Use of non-CpG phosphorothioate oligonucleotides in preparation of antiplatelet drugs
阅读说明：本技术 非CpG的硫代磷酸寡核苷酸在制备抗血小板药物中的用途 (Use of non-CpG phosphorothioate oligonucleotides in preparation of antiplatelet drugs ) 是由 夏荣 韩佳 丁忠仁 潘冠星 陈绍恒 于 2020-05-26 设计创作，主要内容包括：本发明属生物制药技术领域,涉及抗血小板的核酸药物,具体涉及非CpG结构的硫代磷酸寡核苷酸在制药中的新用途,经实验证实Let7b inhibitor-PS静脉给药可抑制FeCl3诱发的血栓形成,且药物浓度增加,抑制效果增强；与阳性药物替罗非班比,在相同的抗栓能力下,Let7b inhibitor-PS出血副作用小；Let7b inhibitor-PS可抑制血小板的聚集、分泌、铺展及血块回缩,未硫代磷酸化修饰的Let7b inhibitor不具有上述功能；同时Let7b inhibitor-PS对血小板活化功能的抑制,不具有序列依赖性,但含有CpG基序的硫代磷酸寡核苷酸除外；所述的非CpG结构的硫代磷酸寡核苷酸可用于制备抗血小板的核酸药物,进一步作为预防和治疗动脉血栓性疾病的药物。(The invention belongs to the technical field of biological pharmacy, relates to a nucleic acid medicament for resisting platelet, and particularly relates to a new application of phosphorothioate oligonucleotide with a non-CpG structure in pharmacy, and experiments prove that the intravenous administration of Let7b inhibitor-PS can inhibit the thrombosis induced by FeCl3, the concentration of the medicament is increased, and the inhibition effect is enhanced; compared with the positive medicament tirofiban, under the same antithrombotic capacity, Let7b inhibitor-PS has small bleeding side effect; the Let7b inhibitor-PS can inhibit the aggregation, secretion, spreading and retraction of blood clots of platelets, and the Let7b inhibitor which is not modified by thiophosphorylation does not have the functions; meanwhile, Let7b inhibitor-PS has no sequence dependence on the inhibition of the platelet activation function, except for phosphorothioate oligonucleotides containing CpG motifs; the phosphorothioate oligonucleotide with the non-CpG structure can be used for preparing a nucleic acid medicament for resisting platelet, and further can be used as a medicament for preventing and treating arterial thrombotic diseases.)
1. Use of a non-CpG phosphorothioate oligonucleotide, which is any phosphorothioate-modified oligonucleotide sequence that does not contain a CpG motif, in the preparation of an antiplatelet agent;
the antiplatelet drug is a nucleotide drug for resisting platelet aggregation.
2. Use according to claim 1, wherein said phosphorothioate oligonucleotide is an aptamer to the platelet surface function receptor GPIb.
3. The use according to claim 1, wherein the platelet aggregation inhibiting nucleotide medicament is for the manufacture of an antithrombotic medicament.
4. The use according to claim 1, wherein the platelet aggregation inhibiting nucleotide medicament is for the manufacture of a medicament for the prevention of arterial thrombotic disorders.
5. The use according to claim 1, wherein the platelet aggregation inhibiting nucleotide medicament is for the manufacture of a medicament for the treatment of arterial thrombotic disorders.
6. Use according to claim 4 or 5, wherein the arterial thrombotic disease is coronary heart disease, ischemic stroke and peripheral arterial occlusive disease.
Technical Field
The invention belongs to the technical field of biological pharmacy, relates to an antiplatelet nucleic acid medicament, and particularly relates to a novel application of phosphorothioate oligonucleotides with non-CpG structures in pharmacy.
Background
The study published in the new england journal in 2015 shows that in nearly 20 years, the number of deaths of cardiovascular diseases in the world is increased by 40.8%, and ischemic heart disease is the largest cause of the increase of the number of cardiovascular deaths (the total number of cardiovascular deaths is increased by 500 ten thousand, wherein the number of ischemic heart disease is 240 ten thousand); ischemic stroke is the second; studies in asian areas have shown that more than 120 million deaths in 2013 increased by 47.2% over 1990. In 2017, the cardiovascular disease reports in China indicate that death of cardiovascular diseases accounts for more than 40% of death of resident diseases, and the death of cardiovascular diseases is the top and higher than that of tumors and other diseases. Clinical studies disclose that arterial thrombosis, manifested as Myocardial Infarction (MI) and ischemic stroke, is a major cause of death and disability worldwide. It is believed that platelets play a fundamental role in mediating atherosclerotic thrombosis and, therefore, antiplatelet agents are central to the prevention and treatment of arterial thrombosis.
