KR20180005149A - Pharmaceutical composition for treating or preventing aging or age­related diseases comprising CD9 antibody - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cellular senescence or senescence-associated diseases, comprising an antibody which specifically binds to CD9 as an effective component, wherein the CD9-specific antibody specifically binds to an extracellular region of CD9 antigen exposed to the surface of cells, thereby exhibiting effects of inhibiting cellular senescence through reducing CD9 expression in aged cells and reducing atherosclerotic lesions, which are a senescence-associated vascular disease. Accordingly, the CD9 of the present invention can be used as a biomarker composition for cellular senescence or senescence-associated diseases, and a composition comprising a CD9-specific antibody as an effective component can be beneficially used as a pharmaceutical composition or a dietary supplement composition capable of inhibiting cellular senescence, or preventing or treating senescence-associated diseases.

Description

Pharmaceutical composition for preventing or treating cell aging or aging-related diseases comprising CD9 antibody as an active ingredient {Pharmaceutical composition for treating or preventing aging or age-related diseases comprising CD9 antibody}

The present invention relates to a pharmaceutical composition for the prevention or treatment of cell aging or aging-related diseases containing a CD9 antibody as an active ingredient, and by using a CD9-specific antibody that inhibits cell aging, the prevention or treatment of cell aging or aging-related diseases I want to.

When the normal somatic cells of mammals are isolated and cultured in a test tube, after a certain number of divisions, they do not divide any more and growth stops. This is called replication aging. Replication aging is known to occur when the telomeres, which are the terminal parts of the chromosome, shorten as cells divide. In addition, cell aging is induced by oxidative stress, increased activity of cancer genes and cancer suppressor genes, and administration of cytotoxic substances such as anticancer drugs. .

Senescent cells are not only morphological features that increase the size of the cells and flatten them, but also molecules such as increased senescence-associated β-galactosidase (SA-β-gal) activity, and increased expression of p16, p53, and Rb known as cancer suppressors. It is accompanied by enemy characteristics. In addition, it is known that the expression of various genes related to inflammatory response, DNA damage, cell growth and cycle regulation in senescent cells is changed, and related genes include oncogenes such as Raf and Ras, and cancer suppressing genes such as p53 and p16. In addition, inflammatory factors such as interleukin-6, interleukin-1β, interferon, and IGFBP5, and cell cycle regulatory genes such as aurora kinase B and polo-like kinase 1 are known to be involved.

Cellular aging contributes to individual and tissue aging, inhibits or promotes cancer, and contributes to tissue repair and etiology of age-related diseases. In particular, cell aging contributes to the etiology of various aging-related diseases such as cancer, atherosclerosis, endovascular proliferation, hepatitis, diabetes, additional plate degeneration, skin aging, degenerative renal disease, sarcopenia, osteoporosis, and enlarged prostate. According to recent research results, it has been reported that selective control of cellular aging can control tissue and organ aging, healthy lifespan, and the occurrence of aging-related diseases.

CD9 antigen is a tetraspanin cell membrane glycoprotein with a molecular weight of 24-27kDa. Cell adhesion and migration, platelet activity and aggregation, fusion of egg and sperm during fertilization in mammals, development and metastasis of cancer, humoral immune response And it is known as an antigen that plays an important role in allergic reactions, HIV-1 and influenza virus replication, and the like. However, studies on the regulation of cellular senescence or senescence of CD9 antigens have only reported that the expression is increased in aging human vascular endothelial cells, and the mechanism of cellular senescence regulation of CD9 and its role in atherosclerosis, a disease related to vascular aging, have been reported. Didn't.

Therefore, the present invention has completed the present invention by discovering the effect of inhibiting the action of CD9 by the CD9 antibody and inhibiting cell aging and atherosclerotic lesions through this, while studying how CD9 affects the regulation of cell aging. .

Korean Patent Publication No. 10-2010-0115157

An object of the present invention is a biomarker composition capable of inhibiting cell aging and diagnosing aging-related diseases by using a composition containing as an active ingredient an antibody that specifically binds to CD9 protein, which is increased in expression in aged cells. And it is intended to provide a pharmaceutical composition and a health food composition capable of preventing or treating cell aging or aging-related diseases.

In order to achieve the above object, the present invention provides a cell aging or aging-related disease biomarker composition containing CD9 as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating cell aging or aging-related diseases containing an antibody that specifically binds to CD9 as an active ingredient.

The present invention provides a health food composition for preventing or improving cell aging or aging-related diseases containing an antibody that specifically binds to CD9 as an active ingredient.

In addition, the present invention provides a method for screening a therapeutic agent for aging-related diseases, comprising the step of selecting a candidate drug that inhibits CD9 expression.

