CN116082511A

CN116082511A - Multi-effect factor composition and application thereof in treatment of arterial plaque

Info

Publication number: CN116082511A
Application number: CN202211325837.4A
Authority: CN
Inventors: 李静; 陈家伟; 刘山胜
Original assignee: Jinan Chengyi Longfengyi Biotechnology Co ltd
Current assignee: Jinan Chengyi Longfengyi Biotechnology Co ltd
Priority date: 2022-10-27
Filing date: 2022-10-27
Publication date: 2023-05-09

Abstract

The present invention relates to a pleiotropic factor composition and its use in the treatment of arterial plaque. According to the invention, umbilical cord mesenchymal stem cell pleiotropic factor is prepared by culturing umbilical cord mesenchymal stem cells, the pleiotropic factor can effectively treat arterial plaque, and meanwhile, HMG-CoA monoclonal antibody is prepared specifically, and the antibody can obviously down regulate the expression of IL-18, so that the antibody can be used for treating plaque. After the two components are combined, the formation and treatment of arterial plaque can be effectively prevented and treated, and the application prospect is good.

Description

Multi-effect factor composition and application thereof in treatment of arterial plaque

Technical Field

The present invention relates to the field of biology, more particularly to a pleiotropic factor composition and its use in the treatment of arterial plaque.

Background

Carotid plaque is a manifestation of carotid atherosclerosis, well occurring at the carotid bifurcation, and is currently thought to be closely related to the occurrence of ischemic stroke in the elderly. The mechanism by which ischemic stroke is caused may be: the carotid artery stenosis caused by plaque enlargement causes intracranial hypoperfusion and plaque shedding to form emboli, which leads to intracranial arterial embolism. Clinically, carotid plaque was evaluated by morphological measurement of its stenosis and plaque, and its harmfulness was judged.

The carotid plaque formation is the result of the combined action of external environmental factors and inherent polygenic regulation abnormality, and the carotid plaque development is a dynamic balance process, namely the balance between plaque caps formed by collagen fibers generated by smooth muscle cells and matrix degradation mediated by metalloproteinases and the like, so that the balance is broken, and the stability of the plaque is reduced, so that the carotid plaque becomes an unstable plaque or a vulnerable plaque. In clinical practice, atherosclerotic plaques are generally classified into two types, stable plaque (hard plaque) and vulnerable plaque (soft plaque/unstable plaque), which are subjects of clinical intervention.

Vulnerable plaque refers to plaque that is prone to thrombus formation or may rapidly progress to a criminal lesion. According to diagnostic criteria set forth in Naghavi M et al, 2003 based on autopsy study data, vulnerable plaque includes five major features and five minor features: the five main features include: plaque-interior active inflammation-plaque mononuclear cell, macrophage infiltration, sometimes T lymphocyte infiltration. Thin fibrous cap and large lipid core: it is believed that when the fibrous cap thickness is less than 100% u3bcm and the lipid core is 40% or more of the plaque volume, the atheromatous plaque is prone to rupture. Vascular endothelial erosion is accompanied by surface platelet aggregation. Slit-like plaques. The lumen stenosis is greater than 90%. While the five secondary features include: plaque surface nodular calcification. Yellow and bright plaques only visible under the endovascular scope. Intra-plaque hemorrhage. Vascular endothelial dysfunction. The blood vessel is reshaped.

Statin is a cornerstone for the prevention and treatment of atherosclerosis, but some patients still have a high risk of cardiovascular events after receiving intensive statin therapy. Thus, there remains a need to explore novel anti-atherosclerosis agents that are highly effective and safe.