Currently, the following 4 antiplatelet drugs are mainly used in clinical practice, and can be used alone or in combination according to the situation: cyclooxygenase1 (COX 1) inhibitor (aspirin), ADPP2Y12Receptor antagonists (cagrelor, clopidogrel, prasugrel and ticagrel), protease-activated receptor 1 (PAR 1) antagonists (vorapasha) and GPIIb/IIIa antagonists (abciximab, eptifibatide and tirofiban). The existing antiplatelet drugs respectively inhibit important targets of a normal hemostasis process, the upper limit of clinical benefit seems to be reached, tolerance and resistance phenomena exist in clinical application practice, and the increase of treatment intensity inevitably leads to the increase of bleeding risk. In summary, there is still a need to develop new antiplatelet drugs that allow safer, more effective, and more extensive platelet inhibition to meet the existing clinical needs.
The new platelet activation theory in the field suggests that there is a great difference between the normal hemostatic process and the pathological mechanism of thrombus formation, and these differences are potential targets for novel antiplatelet drugs. Fully activated platelets at the core of the thrombus, i.e., the site of arterial injury, are highly dependent on soluble agonists, such as thrombin; in contrast, a persistent thrombus is composed of platelets in a poorly activated state, and platelets recruited in this region appear to be less sensitive to traditional antiplatelet drugs; the industry is increasingly aware of the regulatory mechanisms of excessive thrombus growth, and provides the possibility of developing therapeutic drugs with low bleeding risk. Studies have shown that changes in blood flow in atherosclerotic lesions due to stenosis appear to be an important factor in regulating thrombus growth, and the interaction between GPIb and vWF is more potent under high shear stress conditions in stenosed arteries, so that studies on anti-GPIb or vWF antibodies, snake venom-derived GPIb antagonists, anti-vWF aptamers, etc. have been developed at different stages of clinical trials;
at the same time, single-stranded protein-binding oligonucleotides (i.e., aptamers) have made significant new advances in antithrombotic therapy. Preclinical studies of aptamers against von willebrand factor, FVIIa-tissue factor complex, FIXa and thrombin have led to encouraging results, especially the construction of a reversal agent of pharmaceutical activity, which can provide targeted and controlled therapy for patients with thrombosis. Although aptamer drugs have many advantages over protein antibodies, to date, no report has been made on aptamers that target receptors that function on platelet surface.
Based on the current situation of the prior art, aiming at the defects of the existing antiplatelet drugs, a novel antiplatelet nucleic acid drug is provided, and the novel application of the phosphorothioate oligonucleotide with a non-CpG structure in pharmacy is related, so that the phosphorothioate oligonucleotide is further used for preventing and treating arterial thrombotic diseases.
Disclosure of Invention
The invention aims to provide a novel antiplatelet nucleic acid medicament based on the current situation of the prior art and aims at overcoming the defects of the conventional antiplatelet medicament, and relates to a novel application of a non-CpG-structure phosphorothioate oligonucleotide in pharmacy.
The invention provides a new application of phosphorothioate oligonucleotide with non-CpG structure in preparing antiplatelet nucleic acid medicine, the invention carries out in vitro experiment and in vivo experiment, the in vitro experiment proves that Let7b inhibitor-PS can inhibit the aggregation, secretion, spreading and blood clot retraction of platelets, and Let7b inhibitor which is not modified by phosphorothioate has no function; meanwhile, Let7b inhibitor-PS is found to inhibit the platelet activation function, and has no sequence dependence except phosphorothioate oligonucleotide containing CpG motif; in vivo experiments, a mouse mesenteric artery thrombus model is induced by FeCl3, and the fact that the thrombus formation induced by FeCl3 can be inhibited after Let7b inhibitor-PS is administrated intravenously is proved, and the inhibition effect is enhanced along with the increase of the drug concentration; the result of a mouse truncation experiment shows that the Let7b inhibitor-PS has small bleeding side effect under the same antithrombotic capacity compared with the positive medicament tirofiban.
