CN111879949B - Application of substance for detecting or regulating lactoferrin expression quantity in preparation of medicine or kit for preventing and/or treating cardiovascular and cerebrovascular diseases - Google Patents

Application of substance for detecting or regulating lactoferrin expression quantity in preparation of medicine or kit for preventing and/or treating cardiovascular and cerebrovascular diseases Download PDF

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CN111879949B
CN111879949B CN202010776990.3A CN202010776990A CN111879949B CN 111879949 B CN111879949 B CN 111879949B CN 202010776990 A CN202010776990 A CN 202010776990A CN 111879949 B CN111879949 B CN 111879949B
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lactoferrin
substance
regulating
antibody
cerebrovascular diseases
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CN111879949A (en
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赖仞
唐小芃
程若梅
吕秋敏
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Kunming Institute of Zoology of CAS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/323—Arteriosclerosis, Stenosis

Abstract

The invention relates to an application of a substance for detecting or regulating the expression level of lactoferrin in the preparation of a medicament or a kit for preventing and/or treating cardiovascular and cerebrovascular diseases, belonging to the technical field of medical molecular biology. The invention provides the application of a substance for detecting or regulating the expression quantity of lactoferrin, and the lactoferrin has good effect on treating stroke, atherosclerosis or other cardiovascular and cerebrovascular diseases by taking the lactoferrin as a drug target and/or a treatment target, thereby indicating good prospect of being applied to medicines.

Description

Application of substance for detecting or regulating lactoferrin expression quantity in preparation of medicine or kit for preventing and/or treating cardiovascular and cerebrovascular diseases
Technical Field
The invention relates to the technical field of medical molecular biology, in particular to application of a substance for detecting or regulating the expression level of lactoferrin in preparation of a medicament or a kit for preventing and/or treating cardiovascular and cerebrovascular diseases.
Background
Lactoferrin (Lf) is an important non-heme iron-binding glycoprotein expressed and secreted mainly by mammary epithelial cells, has a molecular weight of 80kDa, and belongs to a member of the transferrin family. Lactoferrin is a natural protein in animal colostrum, is a multifunctional protein, has the functions of broad-spectrum antibiosis and virus infection resistance, can regulate the balance of iron in a body, regulate the immune function of an organism, inhibit tumor cells of the human body and the like, but the research report and the application of lactoferrin in cardiovascular diseases are not seen yet at present.
Disclosure of Invention
The invention aims to provide application of a substance for detecting or regulating the expression level of lactoferrin in preparation of a medicament or a kit for preventing and/or treating cardiovascular and cerebrovascular diseases. The invention provides the application of a substance for detecting or regulating the expression quantity of lactoferrin, and the lactoferrin has good effect on treating stroke, atherosclerosis or other cardiovascular and cerebrovascular diseases by taking the lactoferrin as a drug target and/or a treatment target, thereby indicating good prospect of being applied to medicines.
The invention provides application of a substance for detecting or regulating the expression level of lactoferrin in preparation of a medicament or a kit for preventing and/or treating cardiovascular and cerebrovascular diseases.
Preferably, the cardiovascular and cerebrovascular diseases comprise cardiovascular and cerebrovascular diseases caused by lactoferrin overexpression.
Preferably, the cardiovascular and cerebrovascular diseases comprise ischemic cerebral apoplexy or atherosclerosis.
The invention also provides application of the substance for regulating the expression level of lactoferrin in preparing a medicament for inhibiting the generation of atherosclerotic plaques.
The invention also provides application of the substance for regulating the expression quantity of lactoferrin in preparing a medicine for relieving over-activation of coagulation factors thrombin and FXIIa.
The invention also provides application of the substance for regulating and controlling the expression quantity of the lactoferrin in preparing the medicine for relieving the excessive activation of the blood coagulation function.
Preferably, the substance for detecting or regulating the expression level of lactoferrin comprises a lactoferrin antibody.
Preferably, the lactoferrin antibodies comprise anti-lactoferrin specific rabbit polyclonal antibodies.