Recent clinical trials and basic studies show that 3 hydroxy-3 methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors (statins) except for affirmative Soma and the like observe the inhibition effect of different statins on the neointima of cholesterol normal rabbit carotid artery caused by endothelial injury, and the results of the study show that the formation of neointima on endothelial injury side is 12 times that on control side, fluvastatin (fluvastatin) is significantly reduced from that of simvastatin-treated animals, and pravastatin is ineffective at the same dose. Studies by Corsini et al have shown that fluvastatin, simvastatin, lovastatin, cerivastatin, atorvastatin (atorvastatin) all inhibit Platelet Derived Growth Factor (PDGF) and fibrinogen mediated proliferation and migration of rat aortic vascular smooth muscle cells and human femoral vascular smooth muscle cells dose dependently on their cholesterol lowering effects, but pravastatin alone does not. However, hidaka et al have shown earlier that pravastatin also inhibits PDGF and fibrinogen-stimulated vascular smooth muscle cell migration and prevents intimal hyperplasia, but rather has a much weaker effect than simvastatin, perhaps because pravastatin is less lipophilic and does not readily penetrate the cell membrane. The mechanism by which statins inhibit vascular smooth muscle cell proliferation is not well understood, and some studies are thought to be in that they significantly reduce or lose mevalonate, thereby resulting in inhibition of deoxyribonucleic acid synthesis by vascular smooth muscle cells. HMG-CoA reductase inhibitors have also been reported to inhibit the expression of oncogene proteins associated with cell proliferation (e.g., P21 ras). In addition to lowering plasma cholesterol (the primary role), non-lipid-regulating anti-motor mechanisms independent of cholesterol lowering such as improving endothelial function, inhibiting vascular smooth muscle cell proliferation and migration, anti-inflammatory response, promoting plaque stabilization, inhibiting platelet aggregation, etc. may still be involved.

HMG-CoA reductase catalyzes the production of dimethylolvalerate (MVA) from HMG-CoA, which is a necessary precursor for the synthesis of cholesterol, and therefore HMG-CoA reductase is the rate-limiting enzyme that catalyzes the synthesis of cholesterol. In addition to being involved in cholesterol metabolism, HMG-CoA reductase is also involved in metabolic pathways of other compounds, such as the synthesis of Ras proteins. HMG-CoA reductase is the rate-limiting enzyme for cholesterol synthesis, so that its inhibitor can reduce the level of plasma cholesterol, and currently, cholesterol-lowering drugs-statins, which are widely used clinically, are inhibitors of HMG-CoA reductase. They block the formation of isoprenoids, i.e., HMG-CoA+2NADPH+2H+ & fwdarw (R) isoprene+2NADP++ CoASH, thereby reducing cholesterol synthesis. The reduction of endogenous cholesterol levels promotes the hydrolysis of SREBP bound to the plasma omentum, which converts the latter into active protein with helix-loop-helix-leucine zipper, which breaks away from the plasma omentum and moves into the nucleus, binds to the promoter of the low density lipoprotein receptor gene (LDLR), activates transcription of the LDLR gene, increases cellular LDLR levels, enhances LDLR binding to Low Density Lipoprotein (LDL) in plasma, and thereby reduces plasma LDL levels. In addition, statins also promote LDLR synthesis, increase circulating High Density Lipoprotein (HDL) and reduce Triglyceride (TG) levels. HMG-CoA reductase is a key enzyme in the cholesterol synthase family, and statins inhibit liver synthesis of cholesterol by their inhibitory action. Lovastatin and simvastatin are orally administered and require activation in vivo to be bioactive. Atorvastatin and cerivastatin are biologically active upon oral absorption. The medicine can reduce lipid infiltration and foam formation, reduce the volume of atherosclerosis plaque, induce the reversion of atherosclerosis plaque, improve vascular endothelial function, weaken the vasoconstriction and spasm induced by acetylcholine, thereby improving myocardial ischemia, and also can inhibit platelet adhesion, aggregation, regulate coagulation-anticoagulation system in blood, etc. When an arteriosclerotic plaque is ulcerated, normal repair processes require smooth muscle cell proliferation, but excessive proliferation can have adverse effects. Lovastatin, simvastatin and fluvastatin all have strong effects of inhibiting vascular smooth muscle cell proliferation. This effect may delay or inhibit the progression of early atherosclerosis.

However, at present, there are few corresponding drugs for HMG-CoA reductase inhibitors, and thus, the focus of research is now on.

Disclosure of Invention

The present invention provides an improved HMG-CoA inhibitor and stem cell cytokine for the treatment of arterial plaque-related diseases.

In one aspect, the invention provides a pleiotropic factor, also known as a stem cell factor, extracted from umbilical cord mesenchymal stem cells.