According to the present invention, the CD9-specific antibody specifically binds to the extracellular region of the CD9 antigen exposed on the cell surface and reduces the expression of CD9 in senescent cells, thereby inhibiting cell senescence and inhibiting aging and atherosclerosis, a vascular disease related to aging. It showed the effect of reducing sclerosing lesions. Accordingly, the CD9 of the present invention can be used as a biomarker composition for cell aging or aging-related diseases, and a pharmaceutical composition capable of inhibiting cell aging or preventing or treating aging-related diseases by using a composition containing a CD9-specific antibody as an active ingredient. Or it can be usefully used as a health food composition.

1 is a result of confirming the increase in CD9 expression according to aging in cells inducing replication aging or human fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs) induced senescence by adriamycin treatment, FIG. 1A is Western blot analysis and real-time PCR results confirming the level of CD9 protein and mRNA expression in young cells and each cell in which replication senescence was induced. FIG. 1B shows the level of CD9 expression in each cell in which senescence was induced by adriamycin treatment at RT. -Results confirmed by PCR (Y=young cells; O=senescent cells; adr=cells in which senescence was induced by adrimycin).
FIG. 2 shows recombinant CD9 adenovirus (Ad/CD9) and control adenovirus (Ad/Cont) in young cells of human fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs), respectively, 0, 5, 10 or 15. As a result of confirming cell aging induced by CD9 overexpression after infection with MOI (multiplicity of infection) and culture for 24 hours, FIG. 2A is a result of analyzing SA-β-gal activity in human umbilical vein endothelial cells, 2B is a result of analyzing SA-β-gal activity in human fibroblasts, FIG. 2C is a result of Western blot and RT-PCR analysis of each cell, and FIG. 2D is a result of confirming cell growth after CD9 overexpression (Y =Young cells; O=aging cells; PDL (population doubling level) = cell population doubling number) *P<0.05, **P<0.01.
3 is a result of confirming the degree of cell aging after reducing the expression of CD9 by transforming aging human fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs) with control siRNA or CD9 siRNA. Blot analysis results, Figure 3B is a result of confirming the cell growth due to the decrease in the expression of CD9, Figure 3C is the result of analyzing the SA-β-gal activity.
4 is a result of confirming the expression level of CD9 in human spleen vascular tissue, 20 vascular tissue samples of human spleen were prepared by age at 10-year intervals from 0 to 89 years old, and then the expression of CD9 was confirmed by immunostaining. The degree of positive expression of CD9 in vascular tissues for each age group was expressed as% (NC=negative control).
Figure 5 is after administration of a CD9 human antibody (CD9 hAb) or a control human IgG to aged human fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs) at 0, 1, 3 and 10 μg/ml, respectively. As a result of confirming the cell membrane binding of CD9 human antibody and the effect of inhibiting cell aging, FIG. 1A is the result of observing the cell membrane binding of CD9 human antibody by immunofluorescence staining, and FIG. 1B is the result of confirming cell proliferation by administration of CD9 human antibody And Figure 1C is a result of analysis of SA-β-gal activity *P<0.05, **P<0.01.
6 is a result of confirming the level of CD9 expression in atherosclerotic lesions of the aortic sinus and atherosclerotic lesions of human vascular tissues in ApoE-deficient mice and LDLR-deficient mice to confirm the level of CD9 expression in atherosclerotic lesion tissue. , FIG. 1A is an immunostaining result confirming CD9 expression in atherosclerotic lesions of ApoE-deficient mice, SA-β-gal staining result and Oil-red O staining result confirming SA-β-gal activity, and FIG. 1B is an LDLR-deficient mouse It is the immunostaining result confirming the expression of CD9 in atherosclerotic lesions of, Figure 1C is the immunostaining result confirming the expression of CD9 in atherosclerotic lesions of human vascular tissues (L = lumen; A = atherosclerotic lesions; ApoE-/- =ApoE-deficient mice; LDLR-/-=LDLR-deficient mice; WT=wild type mice).
Figure 7 is a high-fat diet to confirm the effect of the CD9 antibody on atherosclerotic lesions of ApoE-deficient mice, CD9 rat antibody (αmCD9) or rat IgG (mIgG) to the ApoE-deficient mice, each 100 ㎍ at 3.5 days interval 15 After weekly intraperitoneal administration, as a result of confirming the weight, food intake, and atherosclerotic lesions of the aorta and aortic tunnel, FIG. 7A is a graph confirming the change in body weight according to administration of the CD9 rat antibody to ApoE-deficient mice, and FIG. 7B is an ApoE defect. It is a graph confirming the food intake according to the administration of CD9 rat antibody to mice, and FIG. 7C is a graph showing serum triglyceride (TG), total cholesterol (T-Chol), high density lipoprotein (HDL) and low density according to the administration of CD9 rat antibody to ApoE-deficient mice. It is a graph showing the change in the concentration of lipoprotein (LDL), Figure 7D is the result of confirming the change in atherosclerotic area by performing Oil-red O staining and SA-β-gal staining in the aorta of ApoE-deficient mice, and Figure 7E is ApoE Hematoxylin-eosin (H/E) staining, CD9 immunostaining, Oil-red O staining, and SA-β-gal staining of aortic sinus tissue specimens from defective mice, and volume values of aortic sinus and atherosclerotic lesions It is a graph showing the mean and standard deviation of *P<0.05, **P<0.01.