The invention also provides a diagnostic kit for preventing and/or treating cardiovascular and cerebrovascular diseases based on the application of the technical scheme, wherein the kit comprises a lactoferrin antibody.
Preferably, the kit further comprises a 96-well plate, a coating solution, a blocking solution, a washing solution, a secondary antibody, a chromogenic substrate and a stop solution.
The invention provides application of a substance for detecting or regulating the expression level of lactoferrin in preparation of a medicament or a kit for preventing and/or treating cardiovascular and cerebrovascular diseases. The invention utilizes substances (including lactoferrin antibody) for detecting or regulating the expression quantity of lactoferrin to inhibit atheromatous plaque formation and achieve the purpose of treating atherosclerosis. The test result shows that the substance for detecting or regulating the expression quantity of the lactoferrin can inhibit the over-activation of the blood coagulation factors thrombin and FXIIa of the atherosclerosis patients caused by the over-expression of the lactoferrin, inhibit the over-activation of the blood coagulation function and improve the plaque generation caused by the atherosclerosis.
The kit provided by the invention takes lactoferrin as a marker of ischemic stroke and atherosclerosis, has high detection specificity and sensitivity, and is simple in detection method. Meanwhile, the lactoferrin is used as a drug target and/or a treatment target, has good effect on treating apoplexy, atherosclerosis or other cardiovascular and cerebrovascular diseases, and indicates good prospect of being applied to drugs.
Drawings
FIG. 1 is the lactoferrin content in plasma of a lactoferrin antibody-mediated atherosclerosis model mouse provided by the present invention;
FIG. 2 is a graph showing the experimental results of the lactoferrin content in plasma of a patient with ischemic stroke according to the present invention;
FIG. 3 is a graph showing the results of an experiment on the lactoferrin content in plasma of an atherosclerotic patient according to the present invention;
FIG. 4 shows FeCl provided by the present invention3Model map of induced carotid artery thrombosis in mice;
FIG. 5 is a graph of the results of a cerebral ischemia model provided by the present invention;
FIG. 6 is a graph showing the results of oil red O staining of thoracic aortic plaques in a lentivirus-intervened atherosclerosis model mouse provided by the present invention;
FIG. 7 is a graph showing the results of oil red O staining of thoracic aortic plaque of a mouse model of atherosclerosis by the antibody provided by the present invention;
FIG. 8 is a statistical plot of the percentage of the total arterial root section area occupied by the oil red-O staining area provided by the present invention;
FIG. 9 is a graph of the effect of lactoferrin antibodies on plasma relative to the activity of thrombin provided by the invention;
FIG. 10 is a graph showing the effect of lactoferrin antibodies provided by the present invention on plasma relative FXIIa activity;
FIG. 11 is a graph of the effect of lactoferrin antibodies on APTT provided by the invention;
FIG. 12 is a graph of the effect of lactoferrin antibodies on PT provided by the invention;
FIG. 13 is a graph showing the effect of lactoferrin antibodies of the present invention on tailgating bleeding time.
Detailed Description
The invention provides application of a substance for detecting or regulating the expression level of lactoferrin in preparation of a medicament or a kit for preventing and/or treating cardiovascular and cerebrovascular diseases. In the present invention, the cardiovascular and cerebrovascular diseases include cardiovascular and cerebrovascular diseases caused by lactoferrin overexpression. In the present invention, the cardiovascular and cerebrovascular diseases include ischemic cerebral stroke or atherosclerosis, and more preferably ischemic cerebral stroke or atherosclerosis caused by lactoferrin overexpression. In the present invention, the lactoferrin preferably comprises plasma lactoferrin, more preferably comprises human plasma lactoferrin; the human plasma lactoferrin is conventionally commercially available.
The invention also provides application of the substance for regulating the expression level of lactoferrin in preparing a medicament for inhibiting the generation of atherosclerotic plaques. In the present invention, the formation of atherosclerotic plaques is preferably the formation of atherosclerotic plaques resulting from lactoferrin overexpression.