Specifically, the stem cell factor is prepared by the following method: inoculating human umbilical cord mesenchymal stem cells into a culture container, adding a culture medium, and then carrying out subculture; wherein the medium comprises the following components: FBS at 20% by volume and GlutaMAXTM-I at 2% by volume, the remainder DMEM; after the fusion degree of the human umbilical cord mesenchymal stem cells reaches 80-90%, adjusting the oxygen concentration in a culture container to 1% (v/v), and then continuously culturing the cells overnight; the cells after overnight were digested with 0.25% pancreatin-EDTA solution for 2-3min, then centrifuged, the supernatant was removed, and the cells were collected after washing the precipitate with physiological saline and examined for cell viability, and cell counting was performed. Regulating cell concentration with physiological saline, adding EDTA and Vc, mixing, transferring into freezing tube, freezing in gas phase liquid nitrogen for 1 time, resuscitating in constant temperature water bath, and repeating the above steps. Transferring the frozen and resuscitated cell lysate into a centrifuge tube, centrifuging, filtering the lysate supernatant by a 0.22 mu m filter, and finally adding physiological saline, EDTA and Vc to prepare the mesenchymal stem cell factor.

Further, the invention provides the use of stem cell factor in preparing medicine for treating arterial plaque.

Specifically, according to the invention, a specific HMG-CoA monoclonal antibody HMG-CoA-3D8 is obtained by immunizing a mouse and screening after cell fusion, and the light chain and heavy chain sequences of the antibody are identified, and the variable region sequences of the light chain and the heavy chain are shown as SEQ ID NO:1, the heavy chain variable region sequence of which is shown in SEQ ID NO: 2.

Further, there is also provided the use of an HMG-CoA monoclonal antibody in the manufacture of a medicament for inhibiting HMG-CoA activity.

Furthermore, the invention also provides the application of the stem cell factor combined with the HMG-CoA monoclonal antibody in preparing a pharmaceutical composition for treating arterial plaque.

Further, pharmaceutical compositions and pharmaceutical packages "pharmaceutical compositions" refer to pharmaceutical formulations for use in humans. The pharmaceutical composition comprises a suitable formulation of the humanized monoclonal antibody or antigen binding fragment thereof of the invention, and a carrier, stabilizer and/or excipient. The present invention provides pharmaceutical formulations comprising the monoclonal antibodies or antigen-binding fragments thereof of the invention. To prepare a pharmaceutical or sterile composition, the antibody or antigen-binding fragment thereof is admixed with a pharmaceutically acceptable carrier or excipient. Formulations of therapeutic and diagnostic agents in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions may be prepared by mixing with physiologically acceptable carriers, excipients or stabilizers.

Suitable routes of administration include parenteral (e.g., intramuscular, intravenous, or subcutaneous) administration and oral administration. Antibodies for use in pharmaceutical compositions or for practicing the methods of the invention may be administered in a variety of conventional ways, such as oral ingestion, inhalation, topical administration, or transdermal, subcutaneous, intraperitoneal, parenteral, intraarterial, or intravenous injection. In one embodiment, the binding compounds of the invention are administered intravenously. In another embodiment, the binding compounds of the invention are administered subcutaneously. Alternatively, one may administer the antibody in a local rather than systemic manner (typically as a depot or slow release formulation), for example via injection of the antibody directly into the site of action. Furthermore, one can administer antibodies in a targeted drug delivery system.

The pharmaceutical compositions of the present invention may also contain other agents including, but not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic or antimetabolite agents, targeted tumor agents, immunostimulants or immunomodulators, or antibodies conjugated to cytotoxic agents, cytostatic agents or other toxic agents. The pharmaceutical compositions may also be administered with other forms of treatment, such as surgery, chemotherapy, and radiation.

Therapeutically effective amount as used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount effective to prevent or slow down a disease or disorder to be treated when a bispecific antibody of the present invention, or fragment thereof, is administered alone or in combination with another therapeutic agent to a cell, tissue or subject. A therapeutically effective dose further refers to an amount of the compound sufficient to cause a alleviation of symptoms, such as treatment, cure, prevention, or alleviation of the relevant medical condition, or increase the rate of treatment, cure, prevention, or alleviation of the condition. When administered to an individual an active ingredient administered alone, a therapeutically effective amount refers to the individual ingredient. When a combination is administered, a therapeutically effective amount refers to the amount of the combination of active ingredients that produces a therapeutic effect, whether administered in combination, serially or simultaneously. A therapeutically effective amount will alleviate symptoms, typically by at least 10%; typically at least 20%; preferably at least about 30%; more preferably at least 40% and most preferably at least 50%.