The present invention provides a cell aging or aging-related disease biomarker composition containing CD9 as an active ingredient.

According to an embodiment of the present invention, the expression of the CD9 protein was increased in aged cells than in young cells. When the expression of the CD9 protein was reduced in senescent cells, the growth of senescent cells was improved, and the activity of SA-β-gal, which indicates senescent activity, was decreased. On the other hand, when overexpressing CD9 in young cells, it was confirmed that cell growth decreased and senescence was promoted. From the above results, it was confirmed that the CD9 protein is an important biomarker composition capable of controlling and diagnosing cell aging.

The CD9 protein has a cell membrane region, a cytoplasmic region, and an extracellular region, and the extracellular region of CD9 is exposed on the cell surface, and the CD9 antibody of the present invention specifically binds to the extracellular region of CD9 and acts on CD9. By inhibiting, it exhibited cell growth and aging recovery effects of aged cells, and reduced atherosclerotic lesions, which are vascular diseases associated with cell aging.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating cell aging or aging-related diseases containing an antibody that specifically binds to CD9 as an active ingredient.

The CD9 antibody of the present invention may be represented by the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, and may be selected from the group consisting of human, mouse, rat, and goat. . More specifically, it may be a CD9 human monoclonal antibody or a CD9 rat monoclonal antibody, more preferably a CD9 human monoclonal antibody, but is not limited thereto.

In the present invention, "antibody" refers to a specific protein molecule directed against an antigenic site. The antibody of the present invention may include both a polyclonal antibody and a monoclonal antibody, and the antibody may be prepared using techniques well known in the art.

As described above, the antibody specifically binding to CD9 of the present invention can effectively prevent or treat diseases related to cell aging or aging.

In the present invention, cell aging may be aging or replication aging of vascular endothelial cells or fibroblasts, and aging of vascular endothelial cells or fibroblasts may be induced by adriamycin, but is not limited thereto.

In addition, the age-related diseases in the present invention may be selected from the group consisting of atherosclerosis, skin aging, osteoporosis, rheumatism, and degenerative osteoarthritis, but is not limited thereto.

The composition for preventing or treating cell aging or aging-related diseases of the present invention may further include pharmaceutically acceptable excipients, carriers, diluents, and the like. Carriers that can be used in the present invention include proteins, polypeptides, liposomes, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and slowly metabolized macromolecules such as inactive virus particles. Salts of inorganic acids such as, for example, hydrochloride, hydrobromide, phosphate and sulfate; Pharmaceutically acceptable salts such as salts of organic acids such as acetate, propionate, malonate and benzoate; Liquids such as water, brine, glycerol and ethanol; And auxiliary substances such as hydrating agents, emulsifying agents or pH buffering substances.

In addition, the composition is formulated in a unit dosage form suitable for intra-body administration of a patient according to a conventional method in the pharmaceutical field, preferably in a form useful for administration of protein drugs, and is administered commonly used in the art. Oral, or intravenous, intramuscular, intraarterial, intramedullary, meningeal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, gastrointestinal, topical, sublingual, intravaginal or rectal using the method. It may be administered by a parenteral route including the route, but is not limited thereto.

Formulations suitable for this purpose include various preparations for oral administration such as tablets, pills, dragees, powders, capsules, syrups, solutions, gels, suspensions, emulsions, microemulsions, and injections such as ampoules for injection, Formulations for parenteral administration such as injections and sprays such as hypospray are preferred. In the case of a formulation for injection or infusion, it may take the form of a suspension, a solution, or an emulsion, and may include a formulation agent such as a suspending agent, a preservative, a stabilizer and/or a dispersing agent. In addition, the antibody molecule may be formulated in a dried form that can be readjusted to an appropriate sterile liquid prior to use and used.