The invention also provides application of the substance for regulating the expression quantity of lactoferrin in preparing a medicine for relieving over-activation of coagulation factors thrombin and FXIIa. In the present invention, the excessive activation of the coagulation factors thrombin and FXIIa is preferably the excessive activation of the coagulation factors thrombin and FXIIa caused by lactoferrin overexpression.
The invention also provides application of the substance for regulating and controlling the expression quantity of the lactoferrin in preparing the medicine for relieving the excessive activation of the blood coagulation function. In the present invention, the coagulation function overactivation preferably includes coagulation function overactivation caused by lactoferrin overexpression. The substance for regulating the expression quantity of lactoferrin can reduce the tendency of hypercoagulability when being used as a medicine.
In the present invention, the substance for detecting or regulating the expression level of lactoferrin comprises a lactoferrin antibody. The method for producing the lactoferrin antibody of the present invention is not particularly limited, and the lactoferrin antibody can be produced by a conventional method known to those skilled in the art, or can be produced by a conventional commercially available product of a lactoferrin antibody known to those skilled in the art. In the present invention, the lactoferrin antibodies preferably comprise rabbit polyclonal antibodies specific for lactoferrin.
The invention also provides a diagnostic kit for preventing and/or treating cardiovascular and cerebrovascular diseases based on the application of the technical scheme, wherein the kit comprises a lactoferrin antibody. The kit provided by the invention can realize early diagnosis of ischemic stroke and atherosclerosis.
In the invention, the kit further comprises a 96-well plate, a coating solution, a sealing solution, a washing solution, a secondary antibody, a chromogenic substrate and a stop solution. When the lactoferrin antibody is an anti-lactoferrin specific rabbit polyclonal antibody, the secondary antibody is preferably a horseradish peroxidase-labeled anti-rabbit IgG antibody secondary antibody. The sources of the 96-well plate, the coating solution, the confining solution, the washing solution, the secondary antibody, the chromogenic substrate and the stop solution in the kit are not particularly limited, and a common enzyme-linked immunosorbent reagent is adopted.
In the present invention, the method of using the kit preferably comprises the steps of: diluting the collected plasma sample of the patient with a coating solution, coating the diluted plasma sample in a 96-well plate, sealing the sample well, adsorbing the sample well on lactoferrin in the sample by using a specific antibody of the lactoferrin, and finally establishing a color reaction by using a horseradish peroxidase-labeled secondary antibody. The concentration of lactoferrin in the samples tested was calculated using a standard curve. According to the concentration of the lactoferrin, the severity of the disease is diagnosed, and the purpose of early diagnosis of ischemic stroke, atherosclerosis or other cardiovascular and cerebrovascular diseases is achieved.
The invention finds that the lactoferrin is up-regulated in the plasma of patients suffering from ischemic stroke and atherosclerosis, and the diseases are related to the expression of the lactoferrin by constructing a mouse thrombus model, a cerebral stroke model and an atherosclerosis model. The invention relates to a method for detecting the concentration of lactoferrin in blood plasma of a patient, which is used as an early diagnosis and monitoring marker of cardiovascular and cerebrovascular diseases such as stroke and atherosclerosis. Meanwhile, lactoferrin is used as a drug target and/or a treatment target, and the incidence or further deterioration of the cardiovascular and cerebrovascular diseases in the incidence process of the cardiovascular and cerebrovascular diseases is relieved by preparing a lactoferrin antibody and adopting an antibody treatment mode or other modes for reducing the expression of the lactoferrin.
The application of the substance for detecting or regulating the expression level of lactoferrin in the invention in the preparation of a medicament or a kit for preventing and/or treating cardiovascular and cerebrovascular diseases is described in further detail with reference to the following specific examples, and the technical scheme of the invention includes but is not limited to the following examples.
Example 1
Lactoferrin detection kit.