Advantageous effects

According to the invention, umbilical cord mesenchymal stem cell pleiotropic factor is prepared by culturing umbilical cord mesenchymal stem cells, the pleiotropic factor can effectively treat arterial plaque, and meanwhile, HMG-CoA monoclonal antibody is prepared specifically, and the antibody can obviously down regulate the expression of IL-18, so that the antibody can be used for treating plaque. After the two components are combined, the formation and treatment of arterial plaque can be effectively prevented and treated, and the application prospect is good.

Drawings

FIG. 1 influence of HMG-CoA-3D8 mab on the expression level of IL-18 in cells

FIG. 2 influence of HMG-CoA-3D8 mab on cell proliferation

FIG. 3 Effect of Stem cell factor combination on arterial plaque

Detailed Description

The invention may be understood more readily by reference to the following detailed description of some embodiments of the invention and the examples included therein. Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

EXAMPLE 1 preparation of HMG-CoA monoclonal antibodies

Female BALB/c mice at 8 weeks of age were injected back subcutaneously with HMG-CoA recombinant protein (Zeolite, cat# ZY6175Hu 014). Immunization 1, wherein the immunization dose is 80 mug/dose, diluted to a proper volume by normal saline, added with equal volume Freund's Complete Adjuvant (FCA), emulsified into a water-in-oil (W/O) state by adopting a syringe push-pull method, and the emulsified antigen is injected subcutaneously into the back (80 mug/dose); after 2 weeks, the same dose of immunogen was mixed and emulsified with Freund's Incomplete Adjuvant (FIA) for booster immunization, after which the same procedure was used for immunization 3 and 4 times, each at 14d intervals. Serum was collected from the tail vein of the mice 1 week after each immunization, and serum titers were measured. Spleen cells of the highest titer mouse No. 3 were fused with mouse myeloma cell line SP 2/0. 3d before fusion, no adjuvant is added, and 100 mug of antigen is directly injected into the abdominal cavity for impact immunization. After fusion, cells were plated on 4 96-well plates and cultured in HAT medium. After 7d, death and lysis of unfused cells were observed, and a round and transparent cluster of fused cells in the shape of a grape cluster was observed, with 2-5 growing cell clones per 96 wells. And (3) taking culture supernatant with cell clone holes for indirect ELISA detection, and screening to obtain 45 positive holes.

3D8 holes with strong positive and better competitive inhibition result are subjected to monoclonalization by a limiting dilution method. Subcloning by 3 times of limiting dilution method, continuing monoclonalization for 3 times, and recovering cells after the last 2 times of freezing and storing. The positive rate of cells in the case of single clone was 100%. After liquid nitrogen cryopreservation and resuscitation, the cell strain can still stably secrete specific antibodies, and the monoclonal is successful, and is named as HMG-CoA-3D8 hybridoma cell strain.

Adopting an in-vivo induction method to prepare a large amount of ascites. Taking 12-week-old BALB/c mice, and intraperitoneally injecting Freund's incomplete adjuvant into each of the mice at 0.2mL, and intraperitoneally injecting 1×10 into each of the mice after 1 week ⁶ And collecting ascites when the abdomen of the 10D mice obviously rises after injection of HMG-CoA-3D8 hybridoma cells, centrifuging for 10min at 4000r/min, sucking the supernatant, sub-packaging, and freezing at-20 ℃. Purifying by caprylic acid-saturated ammonium sulfate precipitation to obtain purified HMG-CoA-3D8, and detecting by SDS-PAGE, wherein the impurity proteins are largely removed, and only IgG heavy chain and light chain are remained. The purity of the purified McAb reaches 99.3 percent, and the recovery rate reaches 84 percent.

Example 2 determination of HMG-CoA monoclonal antibody HMG-CoA-3D8 specificity

And (3) diluting BSA, OVA, PD-1 and gelatin to the same concentration as the original HMG-CoA for coating, adding an HMG-CoA-3D8 antibody, and testing the binding color development condition. The cross-reaction between them and mab was determined. The results of the specific identification of HMG-CoA-3D8 are shown in Table 1.