As an active ingredient of the composition or pharmaceutical formulation of the present invention, the antibody is divided into one or several times by dividing 0.01 to 50 mg/kg body weight, preferably 0.1 to 20 mg/kg body weight per day, for mammals including humans. Can be administered. However, the actual dosage of the active ingredient is in light of various related factors such as the disease to be prevented or treated, the severity of the disease, the route of administration, the patient's weight, age and sex, drug combination, reaction sensitivity, and resistance/response to the treatment. It is to be understood that it is to be determined, and therefore, the dosage is not in any way limiting the scope of the invention.

In addition, the present invention provides a health food composition for preventing or improving cell aging or aging-related diseases containing an antibody that specifically binds to CD9 as an active ingredient.

The CD9 antibody of the present invention may be represented by the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, and may be selected from the group consisting of human, mouse, rat, and goat. . More specifically, it may be a CD9 human monoclonal antibody or a CD9 rat monoclonal antibody, more preferably a CD9 human monoclonal antibody, but is not limited thereto.

In the present invention, cell aging may be aging or replication aging of vascular endothelial cells or fibroblasts, and aging of vascular endothelial cells or fibroblasts may be induced by adriamycin, but is not limited thereto.

In addition, the age-related diseases in the present invention may be selected from the group consisting of atherosclerosis, skin aging, osteoporosis, rheumatism, and degenerative osteoarthritis, but is not limited thereto.

The health food may be provided in the form of powder, granule, tablet, capsule, syrup or beverage, and the health food may be provided with other foods or food additives in addition to the antibody specifically binding to CD9 according to the present invention, which is an active ingredient. Used, and can be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use, for example, prevention, health or therapeutic treatment.

The effective dose of the antibody specifically binding to CD9 contained in the health food can be used in accordance with the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control In the above range, it is clear that the active ingredient can be used in an amount above the above range because there is no problem in terms of safety.

There is no particular limitation on the kind of health food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, Drinks, alcoholic beverages, and vitamin complexes.

In addition, the present invention provides a method for screening a therapeutic agent for aging-related diseases, comprising the step of selecting a candidate drug that inhibits CD9 expression.

The screening method may be used as a screening method for a therapeutic agent for age-related diseases including atherosclerosis, skin aging, osteoporosis, rheumatism, and degenerative osteoarthritis.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.

< Experimental example 1> Cell culture

Human umbilical vascular endothelial cells (HUVECs) and human dermal fibroblasts (HDFs) were purchased from LONZA Inc. (Walkersville, MD). HUVECs cells are in EGM-2 medium, HDFs cells are Dulbecco's. ^{Each 2×10 5} cells in Modified Eagle Medium (DMEM) medium were dispensed into a 100 mm culture dish, and 37°C, 5% CO ₂ It was cultured in an incubator. When the cells were present in about 80 to 90% of the culture dish, trypsin was treated to separate them from the culture dish, and cell growth was confirmed by cell population doubling (PDs) as shown in the following formula.

PD=log ₂ F/log ₂ I (F=number of cells collected last, I = number of cells dispensed for the first time)

After confirmation, cells with PD<28 were used as young cells and cells with PD>50 as senescent cells.

< Experimental example 2> RNA Extraction and Reverse transcription Polymerase chain reaction ( RT - PCR )

Human umbilical endothelial cells and human fibroblasts were aliquoted ^{into 4×10 5 cells in each 100 mm culture dish.} RNA was extracted from each cell using Trizol reagent. DNTP, RNase inhibitor, AMV RTase, and oligo-d(T) were mixed with 1 μg of the isolated RNA and reacted to obtain cDNA. Using cDNA, each primer and Taq polymerase as shown in Table 1, 25 to 30 cycles were performed with a thermocycler to obtain a PCR product. It was confirmed by electrophoresis on 1% agarose gel. Using GAPDH as a control, PCR was performed under the same conditions, and then confirmed by electrophoresis.

Gene name Sequence list Base sequence CD9
One Forward CATGATGCTGGTGGGCTTCC 2 Reverse CCAAACCACAGCAGTTCAAC p53
3 Forward GCCTGAGGTTGGCTCTGA 4 Reverse GTGGTGAGGCTCCCCTTT IL6
5 Forward TGAGGAGACTTGCCTGGTG 6 Reverse TGCCCAGTGGACAGGTTT IL1beta
7 Forward TCTCCACCTCCAGGGACA 8 Reverse CTGCCAGCCCTAGGGATT GAPDH
9 Forward CGACCACTTTGTCAAGCTCA 10 Reverse AGGGGTCTACATGGCAACTG

< Experimental example 3> Real-time cell growth check

The cells were aliquoted into a 96-well at 1×10 ³ and cultured for 24 hours, and then treated with CD9 adenovirus or transfected with CD9 siRNA and cultured for an additional 24 hours. After replacing the culture medium, cells were photographed with a Leica ASMDW confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) every 2 hours for 96 hours. Cell growth was analyzed by counting the number of cells directly from photo images taken every 6 hours.