The determination method is a conventional enzyme-linked immunosorbent assay (ELISA) and comprises the following brief steps: mu.l of 1000-fold diluted plasma of the atherosclerotic mice were mixed with 99. mu.l of a coating solution (50mM carbonate buffer, pH 9.6), added to wells of a 96-well microplate (Nunc, Denmark), and coated overnight at 4 ℃. The next day, the well contents were discarded, washed 3 times with 0.1M phosphate buffer and then blocked, after which the rabbit polyclonal anti-lactoferrin antibody (10. mu.g/ml) was added and incubated at 37 ℃ for 60 minutes. After washing to remove unbound antibody, horseradish peroxidase-labeled anti-rabbit IgG secondary antibody (KPL, USA) was added, and finally, color development was performed with Tetramethylbenzidine (TMB). The concentration of lactoferrin in the plasma samples assayed was calculated using a series of standard lactoferrin dilutions in gradient as a standard curve (see fig. 1 for results showing the lactoferrin antibody levels in plasma of mice model atherosclerosis prognosis, wherein HFD: high fat diet induction; HFD + IgG: high fat diet induction and IgG intervention; HFD + LfAB: high fat diet induction and lactoferrin antibody intervention). The above results show that lactoferrin levels are elevated during the development of atherosclerosis and that intervention with lactoferrin antibodies can effectively down-regulate lactoferrin levels (/ p <0.05,/p < 0.01).
Example 2
Lactoferrin detection kit.
The determination method is a conventional enzyme-linked immunosorbent assay (ELISA) and comprises the following brief steps: mu.l of 1000-fold diluted plasma (Normal) of ischemic stroke patient (IS), atherosclerotic patient (CHD) and Normal human respectively was mixed with 99. mu.l of coating solution (50mM carbonate buffer, pH 9.6), added to a well of a 96-well microplate (Nunc, Denmark), and coated overnight at 4 ℃. The next day, the well contents were discarded, washed 3 times with 0.1M phosphate buffer and then blocked, after which the rabbit polyclonal anti-lactoferrin antibody (10. mu.g/ml) was added and incubated at 37 ℃ for 60 minutes. After washing to remove unbound antibody, horseradish peroxidase-labeled anti-rabbit IgG secondary antibody (KPL, USA) was added, and finally, color development was performed with Tetramethylbenzidine (TMB). The concentration of lactoferrin in the plasma samples determined was calculated using a series of standard lactoferrin dilutions in gradients as a standard curve (see results a in fig. 2 and a in fig. 3). The results show that lactoferrin in the plasma of IS and CHD patients showed up-regulation trend compared with that of normal persons (fig. 2 IS a graph of experimental results of lactoferrin content in plasma of ischemic stroke patients, and fig. 3 IS a graph of experimental results of lactoferrin content in plasma of atherosclerotic patients).
Example 3
Determination of the lactoferrin content in the plasma of patients with Ischemic Stroke (IS), atherosclerotic (CHD) and Normal (Normal).
3.8% sodium citrate 1 collected from hospital: the anticoagulated plasma of the above patients (ischemic stroke patient, n-187, normal, n-92; atherosclerotic, n-40, normal, n-40) was assayed for lactoferrin content as in example 2. As a result, as shown in a in fig. 2 and a in fig. 3, respectively, the plasma concentration levels of lactoferrin exhibited a significant up-regulation trend (× p <0.01) in ischemic stroke patients and atherosclerotic patients.
Example 4
Western blot assay of lactoferrin content in plasma of ischemic stroke patients, atherosclerotic patients and normal persons.
After the blood plasma of the patients is diluted by 10 times, the ordinary protein electrophoresis (SDS-PAGE) is carried out, and then the western blot detection is carried out. The experiment uses a self-made lactoferrin antibody to carry out western blot detection. And analyzing the Image obtained after the Western blot result is photographed by using Image J software. The results are shown in B, C in fig. 2 (B in fig. 2 shows western blot results of lactoferrin concentration in plasma of ischemic stroke patients; C in fig. 2 shows statistical results for B in fig. 2 from experimental results of 5 independent repetitions p <0.01), B, C in fig. 3 (B in fig. 3 is western blot results of lactoferrin concentration in plasma of atherosclerotic patients; C in fig. 3 shows statistical results for B in fig. 3 from experimental results of 5 independent repetitions p <0.01), respectively, and the concentration levels of lactoferrin in plasma of ischemic stroke patients and atherosclerotic patients show a clear up-regulation trend (p < 0.01).