TABLE 1 identification of HMG-CoA-3D8 specificity

Coating material	OD value	Cross-reactivity/%
			BSA	0.06	＜0.01
OVA	0.07	＜0.01
			PD-1	0.04	＜0.01
Gelatin	0.05	＜0.01
			HMG-CoA	0.63	100

The data in Table 1 show that none of the relevant proteins involved in the monoclonal antibody preparation process, such as BSA, OVA, gelatin for blocking, etc., reacted with HMG-CoA-3D8 antibody, indicating that HMG-CoA-3D8 antibody was specific for HMG-CoA only. HMG-CoA-3D8 does not cross react with other proteins or substances.

Example 3 determination of HMG-CoA monoclonal antibody HMG-CoA-3D8 affinity

HMG-CoA monoclonal antibody HMG-CoA-3D8 was diluted to 10ug/ml with PBST using AMC sensor, and HPGII (fitzgerald, 30R-AP 039) was gradient diluted with PBST: 444.4nmol/ml, 222.2nmol/ml, 111.1nmol/ml, 55.6nmol/ml, 27.8nmol/ml, 0nmol/ml; the operation flow is as follows: buffer 1 (PBST) was equilibrated for 50s, antibody 350s was immobilized in antibody solution, PBST buffer was incubated for 200s, antigen solution was bound for 450s, dissociated for 1200s, and sensor regeneration was performed with 10mM pH1.69 GLY solution, and data was output. (KD represents equilibrium dissociation constant, i.e., affinity) results show that the dissociation constant of HMG-CoA monoclonal antibody HMG-CoA-3D8 is (10.58.+ -. 0.14) nM.

Example 4 identification of HMG-CoA monoclonal antibody HMG-CoA-3D8 Activity

Resuscitating and passaging frozen human HUVEC strain, and placing in 37% CO and 5% ₂ Culturing in incubator, and the 4 th generation is used for experiment. Cells were seeded in 12-well and 96-well plates with DMEM medium (20% fetal bovine serum). After the cells were grown to confluence for 24h, the cells were further cultured with serum-free medium for 24h. The experiments were divided into 4 groups: (1) a blank; (2) OX-LDL group (100 mg/L); (3) HMG-CoA-3D8 mab-treated group: monoclonal antibodies 10, 50, 100 and 200mg/L are firstly acted on endothelial cells for 4 hours respectively, and then ox-LDL oxidized low density lipoprotein (100 mg/L) is added to act on the cells for 24 hours. (4) positive control group: atorvastatin 100mg/L was first allowed to act on endothelial cells for 4h, respectively, followed by ox-LDL (100 mg/L) for 24h.

And (3) measuring the IL-18 content of the treated cells by adopting a cell ELISA (enzyme-linked immunosorbent assay). The supernatant from the 12-well plates was collected and assayed for IL-18 content. 100 μl of detection diluent is added to the microwell plate coated with anti-IL-18 monoclonal antibody, 50 μl of standard rlL-18 serum sample to be detected is added respectively, and the microwell plate is subjected to 200r/min micro-shaking and incubated for 2h at room temperature. After 3 washes, 100. Mu.l of biotin was added, and the mixture was subjected to 200r/min micro-shaking and incubated at room temperature for 1h. After washing 3 more times, 100. Mu.l of polyclonal anti-IL-18 antibody labeled with horseradish peroxidase (HRP) was added, and the mixture was subjected to 200r/min micro-shaking and incubated at room temperature for 1h. Finally, the mixture was washed 3 times, 100. Mu.l of TBM substrate solution was added, and the mixture was subjected to micro-shaking at 200r/min and at room temperature for 10min. The reaction was terminated by adding an end-point solution. The absorbance (A) was measured at a wavelength of 630nm, and a standard curve was obtained to calculate the sample content.

IL-18 also activates monocytes directly to promote secretion of MMPs, and IL-18 promotes expression of ILB, IL-8, monocyte chemotactic protein-1 (MCP.1), adhesion molecules, etc. in cells of blood vessel wall, and plays an important role in plaque rupture. As a result, IL-18 was not secreted in normal HUVEC cells, as shown in FIG. 1. While ox-LDL induces HUVEC to secrete IL-18, the monoclonal antibodies of the invention can obviously down regulate the expression of IL-18 as the positive control group, so that the monoclonal antibodies can be used for treating plaque.