< Experimental example 4> Real-time polymerase chain reaction ( real - time quantitative PCR analysis )

MRNA expression was measured using SYBR Green (Applied Biosystems, USA). 2×DNA master (SYBR Green) 10 μl of sterile distilled water 6 μl, cDNA 2 μl, 2 μM CD9 (SEQ ID NO: 1) or GAPDH (SEQ ID NO: 9) forward primer and 2 μM CD9 (SEQ ID NO: 2) Alternatively, each 1 µl of GAPDH (SEQ ID NO: 10) reverse primer was added and the total reaction volume was 20 µl. PCR was performed using a 7500 Real Time PCR System (Applied Biosystems, USA). The reaction process was carried out 38 times at 50°C for 2 minutes and 94°C for 10 minutes, 95°C for 15 seconds and 54°C for 2 minutes, and the results were analyzed by the LightCycler program (Applied Biosystems, USA).

< Experimental example 5> protein extraction and Western Blot analysis

^{After dispensing 4×10 5} cells in a 100 mm culture dish and washing with cold PBS, 100 μl RIPA buffer [25 mM Tris-HCl, pH 7.4, 150 mM KCl, 5 mM EDTA, 1% NP-40, Cells were lysed by adding 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na ₃ VO ₄ , 5 mM NaF, 1 mM phenylmethyl sulfonylfluoride]. Then, centrifugation was performed at 12,000 g for 10 minutes, and the concentration of the protein was measured by the bicinchoninic acid (BCA) method. 30 μg of protein was transferred to a nitrocellulose membrane after 10% SDS-PAGE. The membrane was treated with 5% skim milk for 30 minutes and reacted with the primary antibody for at least 4 hours. After washing for 30 minutes using 1×TBS, reacting for 90 minutes with a secondary antibody to which horseradish peroxidase is attached, washing, and then reacting with ECL solution for 2 minutes and with LAS-3000. Confirmed. The amount of protein was compared using the GAPDH antibody.

< Experimental example 6> CD9 Adenovirus Production and overexpression

CD9 overexpressing adenovirus was constructed using pAdEasy-1 adenovirus vector (adenoviral vector). CD9 cDNA was obtained by performing a polymerization chain reaction from pSM2/CD9, and the nucleotide sequence of CD9 cDNA was confirmed by dideoxy sequencing. CD9 cDNA was inserted into the pShuttle vector. The pShuttle/CD9 recombinant vector was treated with Pme I restriction enzyme and alkaline phosphatase and precipitated with ethanol. After transforming the DNA and pAdEasy vector into BJ5183 cells, a pAd/CD9 recombinant vector was prepared. After the pAd/CD9 recombinant vector was treated with Pac I, AD293 cells were transformed. Virus titer was measured using the pAdEasy titer kit.

< Experimental example 7> In cells and tissues senescence - associated β- galactosidase ( SA -β- gal ) Dyeing check

Cells and frozen tissue sections were washed with 1×PBS, and fixed with PBS containing 3.7% (v/v) paraformaldehyde. Then 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactoside, 40 mM citric acid -Sodium phosphate (citric acid-sodium phosphate; pH 6.0), 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl ₂ were reacted at 37° C. for 17 hours and 30 minutes, and then blue using an optical microscope. The cells stained with were confirmed.

< Experimental example 8> human spleen vascular tissue specimen and Atherosclerosis Lesion tissue specimen

Normal vascular tissues by age were 20 samples under 10 years old, 20 samples aged 11-20 years old, 20 samples aged 21-30 years old, from normal tissue around the spleen and testis surgically excised from 1995 to 2012 at Yeungnam University Hospital. 20 samples, 41-50 years old, 20 samples, 51-60 years old, 20 samples, 61-70 years old, 20 samples, and 71 years old and over, 20 samples each. Six samples diagnosed with atherosclerosis after carotid artery resection were included. The resected spleen and carotid artery tissues were fixed in 10% neutral formalin, embedded in paraffin, and observed by performing hematoxylin-eosin staining. Histological findings were confirmed on the hematoxylin-eogene stained slides, representative sites were marked, and corresponding sites were selected in the paraffin block. Using a tissue microarray block manufacturing apparatus, the selected area was punched in a size of 5 mm, and then planted in a total of 20 recipient blocks in 4 columns and 5 rows with a diameter of 5 mm, thereby producing a total of 8 blocks.