Example 5
In order to further verify the correlation between lactoferrin and thrombosis, the lactoferrin content in the plasma of a mouse (C57BL/6J mic, 28-30 g) is reduced in a lactoferrin level increasing and lactoferrin knocking-down mode by the constructed lactoferrin lentivirus overexpression vector. Experiment by detecting FeCl3The induced thrombus model detects the influence of lactoferrin on the thrombus tendency of mice, and the specific grouping and administration modes of samples are as follows: a control group (NC, normal saline group), an overexpression control (PLP), a lactoferrin overexpression group (PLP-Lf), a knock-down control (RNR), and a lactoferrin knock-down group (RNR-Lf). Wherein FeCl3The induced thrombus model is respectively in the use of 10% FeCl3(w/v) inducing the neck artery for 5min, and detecting the blood of the part by laser Doppler after 10minAnd (4) streaming. The results were: compared with the control group (saline group, NC), the lactoferrin over-expression group (PLP-Lf) has significantly increased cervical arterial thrombosis and decreased blood flow, while the lactoferrin knock-down group (RNR-Lf) has the opposite (see FIG. 4: FeCl for results)3Map of induced carotid artery thrombosis model in mice, wherein A in FIG. 4 shows lactoferrin overexpression and lactoferrin knock-down vs FeCl3Blood flow monitoring experiment of the induced mouse carotid artery thrombosis model; b in fig. 4 shows a in fig. 4 being counted on a perfusion unit basis of blood flow (n-6); p<0.01)。
Example 6
Effect of lactoferrin on mouse cerebral ischemia model.
According to the method of the embodiment 5, the invention increases the lactoferrin level in the plasma of a mouse (C57BL/6J mica, 28-30 g) through the constructed lactoferrin lentivirus overexpression vector and increases the lactoferrin content in the plasma of the mouse in a mode of tail vein injection of the mouse lactoferrin (2.4 mg/kg). After anesthesia, the mouse skin and subcutaneous tissue were dissected layer by layer through the median cervical incision, the sternocleidomastoid muscle was isolated, the anterior abdomen of the two abdominal muscles was dissected, the right Common Carotid Artery (CCA), Internal Carotid Artery (ICA) and External Carotid Artery (ECA) were exposed, and the upper thyroid and pharyngeal arteries of ECA were coagulated using an electrocoagulator and cut. Ligation of the distal end of the ECA and its proximal end, temporary closure of the CCA and ICA, cutting of the ECA, insertion of a plug of thread from the ECA stump into the ICA, ligation of the ECA stump, removal of the ICA arterial clip, and slow advancement from the ICA up and inward. The orientation was adjusted appropriately, the plug was inserted at the plug mark (10 mm from the bifurcation of the CCA), 30min later the plug was removed and the incision was closed after observation for inactive bleeding. The whole process is insulated by an electric blanket, and the temperature is 36-37 ℃. The administration time was 20min after insertion of the wire plug, and the drugs were administered to the tail vein (control group (NC, saline group), overexpression control (PLP), lactoferrin overexpression group (PLP-Lf), and lactoferrin tail vein injection group (2.4 mg/kg)). Brains were taken 12 hours later for 2% TTC staining: carrying out intraperitoneal injection of 3% pentobarbital sodium to deeply anaesthetize a mouse, cutting off the head and stripping the brain, removing olfactory bulbs and brainstem, immediately putting the mouse into a mold with normal saline for freezing for 10-30 min at-20 ℃, taking out the mouse, putting the mouse into a brain mold, transversely cutting 4-5 slices, immersing the mouse into TTC (thermal shock temperature) with the temperature of 37 ℃ for dark dyeing for 10-30 min, fixing by 4% paraformaldehyde, photographing, and calculating the cerebral infarction volume. The results were: the mice in the lactoferrin overexpression group and the lactoferrin tail vein injection group had larger cerebral infarction areas relative to the control group (saline group, NC) (see fig. 5: cerebral ischemia model result graph, wherein a in fig. 5 is a result graph of the effect of lactoferrin overexpression and tail vein injection (2.4mg/kg) on the mice cerebral ischemia model, B in fig. 5 is a statistical result graph of a in fig. 5 based on the percentage of cerebral infarction area (n ═ 6), and C in fig. 5 is a result graph of bedder son (Bederson) scoring of the mice in each group.