Tetrazolium salt colorimetric method (MTT method) to evaluate cell proliferation: taking 4 blocks of 96-well plates to 1×10 ⁴ Cell seed plates with density of/ml, 200 mu l per well, and changing culture medium containing 2% fetal bovine serum and corresponding experimental reagent when the cells are in a subfusion state. Mu.l of MTT solution (5 g/L) was added to each well, followed by placing at 37℃and 5% CO ₂ After incubation for 4 hours in the incubator, the culture supernatant in each well is sucked and removed, 150 μl of dimethyl sulfoxide (DMSO) is added into each well, after shaking for 10min, 490nm wavelength is selected on an enzyme-labeled instrument for colorimetry, and finally blank wells (no cell wells) are used for zeroing during colorimetry.

As can be seen from FIG. 2, the HMG-CoA inhibitor-monoclonal antibody HMG-CoA-3D8 prepared by the invention can promote proliferation of human HUVEC cultured in vitro, and can effectively form cell surrounding winding around rupture plaque through the proliferation, thereby stabilizing the risk of apoplexy caused by plaque rupture.

Example 5 preparation of umbilical cord mesenchymal Stem cell pleiotropic factor (Stem cell factor)

Human umbilical cord mesenchymal stem cells (scientific cells)Cargo number: 7530 Inoculating into culture container, adding culture medium, and placing at 37deg.C and 5% CO ₂ Subculturing under the condition; wherein the medium comprises the following components: FBS at 20% by volume and GlutaMAXTM-I at 2% by volume, the remainder DMEM; after the fusion degree of the human umbilical cord mesenchymal stem cells reaches 80-90%, the oxygen concentration in the culture container is adjusted to 1%

(v/v) and then continuing to culture the cells overnight; the overnight cells were digested with 0.25% pancreatin-EDTA solution for 2-3min, then centrifuged at 2000r/min at 20℃for 3min, the supernatant was removed, and the pellet was washed 3 times with 0.9% physiological saline to collect cells, and the cell viability was measured and counted. Cell concentration was adjusted to 1X 10 with 0.9% physiological saline ⁸ Adding 3mg/mL of EDTA and 10mg/mL of Vc into each cell/mL, uniformly mixing, transferring the mixture into a 5mL freezing tube, freezing the mixture in gas-phase liquid nitrogen for 10min, resuscitating the mixture in a constant-temperature water bath kettle at 37 ℃ for 5min, and finally, freezing and resuscitating the mixture for 4 times respectively. Transferring the frozen and resuscitated cell lysate into a 50mL centrifuge tube, centrifuging at 20deg.C and 5000r/min for 10min, filtering the lysate supernatant with a 0.22 μm filter, adding 0.9% physiological saline, 3mg/mL EDTA and 10mg/mL Vc, and adjusting the supernatant to 30mL to obtain mesenchymal stem cell factor. ELISA method is adopted to detect various cytokines (EGF, FGF, IGF-1, HGF, PDGF-AA, VEGF) according to the specification, and the kit is purchased from R&The detection result shows that the concentration of HGF (hepatocyte factor) secreted by placenta mesenchymal stem cells reaches 15.6ng/ml, the concentration of PDGF-AA (platelet growth factor AA) is 3.3ng/ml, the concentration of bFGF (basic fibroblast growth factor) is 7.2ng/ml, the concentration of VEGF (vascular endothelial growth factor) is 5.8ng/ml, the concentration of IGF-1 (insulin-like growth factor) is 1.1mg/ml, and the concentration of EGF (epidermal growth factor) is 1.6mg/ml. And freeze-drying the cytokine solution for later use to obtain the stem cell factor.

Example 6 Effect of umbilical cord mesenchymal Stem cell Multi-effect factor (Stem cell factor) combination on arterial plaque

Healthy new zealand rabbits were offered by the laboratory animal center at the university of guangxi traditional chinese medicine. 3% pentobarbital sodium (30 mg/kg) was anesthetized in rabbit ear margin vein, right common carotid artery was isolated under aseptic conditions, arterial clip blocked blood flow, normal saline was flushed and vessel lumen was evacuated, and 1ml syringe drawing liquid nitrogen was rapidly injected into right common carotid artery, repeated 3 times to cause vascular endothelial frostbite.