< Experimental example 9> Oil- Red O( Oil - red O) and hematoxylin-eosin ( Hematoxylin -eosin) dyeing

After dissolving Oil-red O in 3% in 2-propanol, the precipitate was removed with a 0.45 μm syringe filter. A 3% Oil-red O solution was mixed with distilled water in a ratio of 6:4 and used for tissue staining. In addition, for hematoxylin-eosin staining, a tissue sample was immersed in a hematoxylin solution for 7 minutes, washed for 3 minutes with tap water, immersed twice in 1% HCl, and then 3 minutes with tap water. During washing.

Then, it was neutralized with 1% ammonia water, washed with tap water for 3 minutes, dyed in eosin solution for 2 minutes, and dehydrated with alcohol.

< Example 1> in senescent cells CD9 Confirmation of expression

To confirm the expression of CD9 antigen according to the degree of cell aging, RT-PCR, realtime PCR in HUVEC and HDF cells induced replication aging through passage and HUVEC and HDF cells induced senescence by adriamycin. , Through Western blot analysis, the level of expression of the CD9 antigen was confirmed.

After inducing cell senescence by culturing the cells in the same manner as in Experimental Example 1, the cells were subjected to real-time PCR and Western blot analysis to confirm the expression level of CD9.

In addition, cell senescence induced by adrimycin was divided into 2×10 ⁵ cells in a 60 mm culture dish and cultured for 24 hours. After washing three times using Dulbecco's Modified Eagle Medium (DMEM), 500 nM of adriamycin was treated on the cells for 4 hours. After removing adriamycin, wash 3 times with DMEM, and human umbilical vein endothelial cells contain 10% fetal bovine serum, 100 U/ml penicillin, and 100 ug/ml streptomycin. EGM-2 medium, human fibroblasts were cultured for 4 days in DMEM medium containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 ug/ml streptomycin. After that, RNA was extracted from each cell, and expression of CD9 and p53 was confirmed by RT-PCR.

As a result, it was confirmed that the expression of CD9 antigen was increased in each HUVEC and HDF cells in which senescence was induced by passage or adriamycin as shown in FIG. 1.

< Example 2> CD9 Confirmation of cell changes according to expression

As shown in the results of Example 1, as it was confirmed that the expression of CD9 was increased in senescent cells than in young cells, it was confirmed whether CD9 is involved in the regulation of cell aging.

One. CD9 Confirmation of increase in cellular senescence due to overexpression

Young cells were treated with CD9 adenovirus to increase the expression of CD9 and confirm the degree of cellular senescence.

In a 100 mm culture dish, HUVEC and HDF young cells were ^{divided into 4×10 5} cells, respectively, and 37° C., 5% CO _{2 for 24 hours.} It was cultured in an incubator. Thereafter, in order to induce overexpression of CD9, the CD9 adenovirus prepared as in Experimental Example 6 was treated and cultured for 24 hours, and then the culture medium was replaced and the cells were further cultured for 4 and 6 days, and the cells were harvested.

In order to confirm whether or not cell senescence progressed in the cells overexpressing CD9, the degree of cell senescence was confirmed through SA-β-gal staining in the same manner as in Experimental Example 7.

As a result, it was confirmed that the expression of p53, p21, IL-6 and IL-1β was increased in UVEC and HDF cells overexpressing CD9 as shown in FIGS. 2A, 2B and 2C, and pRb expression and cell growth were observed in FIGS. 2C and 2D. A decrease was confirmed. On the other hand, referring to FIG. 2C, ATM activity, which plays an important role in DNA damage, did not show any change.

From the above results, it was confirmed that when the expression of CD9 in young cells is increased, cell senescence is promoted.

2. CD9 Confirmation of inhibition of cellular senescence by reduction

After reducing the expression of CD9 in senescent cells, the degree of cellular senescence was confirmed.

To reduce the expression of CD9 in senescent cells, Life Technologies' Stealth ^TM CD9 siRNA [sense, 5'-AAGGUUUCGAGUACGUCCUUCUUGG-3' (SEQ ID NO: 11); and antisense, 5'-CCAAGAAGGACGUACUCGAAACCUU-3' (SEQ ID NO: 12)] Was used. ^{Dispensing senescent cells into 4×10 5} cells in a 100 mm culture dish, 37℃, 5% CO ₂ After incubation for 24 hours in an incubator, CD9 siRNA and control siRNA were transfected using Lipofectamine 2000, and cultured for 4 days. After cultivation, the level of CD9 expression was confirmed by Western blot, and the degree of aging was confirmed through SA-β-gal active staining and cell growth measurement.

As a result, as the expression of CD9 was decreased, the expression of p53 and p21 proteins was decreased as shown in FIG. 3A, and cell growth was promoted as shown in FIG. 3B. In addition, as shown in Figure 3C, the activity of SA-β-gal was reduced.