Example 7
To further verify the relevance of lactoferrin to the pathogenesis of atherosclerosis, the present invention is directed to Apoe-/-Mice were fed with high-fat diet while intervening by over-expressing or knocking down the relevant virus, and the specific grouping and administration of samples were as follows: the virus dose of tail vein injection in virus administration modes of a control group (NC, normal saline group), an overexpression control (PLP), a lactoferrin overexpression group (PLP-Lf), a knock-down control (RNR) and a lactoferrin knock-down group (RNR-Lf) is 107TU (transducing units) and a high lipid induction time of 6 weeks. The experimental mice were sectioned from the arterial root and stained with oil red-O to mark the fat in the aortic root tissue sections of the mice. The size of the oil red-O stained area represents the size of the plaque and the severity of the atherosclerotic event. And (3) measuring the area of the atheroma and the total area of the cross section of the aortic root vessel by using Image analysis software Image J, and calculating the ratio of the area of the atheroma to the total area of the cross section of the aortic root vessel. The results were: compared with a control group (physiological saline group, NC), the lactoferrin overexpression group (PLP-Lf) can obviously promote the atheroma enlargement, the lactoferrin knockdown group (RNR-Lf) can obviously inhibit the atheroma enlargement (the result is shown in figure 6: the result graph of the oil red O staining graph of the thoracic aorta plaque of a mouse with a lentivirus intervention atherosclerosis model, wherein the result graph of the oil red O staining graph of the thoracic aorta section of an A mouse in figure 6, and the B graph in figure 6 is a statistical result graph of the percentage of the oil red O staining area of the A result graph in figure 6 to the whole arterial root section area (n ═ O staining graph6)。**p<0.01)。
Example 8
In one aspect, Apoe-/-The mice were fed with high-fat diet for 2, 4 and 6 weeks (2WK, 4WK, 6WK) to induce the development of atherosclerosis in the group of mice, and on the other hand, while continuously fed with high-fat diet, a treatment with an anti-lactoferrin antibody (HFD + LfAB, 50 μ g/mouse, twice a week) was administered by tail vein injection using lactoferrin as a therapeutic target, while an equivalent amount of IgG isotype control (HFD + IgG) was administered, and only high-fat diet was used as a control (HFD). After the antibody treatment is finished, the experimental mouse is subjected to oil red-O staining after the arterial root section is taken so as to mark fat in the aortic root tissue section of the mouse. The size of the oil red-O stained area represents the size of the plaque and the severity of the atherosclerotic event. And (3) measuring the area of the atheroma and the total area of the cross section of the aortic root vessel by using Image analysis software Image J, and calculating the ratio of the area of the atheroma to the total area of the cross section of the aortic root vessel. To compare the severity of disease onset in mice. (results see FIGS. 7 and 8: FIG. 7 is a graph showing the results of oil red O staining of the thoracic aortic plaque of an antibody-mediated atherosclerosis model mouse, FIG. 8 is a statistical graph showing the percentage of the oil red-O stained area in the section of the entire arterial root, FIG. 7 is a graph showing the results of oil red-O staining of the thoracic aortic section by three sets of HFD, HFD + IgG and HFD + Lf AB, and FIG. 8 is a graph showing the statistics of the percentage of the oil red-O stained area in the section of the entire arterial root for FIG. 7 (n 6)<0.01). The results were: compared with the HFD group, the area of the plaque at the aortic root of the mouse of the HFD + LfAB group is greatly reduced. The results clearly prove that the lactoferrin serving as a drug target and/or a therapeutic target can obviously delay or inhibit the onset of the atherosclerotic disease after the atherosclerotic disease is treated by an antibody therapy.