Post-operative stem cell factor treatment group: high fat feed (containing 1% cholesterol, 3% lard, 15% egg yolk, and 81% normal feed) was used for 8 weeks. 100mg of the mesenchymal stem cell factor prepared in example 5 was implanted in the right common carotid artery by a syringe, dissolved in physiological saline, and incubated for 10min. The administration was 1 time per week.

The stem cell factor combined with atorvastatin treatment group is that 100mg of mesenchymal stem cell factor prepared in the example 5 is implanted in the right common carotid artery by a syringe, dissolved by normal saline, incubated for 10min, and simultaneously injected with 5mg/d of HMG-CoA inhibitor atorvastatin; the administration was 1 time per week. And feeding with high-fat feed (containing 1% cholesterol, 3% lard, 15% egg yolk, and 81% common feed) for 8 weeks.

The control group was fed with high fat diet (1% cholesterol, 3% lard, 15% egg yolk, and 81% normal diet) for 8 weeks. Physiological saline is injected.

The normal group was fed with normal feed for 8 weeks. Physiological saline is injected.

Each of the above groups, 10.

After 8 weeks, the corresponding arterial plaque was removed and weighed, and the specific results are shown in fig. 3.

From the results shown in fig. 3, it can be seen that the stem cell factor treatment can inhibit the formation of arterial plaque to a certain extent, but no stem cell factor combined with the HMG-CoA inhibitor has a remarkable inhibiting effect, which indicates that the umbilical cord mesenchymal stem cell factor combined with the HMG-CoA inhibitor can effectively inhibit the formation of arterial plaque, and is beneficial to the treatment of atherosclerosis. The monoclonal antibody of the invention is an inhibitor specific to human HMG-CoA, and based on the principle, the combined stem cell factor of the HMG-CoA monoclonal antibody of the invention can also produce the same technical effect in human plaque treatment. Has better application prospect.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

HMG-CoA monoclonal antibody HMG-CoA-3D8 characterized in that: the sequence of the light chain variable region is shown in SEQ ID NO:1, the heavy chain variable region sequence of which is shown in SEQ ID NO: 2.
2. Use of the monoclonal antibody of claim 1 for the preparation of a medicament for inhibiting human HMG-CoA activity.
3. Use of umbilical cord mesenchymal stem cell pleiotropic factor and HMG-CoA monoclonal antibody of claim 1 for the preparation of a pharmaceutical composition for treating arterial plaque; wherein the umbilical cord mesenchymal stem cell pleiotropic factor is prepared by the following method: inoculating human umbilical cord mesenchymal stem cells into culture container, adding culture medium, and placing at 37deg.C and 5% CO ₂ Subculturing under the condition; wherein the medium comprises the following components: FBS at 20% by volume and GlutaMAXTM-I at 2% by volume, the remainder DMEM; after the fusion degree of the human umbilical cord mesenchymal stem cells reaches 80-90%, adjusting the oxygen concentration in a culture container to 1% (v/v), and then continuously culturing the cells overnight; digesting overnight cells with 0.25% pancreatin-EDTA solution for 2-3min, centrifuging at 20deg.C and 2000r/min for 3min, removing supernatant, washing precipitate with 0.9% physiological saline for 3 times, collecting cells, detecting cell activity, and counting cells; cell concentration was adjusted to 1X 10 with 0.9% physiological saline ⁸ Adding EDTA 3mg/mL and Vc 10mg/mL, mixing, transferring into 5mL cryopreservation tube, placing into gas phase liquid nitrogen, cryopreserving for 10min, and placing into constant temperature water bath at 37deg.CResuscitating in the pot for 5min, and finally freezing and resuscitating for 4 times; transferring the frozen and resuscitated cell lysate into a 50mL centrifuge tube, centrifuging for 10min at 20 ℃ under 5000r/min, filtering the lysate supernatant by a 0.22 mu m filter, and finally adding 0.9% physiological saline, 3mg/mL EDTA and 10mg/mL Vc, and adjusting the supernatant to 30mL to obtain the mesenchymal stem cell multipotent factor which also becomes the mesenchymal stem cell factor.
4. The use according to claim 2 or 3, wherein the medicament or pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
5. The use according to claim 4, wherein the pharmaceutically acceptable carrier comprises an excipient and a stabilizer.