Specifically, the invention is implemented by the following technical scheme,
firstly, the fact that Let7b inhibitor-PS can be combined with GPIb is verified in a molecular way through a cell Thermal transition analysis (CETSA); secondly, realizing a three-dimensional space mode of interaction between Let7b inhibitor-PS and GPIb and an amino acid binding site by molecular simulation and molecular docking technology by virtue of computer bioinformatics, and simultaneously presuming that Let7b inhibitor-PS and GPIb can be bound because of the loose nucleotide space structure caused by the breakage of molecular hydrogen bonds; the experimental technology proves that the phosphorothioate oligonucleotide with the non-CpG structure can be combined with the platelet surface functional receptor GPIb in a spatial structure;
the invention further passes a series of in vivo functional experiments, such as FeCl3An induced mouse mesenteric artery thrombus model proves that the Let7b inhibitor-PS can inhibit FeCl after intravenous administration3Induced arterial thrombosis and with increasing drug concentration, the inhibitory effect is enhanced; the result of a mouse tail-cutting experiment shows that the bleeding side effect caused by Let7b inhibitor-PS is small compared with the positive medicament tirofiban under the same antithrombotic effect;
experiments prove that the Let7b inhibitor-PS and other phosphorothioate oligonucleotides without CpG motifs can be used for preparing antithrombotic drugs;
the phosphorothioate oligonucleotide with non-CpG structure can be used as aptamer of platelet surface function receptor GPIb to inhibit platelet activation and aggregation, and can be used for preventing and treating arterial thrombotic diseases.
In the invention, the aptamer-non-CpG phosphorothioate oligonucleotide refers to a short-chain nucleotide which is bound with a specific protein in a spatial conformation, wherein the specific protein refers to GPIb, and only the oligonucleotide modified by phosphorothioate can be bound with the specific protein; the binding is not sequence specific; however, phosphorothioate oligonucleotides containing CpG structures cannot bind to them.
In the invention, in vitro experiment results show that Let7b inhibitor-PS (a phosphorothioate modified Let7b inhibitor nucleotide sequence) inhibits the aggregation and secretion of human and mouse platelets; inhibit the spreading of the blood platelet and the retraction of blood clot, and the inhibition effect is enhanced along with the increase of the concentration; however, Let7b inhibitor-PO (the Let7b inhibitor nucleotide sequence which is not modified by phosphorothioation) does not have the inhibition function, which indicates that Let7b inhibitor-PS has the potential of becoming an antiplatelet drug; secondly, the experiment of the invention shows that other sequences of phosphorothioate oligonucleotides can also inhibit the activated aggregation of the platelets, but the phosphorothioate oligonucleotides containing CpG motifs can not inhibit the activated aggregation of the platelets;
the experimental results of the invention show that: the phosphorothioate oligonucleotide without the CpG structure can inhibit platelet aggregation under the action of Thrombin and Ristocetin agonists, and the two agonists can activate platelets through a functional receptor GPIb on the surface of the platelets.
In the invention, the Let7b inhibitor-PS nucleotide sequence is shown as a sequence 1.
In the invention, the nucleotide sequence is shown as a sequence 2-4.
The phosphorothioate oligonucleotide with a non-CpG structure has the following characteristics when being further used as a medicament for preventing and treating arterial thrombotic diseases:
1. the targeted antithrombotic treatment effect is good;
2. the bleeding side effect is small;
3. provides a novel anti-platelet nucleotide drug,
4. the phosphorothioate oligonucleotide with non-CpG structure can be used as aptamer of platelet surface function receptor GPIb to play antithrombotic role.
Drawings
FIG. 1: in vitro experiments show that Let7b inhibitor-PS has inhibition on platelet activation function, while Let7b inhibitor-PO does not have the above function, wherein,
(a) under the action of thrombin and ristomycin agonist, Let7b inhibitor-PS inhibits platelet aggregation of human and mouse, and under the action of other agonists, the inhibitor has no inhibition;
(b) under the action of thrombin, Let7b inhibitor-PS inhibits the secretion of human platelet ATP (graph A);
let7B inhibitor-PS inhibited the spreading of platelets on fibrinogen slides (panel B);
let7b inhibitor-PS showed concentration-dependent inhibition of clot retraction (panel C).