Example 9
Lactoferrin antibody intervention on the plasma activity against thrombin and FXIIa.
Specifically, 10. mu.L of the obtained mouse plasma was added to 20mM Tris-HCl buffer solution, and 40. mu.L of each of the small peptide luminescent substrates of thrombomin and FXIIa was added thereto, using the same amount of enzyme as that used in the in vitro assay. Absorbance at 405nm was immediately measured on a microplate reader. The total detection time is 30min, the detection is carried out once every 30s, the change of an enzyme kinetic curve is recorded, and the result is counted. The results were: the lactoferrin antibody group significantly attenuated the plasma relative thrombomin and FXIIa activities compared to the control group. (results see FIGS. 9 and 10, FIG. 9 is a graph showing the effect of lactoferrin antibodies on plasma activity against thrombomin, and FIG. 10 is a graph showing the effect of lactoferrin antibodies on plasma activity against FXIIa).
Example 10
The effect of lactoferrin antibody intervention on Activated Partial Thromboplastin Time (APTT).
The experiment was performed using plasma by incubating 50 μ L of plasma with 50 μ L of APTT reagent pre-warmed at 37 deg.C for 5min and then adding 50 μ L of 25mM CaCl pre-warmed at 37 deg.C2And quickly placing the sample in a microplate reader for measuring the kinetics at 650 nm. The total detection time is 5min, the detection is carried out once every 10s, the change of an enzyme kinetic curve is recorded, and the result is counted. The results were: the lactoferrin antibody group significantly extended the APTT compared to the control group, showing that lactoferrin antibody intervention reduced the propensity for hypercoagulation (see figure 11 for results, a graph of the effect of lactoferrin antibodies on APTT).
Example 11
Effect of lactoferrin antibodies on plasma Prothrombin Time (PT).
The experiment was performed with plasma, 50 μ L pre-heated at 37 ℃ for 3min, 100 μ L of PT reagent pre-heated at 37 ℃ was added, and the kinetics was immediately measured at 650nm in a microplate reader. The total detection time is 5min, the detection is carried out once every 10s, the change of an enzyme kinetic curve is recorded, and the result is counted. The results were: the lactoferrin antibody group significantly prolonged PT compared to the control group, showing that lactoferrin antibody intervention reduced the tendency to hypercoagulability (see figure 12 for results, a graph of the effect of lactoferrin antibodies on PT).
Example 12
Effect of lactoferrin antibodies on tail-biting bleeding time in mice.
At the induction time node, tail bleeding time measurements were performed. Fixing the tail breaking length to be 0.4cm, quickly placing the tail end of the mouse tail into physiological saline preheated at 37 ℃ to record the tail breaking bleeding time, reducing the time error caused by wall collision due to disorder movement by fixing the mouse tail, and counting the result. The results were: administration of lactoferrin antibody significantly increased the time to tail break bleeding compared to the control group, showing that lactoferrin antibody intervention reduced the propensity for hypercoagulability (see figure 13 for results, a graph of the effect of lactoferrin antibody on time to tail break bleeding).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (4)

1. Application of a substance for down-regulating expression quantity of lactoferrin in preparing a medicament for relieving excessive activation of blood coagulation factors thrombin and FXIIa in high-fat feed-induced atherosclerosis.
2. Application of a substance for reducing the expression quantity of lactoferrin in preparing a medicine for relieving excessive activation of blood coagulation function in high-fat feed-induced atherosclerosis.
3. The use according to claim 1 or 2, wherein the substance which down-regulates the expression of lactoferrin comprises an antibody to lactoferrin.
4. The use according to claim 3, wherein said lactoferrin antibodies comprise anti-lactoferrin specific rabbit polyclonal antibodies.
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