From the above results, it was confirmed that when the expression of CD9 in senescent cells was reduced, cellular senescence was suppressed and rejuvenation was observed.

Therefore, it was confirmed that CD9 plays an important role in the regulation of cell aging.

< Example 3> Age-dependent in human vascular tissue CD9 Confirmation of increased expression

It was confirmed that CD9 expression was not only increased during cellular aging, but also actually increased in aged tissues.

First, spleen samples from humans with vascular tissues with vascular endothelial cells were collected by age from 0 to 90 years old, and tissue samples were prepared by the method of Experimental Example 9, and immunohistostaining was performed.

In order to remove the peroxidase activity of human vascular tissue, _{react with 3% H 2} O ₂ diluted in methanol for 10 minutes, then dilute CD9 antibody (Abcam, ab92726) 1:50 and react overnight at 4°C. Then, the level of CD9 expression was confirmed using the Dako Envision kit, and the nuclei were confirmed by staining with Mayer's hematoxylin.

As a result, as shown in Fig. 4, the staining intensity of CD9 increased as age increased, and CD9 positive expression in vascular tissues of 20 tissue samples for each age was 0% in 0-9 years old, 15% in 10-19 years old, 20 to 20 years old. 10% at 29 years old, 15% at 30 to 39 years old, 25% at 40 to 49 years old, 70% at 50 to 59 years old, 70% at 60 to 69 years old, 60% at 70 to 79 years old, and 80 to 89 years old It was confirmed that 40% was expressed in.

From the above results, it was confirmed that CD9 expression increases with age in human vascular tissues, which may be involved in tissue aging of blood vessels.

< Experimental example 4> CD9 Confirmation of the effect of regulating cell aging by human antibodies

As it was confirmed from the results of the previous experiments that CD9 plays an important role in the regulation of cell aging, the effect of the CD9 human antibody on the regulation of cell aging was confirmed.

One. CD9 Human antibody production

Preparation of anti-CD9 monoclonal human antibody 10E4 (SEQ ID NO: 13 or 14) was performed according to Korean Patent Registration No. 10-1227971. For the production of 10E4, HEK293F cells, a suspension cell line, were inoculated into FreeStyle ^TM 293 expression medium (Life Technology, USA) at 1×10 ⁵ cells/ml, cultured for 24 hours, and transfected with a PEI:DNA ratio of 4:1. (transfection). After culturing in a 37°C, 8% CO ₂ suspension incubator for 6 days, cells and debris were precipitated by centrifugation for 5 minutes at 1,000 × g in a centrifuge, and the culture solution was harvested and sterilized through a 0.22 um top filter. In order to purify the antibody from the culture medium, affinity column chromatography was performed through protein A resin (GE Healthcare, USA), and the antibody was fractionated from the column with 0.2M glycine buffer (pH 2.5) and eluted. At this time, PBS (pH 7.4) was added in 1/4 volume to the eluate to increase the pH of the buffer containing the antibody. Fractions containing a high concentration of antibody in the fractionated eluate were selected using Coomassie (Bradford) Protein Assay kit (Thermo, USA), and all these fractions were combined and concentrated with Centricon (MWCO 10 kDa, Millipore, USA) and added thereto. The process of diluting again with PBS was repeated 3 to 4 times to make the final suspension solution of the antibody into PBS. The toxin level (endotoxin level) of the purified antibody was confirmed using the LAL assay kit (Lonza, USA), and only antibodies with less than 1 EU/mg were used.

The sequence of the anti-CD9 monoclonal human antibody 10E4 is shown in SEQ ID NO: 13 and SEQ ID NO: 14 below.

CD9 10E4 HC: MAQVQLVQSGGGLVQPGRSLRLSCAASGFTFD DFAMH GISWNSGDIRYADSVRGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCAR SPVGTTYFDYWGQGALITVSS WVRQAPGKGLEWVA (SEQ ID NO: 13), and CD9 10E4 LC: DIQMTQSPSSLSASVGDRVTITC RASQGISSYLA WYQQKPGKAPKLLIY AASTLQSEVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQLNIFPLT FGGTKVDIKR (SEQ ID NO: 14).

2. Confirmation of cell aging control effect

Human umbilical vein endothelial cells (HUVEC) and human fibroblasts (HDF) were each subcultured to induce senescence, and then 1×10 ⁵ cells were dispensed into a 60 mm culture dish and cultured for 24 hours. The prepared CD9 human antibody and control human IgG were treated with 1-10 μg/ml, respectively, and 37°C, 5% CO ₂ After culturing in an incubator for 3 days, whether or not aging cells were overcome by treatment with CD9 human antibody was confirmed by SA-β-gal active staining and cell growth.