FIG. 2: let7B inhibitor-PS is sequence-independent with respect to platelet function inhibition, wherein phosphorothioate-oligonucleotides (PS-ODN) (1. mu.M) inhibit platelet aggregation and PO-ODN (1. mu.M) does not inhibit platelet aggregation under the action of thrombin (Panel A) and ristocetin agonist (Panel B); CpG typeA-PS can cause spontaneous platelet aggregation in the absence of agonists.
FIG. 3: the binding of Let7b inhibitor-PS and Poly T-PS to GPIb was confirmed by the cell Thermal transition analysis (CETSA) technique under the temperature gradient and the concentration gradient of the drug; CpG type A-PS cannot bind to GPIb; wherein the content of the first and second substances,
(a) under the action of temperature gradient, Let7b inhibitor-PS and Poly T-PS can delay the denaturation of GPIb protein, GPVI and P2Y12 protein on the surface of platelet are not affected, and CpG type A-PS can not play the role;
(b) under the action of concentration gradient, with the increase of the concentration of Let7b inhibitor-PS and Poly T-PS drugs, the denaturation of GPIb protein is delayed, while GPVI and P2Y12 protein are not affected, and CpG type A-PS can not play the role.
FIG. 4: molecular simulation and molecular docking are carried out on the interaction of the Let7b inhibitor-PS and the GPIb by utilizing a biological information technology, and it is predicted that the Let7b inhibitor-PS can be combined with the GPIb because the effect of intermolecular hydrogen bonds is destroyed after phosphorothioate modification, so that the Let7b inhibitor-PS has a loose molecular structure and is easy to combine, wherein,
molecular modeling of Let7b inhibitor-PO and Let7b inhibitor-PS (Panel A);
let7B schematic diagram of the three-dimensional structure of interaction of inhibitor-PS with GPIb (Panel B);
let7b inhibitor-PS amino acid binding site for interaction with GPIb (panel C).
FIG. 5: utilizing laser confocal to carry out co-localization analysis on Let7b inhibitor-PS and GPIb, wherein,
(A) on the adhesion glass, platelets are respectively incubated with Let7b inhibitor-PO (10 mu M) and Let7b inhibitor-PS (10 mu M) for 5min, the green fluorescence of the PO group drugs is scattered and distributed without aggregation from the enlarged image of a single platelet, and the green fluorescence of the PS group drugs is mostly aggregated on the surfaces of the platelets, so that the false phenomenon that the drug amount of the PS group is more than that of the PO group drugs as shown in the first two groups of pictures is caused;
(B) on the fibrinogen glass, platelets were incubated with Let7b inhibitor-PO (10. mu.M) and Let7b inhibitor-PS (10. mu.M) for 30min, respectively, and both Let7b inhibitor-PO and Let7b inhibitor-PS could enter into the plasma of platelets through endocytosis.
FIG. 6: after the thrombocyte is activated by thrombin, Western blot detects the inhibition of Let7b inhibitor-PS on the activation of GPIb downstream signal molecules, wherein the inhibition on downstream phosphorylated proteins (Syk 1/2(Tyr525/526), PLC2(Tyr525/52, Akt (Ser473) and Erk1/2(T202/Y204) is stronger along with the increase of the concentration of the medicament.
FIG. 7: the effect of Let7b inhibitor-PS at different concentrations on mouse thrombosis and tail bleeding time compared to tirofiban, wherein,
(A) in Fecl3In the mouse arterial thrombosis model, compared with tirofiban, the inhibition of thrombosis of mice by Let7b inhibitor-PS with different concentrations is enhanced along with the increase of the concentration of Let7b inhibitor-PS (figure i)), and the inhibition of thrombosis is realized through>20 statistical analysis by time of thrombosis (panel ii)) and time of complete occlusion of mesenteric arterial vessels (panel iii)), Let7b inhibitor-PS (15mg/kg) and Tirofiban (Tirofiban, 0.5mg/kg) were similar in antithrombotic capacity;
(B) under the same antithrombotic action, Let7b inhibitor-PS (15mg/kg) was injected intravenously for a shorter time than tail bleeding of Tirofiban (Tirofiban, 0.5mg/kg) (. p <0.05 vs. salt,. p <0.01 vs. salt,. p <0.005 vs. salt, NS ═ not significant signent.).
