle and prepare for injection. Place the embryo to be injected under a stereomicroscope, focus the low-power objective lens on the embryo, and gently lower the needle tip using the micromanipulator to center the injection needle in the field of view. Fine-tune the needle position using the micromanipulator until the needle tip is clearly visible. Further adjust the microscope focus and the position of the injection needle and embryo to achieve optimal clarity for both the embryo and the injection needle tip. Insert the injection needle through the yolk sac and inject the sample into the animal.

[0052] 72 hours after microinjection, total RNA was extracted from zebrafish in each group using the Axygen RNA Extraction Kit (AP-MN-MS-RNA-250G), and cDNA was obtained using the Bio-Rad Reverse Transcription Kit (1708891). The relative expression level of the CLCA1 gene was detected using the Bio-Rad qPCR Detection Kit (172-5121). The results are as follows: Figure 1 As shown. By Figure 1 The results showed that the average expression level in the experimental group was 0.0015, while the average expression level in the control group was 0.005. There was a significant difference between the two groups (P<0.05), indicating that the CLCA1 gene was silenced.

[0053] 72 hours after microinjection, images were taken under a fluorescence vertical microscope and a 5-second video was recorded to observe the blood flow velocity throughout the zebrafish and in its heart. The blood flow velocity was analyzed using Danio Scope software, and blood flow curves were plotted. Results are as follows: Figure 2 As shown. By Figure 2 The results showed that the average blood flow activity in the experimental group was 0.5, while that in the control group was 17. There was a significant difference between the two groups (P<0.001), indicating that the blood flow velocity in the CLCA1 gene silencing group was significantly slower.

[0054] 72 hours after microinjection, the zebrafish were stained with a prepared o-anisidine dye solution at room temperature in the dark for 15 minutes. After staining, they were eluted three times with fish solution, each time for 10 minutes. The eluted zebrafish were fixed with 1% low-melting-point agarose and images were taken using a stereofluorescence microscope (Leica M205FA stereo microscope, Germany). The results are as follows: Figure 3 As shown. By Figure 3 It can be seen that, compared with the control, the CLCA1 gene silencing group showed that red blood cells were significantly concentrated in the tail vein and the number of red blood cells in the heart area was significantly reduced.

[0055] After microinjection for 72h, total RNA of zebrafish was extracted by Axygen RNA extraction kit (AP-MN-MS-RNA-250G), and cDNA was prepared by Bio-Rad reverse transcription kit (1708891), and the expression of thrombus related genes was detected by Bio-Rad qPCR detection kit (172-5121). The results are shown in Table 2. Figure 4 Figure 4 It can be seen that after CLCA1 gene silencing, the expression levels of thrombus related genes Fga (P<0.05), f7 (P<0.001) and PAI-1 (P<0.01) can be significantly improved.

[0056] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. Application of CLCA1 as an intervention target for thrombosis in the screening or preparation of antithrombotic drugs.

2. The application according to claim 1, characterized in that, Low CLCA1 expression reduces blood flow velocity in blood vessels.

3. The application according to claim 1, characterized in that, Low expression of CLCA1 promotes the aggregation of erythrocytes in blood vessels.

4. The application according to claim 1, characterized in that, The low expression of CLCA1 upregulates the expression levels of thrombosis-related genes, including Fga, f7, and PAI-1.

5. Application of CLCA1 expression promoters in the preparation of drugs for the prevention and / or treatment of thrombosis.

6. The application according to claim 5, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

7. Application of CLCA1 in the preparation of products for assessing the risk of thrombosis.

8. The application according to claim 7, characterized in that, The products include antibodies, drugs, and reagent kits.

9. Application of CLCA1 expression inhibitors in the construction of animal thrombosis models.

10. The application according to claim 9, characterized in that, The CLCA1 expression inhibitors include CLCA1 gene-specific siRNAs.

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