Detailed Description
Example 1 in vitro experiments verify that Let7b inhibitor-PS inhibits platelet activation function
1) Designing and synthesizing Let7b inhibitor-PS and Let7b inhibitor-PO:
finding an RNA sequence 5 'UGAGGUAGUAGGUUGUGUGGUU 3' of hsa-Let-7b-5p by an online website (http:// mirdb. org/cgi-bin/target _ detail. cgitargetid ═ 1913611), wherein a complementary sequence is an hsa-Let7b-5pinhibitor sequence 5 'AACCACACAACCUACUACCUAC 3', and carrying out phosphorothioate modification on the sequence to obtain Let7b inhibitor-PS, wherein the unmodified sequence is Let7b inhibitor-PO (synthesized by Shanghai Jie Li Co.);
2) preparation of washed platelets:
(1) human platelets: platelets are from healthy volunteers who have signed informed consent and do not take anti-platelet drugs such as aspirin and clopidogrel 20 days before blood drawing, venous blood of healthy donors is collected in a 10% ACD-A anticoagulation tube, centrifuged for 300g × 20 minutes, supernatant is collected to obtain platelet-rich plasma, the platelet-rich plasma is collected for 900g × 10 minutes, platelet-rich precipitates are collected and resuspended in Tyrode's (134mM NaCl, 2.9mM KCL0.34mM Na2PO4, 12mM 3, 20mM HEPES, 1mM MgCl2, 5mM glucose, 0.5mg/mL albumin, pH 7.4) buffer, and the number of platelets is adjusted to 3 × 108/mL;
(2) mouse platelets: anesthetizing by intraperitoneal injection of pentobarbital sodium (60mg/kg), performing abdominal aorta puncture on an anesthetized mouse to collect whole blood, placing the whole blood into a 3.8% ACD anticoagulation tube, adding 0.5ml of physiological saline, taking the supernatant after 270g multiplied by 1 min, repeating the operation for 1 time, centrifuging the supernatant, suspending the obtained precipitate with Tyrode's buffer solution for 420g multiplied by 3 min, and preparing washed platelets;
3) measurement of platelet aggregation and secretion:
platelet aggregation and secretion was measured using a platelet aggregometer (Model 400VS, Chrono-Log, Haverston, PA), with parameters set: the speed of agitation was 900rpm, the temperature was 37 ℃, the platelets were co-incubated with drug at 37 ℃ for 5 minutes prior to agonist addition, the platelet aggregation curve monitoring time was 5 minutes, and the aggregation rate was defined as 100% of the solvent control. In measuring ATP secretion from platelets, luciferin/luciferase (3 μ l) was added to 300 μ l of the washed platelet suspension 2 minutes prior to agonist stimulation, taking care to keep out light. The aggregation results under the action of different agonists are shown in FIG. 1(a), and the secretion results are shown in FIG. 1 (b);
4) spreading of platelets:
coating 100. mu.g/ml fibrinogen on a glass slide with 0.1M NaHCO3(pH8.3), overnight at 4 ℃, platelets (1X 107/ml) adhering to and spreading on the fibrinogen coated slide for 1h, fixing, rupturing the membrane, staining with phalloidin-labeled actin, observing the morphology of the adhering platelets with an Olympus fluoview FV1000 confocal microscope, collecting images using IMAGEJ software, measuring the diffusion area of individual platelets in pixel units, randomly selecting areas from at least three different tests for statistical analysis, and Let7b inhibitor-PS inhibiting the platelet spreading function as shown in FIG. 1 (b);
5) and (3) clot retraction:
washed human platelets (4X 10)8/ml) was resuspended in Tyrode buffer in an unused aggregation tube and mixed with 100. mu.l of human plasma (PPP), 10ul of human alpha-thrombin (10U/ml, physiological saline) was added) And 1.5ul of CaCl2(2mM), observed at room temperature, monitored every 15min and photographed, and the clot size quantified from the photograph using J2X software, the inhibition of clot retraction by Let7b inhibitor-PS is shown in FIG. 1 (b).
Example 2 Let7b inhibitor-PS shows sequence-independent experiments on platelet function inhibition
Phosphorothioate oligonucleotide (PS-ODN) refers to a derivative in which the oxygen atom with double bond on the phosphate is replaced by sulfur atom in the oligonucleotide chain, and is resistant to nuclease, thereby prolonging its action time in vivo; to determine whether other nucleotide sequences can inhibit platelet activation after thio-modification, four broad classes of oligonucleotide sequences were synthesized by shanghai jeli: a. let7b inhibitor without CpG motifs; b. random sequence R22 without CpG; c. CpG type a containing CpG motif; d. a 22-nucleotide polymer (polyA, T, C, U) without CpG motif (as shown in sequence listing); each sequence has two divisions of sulfo-modification and non-sulfo-modification, platelet aggregation rate is monitored under the action of agonists Thrombin, Ristocetin, AYPGKF and SFLLRN, and 8 healthy donors are arranged in each group; the experimental results show that: in addition to phosphorothioate oligonucleotides containing CpG motifs, other sequences inhibited platelet aggregation by Thrombin and Ristocetin, while non-phosphorothioated oligonucleotides did not inhibit platelet aggregation (as shown in FIG. 2).
Example 3 cellular thermal transition analysis verification of phosphorothioate oligonucleotide binding to GPIb
The Cellular Thermal Shift Assay (CETSA) technique, based on the feature of improved Thermal stability when proteins and drugs are combined, directly detects the binding affinity of the drug and the target protein in cells or tissues: the target cells and the drug were incubated at a fixed temperature for a certain period of time, and then the target protein in the cytoplasm was detected by Western blotting (Western Blot), with the results shown in fig. 3(a) and 3 (b).
Example 4 computer modeling of Let7b inhibitor-PS binding to GPIb Using bioinformatics
Repeated and repeated pre-experiment results show that the Let7b inhibitor-PS inhibits the activation of platelets under the action of Thrombin and Ristocetin, the action target of the aptamer Let7b inhibitor-PS is a platelet surface functional receptor GPIb, the combination of the Let7b inhibitor-PS and GPIb is simulated through a bioinformatics computer, and the reason and the combination site of the further conjecture are shown in FIG. 4.
Example 5 Observation of the similarities and differences between Let7b inhibitor-PO, Let7b inhibitor-PS and platelets by immunofluorescence staining of platelets
Platelet TB resuspension (1X 10) was obtained by conventional methods7Ml), incubating the platelet suspension and the drug at 37 ℃ for 5min, sucking 150uL of platelets to drip on an adhesive glass, placing the adhesive glass in an oven at 37 ℃ for 30min, standing and washing 1 × PBS for 3 times, dripping 100uL of fixative at room temperature, standing and washing for 15min, using 1 × PBS again, standing and washing 3 times, dripping 150uL 5% BSA (1 × PBS) to incubate at room temperature for 1h, diluting primary antibody with 1 × PBS according to the proportion of 1:200 after washing, incubating for 12-14h (optionally prolonged time) at 4 ℃ according to the condition) (incubation in a wet box), covering on a glass slide after dripping 20uL of anti-fluorescence quencher after standing and washing for 3 times, diluting secondary antibody with 1 × PBS according to the proportion of 1:200, incubating for 1-1.5h at room temperature for 200 uL/piece in a dark place, finally sealing, smearing a proper amount of anti-fluorescence quencher on the glass slide after dripping 20uL of the anti-fluorescence quencher on the glass slide, and observing the co-localization of the GPIt 7-84 b and the GPIt-84 b of the LEt, the results are shown in FIG. 5 (A);
platelet TB resuspension (1X 10) was obtained by conventional methods7And/ml), co-incubating the platelet suspension and the drug at 37 ℃ for 30min, sucking 150uL of platelets to drip on an adhesive glass, placing the adhesive glass in an oven at 37 ℃ for 30min, and observing that both Let7B inhibitor-PO and Let7B inhibitor-PS can enter platelet cytoplasm through endocytosis by a confocal microscope (as shown in figure 5 (B).
Example 6 inhibition of activation of GPIb downstream signaling molecules by Let7b inhibitor-PS with Thrombin agonists
Detecting the expression of phosphorylated protein in a downstream pathway after GP1b activation by an immunoblotting method (Western blot): incubating Let7b inhibitor-PS with different concentrations and platelets for 5min, and collecting and cracking a sample by using a platelet aggregation instrument under the action of an agonist; preparing Tris-glycine SDS polyacrylamide gel, mixing protein and loading buffer solution, loading the mixture, performing gel electrophoresis, transferring the membrane, placing the mixture into 1 xTBST containing 3% BSA for 2h, sealing the mixture for 2h, adding corresponding primary antibody Syk1/2(Tyr525/526), PLC2(Tyr525/52, Akt (Ser473) and Erk1/2(T202/Y204) for overnight incubation at 4 ℃, adding HRP-labeled secondary antibody (1:1000) for incubation for 1h at room temperature after washing the membrane, exposing and developing by using ECL fluorescence developing solution A and B, and detecting a protein band of Western blot and measuring the spectral density by using a QuantityOne image analysis system, wherein the result is shown in figure 6.
Example 7 demonstration of the arterial antithrombotic effect of Let7b inhibitor-PS and the bleeding tendency after intravenous administration of Let7b inhibitor-PS in mice
This example is via FeCl3Inducing mouse mesenteric artery thrombosis model, separating blood platelet from donor mouse, Calcein (Calcein-AM)5 μ g/ml labeling, and injecting Calcein labeled blood platelet (5 × 10) into mouse tail vein6Perg) and drug, circulation 30min, mesenteric vascular bed everted, thrombotic process observed under fluorescence microscope (Leica DM5500) (10% FeCl)3Soaked 3mm2Filter paper damages vessel wall to induce thrombus), a confocal microscope (Leica TCS SPE) records image data of thrombus formation in real time, and the result is shown in figure 7, that Let7b inhibitor-PS can inhibit FeCl after intravenous administration3Induced thrombosis and increased inhibitory effect with increasing drug concentration;
male mice (BALB/C) aged 6-8 weeks were anesthetized with 10% chloral hydrate (400mg/kg) intraperitoneally, the tail was cut off at 3mm, immediately immersed in physiological saline at 37 ℃, and the bleeding cessation time was recorded, i.e., no rebleeding occurred within 60 seconds, with the experimental results shown in fig. 7.
Sequence listing
>Sequence 1Let7b inhibitor (hsa-Let7b-5p inhibitor) without CpG motif
5’AACCACACAACCUACUACCUAC 3’
>Sequence 2Random sequence R22 without CpG
5’UUGUACUACACAAAAGUACUGC 3’
>Sequence 3CpG type A containing CpG motif
5’GGGGGACGATCGTCGGGGG 3’
>Sequence 422 nucleotide polymers (polyA, T, C, U) without CpG motifs
5’AAAAAAAAAAAAAAAAAAAAAA 3’
5’TTTTTTTTTTTTTTTTTTTTTT 3’
5’CCCCCCCCCCCCCCCCCCCCCC 3’
5’UUUUUUUUUUUUUUUUUUUUUU 3’。
SEQUENCE LISTING
<110> Huashan Hospital affiliated to Fudan university
Application of <120> non-CpG phosphorothioate oligonucleotide in preparation of antiplatelet drugs
<130>
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> RNA
<213> hsa-let7b-5p inhibitor
<400> 1
aaccacacaa ccuacuaccu ac 22
<210> 2
<211> 22
<212> RNA
<213> R22
<400> 2
uuguacuaca caaaaguacu gc 22
<210> 3
<211> 19
<212> DNA
<213> CpG type A
<400> 3
gggggacgat cgtcggggg 19
<210> 4
<211> 22
<212> DNA
<213> poly A,T,C,U
<400> 4
aaaaaaaaaa aaaaaaaaaa aa 22
<210> 5
<211> 22
<212> DNA
<213> poly A,T,C,U
<400> 5
tttttttttt tttttttttt tt 22
<210> 6
<211> 22
<212> DNA
<213> poly A,T,C,U
<400> 6
cccccccccc cccccccccc cc 22
<210> 7
<211> 22
<212> RNA
<213> poly A,T,C,U
<400> 7
uuuuuuuuuu uuuuuuuuuu uu 22