In addition, the binding of the CD9 human antibody to the cells was confirmed by immunofluorescence staining.

First, 5×10 ³ cells were dispensed on a slide and cultured in an incubator at _{37° C. and 5% CO 2 for 24 hours.} The cells were washed three times with PBS and fixed at room temperature for 15 minutes with PBS containing 3.7% (v/v) paraformaldehyde. It was treated for 45 minutes using PBS containing 3% goat serum. Then, fluorescence-conjugated anti-human IgG (Fluorescein-conjugated anti-human IgG) was diluted 150 times in 3% goat serum and reacted at 37° C. for 90 minutes. Thereafter, the cells were washed three times with PBS and incubated with 100 ng/ml DAPI for 10 minutes at room temperature, and then the cells were observed with a fluorescence microscope.

As a result, as shown in Fig. 5A, binding of the CD9 human antibody and cells was confirmed.

In addition, referring to FIG. 5B, it was confirmed that the growth of cells treated with CD9 human antibody was increased than that of cells treated with human IgG as a control, and SA-β-gal activity of cells treated with CD9 human antibody as shown in FIG. 5C. Significantly decreased.

From the above results, it was confirmed that the CD9 human antibody binding to the extracellular region of CD9 has an effect of inhibiting cell senescence.

< Example 5> ApoE With missing mice LDLR Missing mouse Atherosclerosis In the lesion CD9 Confirmation of increased expression

To confirm that CD9 expression was also increased in atherosclerotic lesion tissue, a representative vascular disease related to aging, CD9 expression was confirmed by immunostaining in atherosclerotic lesions of ApoE-deficient mice and LDLR-deficient mice, an atherosclerotic mouse model.

After each mouse was sacrificed, the heart was removed and fixed in a 3.7% paraformaldehyde solution. After reacting each day in 10% sucrose, 20% sucrose, and 30% sucrose solution, it is embedded with OCT compound, and frozen sections are manufactured using a LEICA CM1850 frozen section maker. I did. After the aortic sinus tissue section was prepared, CD9 expression in atherosclerotic lesions was confirmed by immunostaining.

Using a paraffin tissue sample of the prepared mouse atherosclerotic lesion, _{reacted with 3% H 2} O ₂ diluted in methanol for 10 minutes to remove the peroxidase activity of the tissue, and then CD9 antibody (Abcam, ab92726) was added. It was diluted 1:50 and reacted at 4° C. overnight, and the level of CD9 expression was confirmed using a Dako Envision kit, and the nuclei were confirmed by staining with Mayer's hematoxylin.

In addition, CD9 expression in the atherosclerotic lesion was confirmed by immunostaining using the human atherosclerotic lesion tissue sample prepared as in Experimental Example 8.

As a result, as shown in Fig. 6A, as the age of the ApoE-deficient mice increased, the degree of atherosclerotic lesions increased, and SA-β-gal activity in the lesioned tissues increased, and CD9 expression increased. In addition, it was confirmed that CD9 expression was increased in atherosclerotic lesion tissues of LDLR-deficient mice of FIG. 6B. In addition, it was confirmed that CD9 expression was increased in human atherosclerotic lesion tissue as shown in FIG. 6C.

From the above results, it was confirmed that CD9 expression was increased in atherosclerotic lesions, which are vascular aging diseases in animals and humans.

< Example 6> CD9 By rat antibody ApoE Missing mouse Atherosclerosis Confirmation of lesion reduction

From the above results, it was confirmed in ApoE-deficient mice whether the actual CD9 antibody has the effect of inhibiting atherosclerotic lesions.

While feeding ApoE-deficient mice on a high fat diet, CD9 rat antibody and IgG as a control were injected intraperitoneally for 15 weeks each 3.5 days, and the mice were sacrificed, and the concentrations of serum triglyceride, total cholesterol, high-density lipoprotein, and low-density lipoprotein were measured. I did. The concentrations of serum cholesterol, triglycerides, high-density lipoproteins, and low-density lipoproteins are determined by Kyowa medex (Tokyo, Japan) for total cholesterol measurement kit (Cat No. 132614), triglyceride measurement kit (Cat No. 132691), and high density lipoprotein measurement kit (Cat No. 136148) and low-density lipoprotein measurement kit (Cat No. 137520), respectively.

In addition, changes in atherosclerotic lesions in the aorta and aortic tunnel were confirmed.

In order to measure the atherosclerotic lesion of the aorta, the mouse was placed on a surgical microscope, and the aorta was carefully removed using a microsurgical instrument while checking the heart, aorta, carotid artery, thoracic artery, and abdominal aorta with a microscope. After removing the adipose tissue around the aorta, SA-β-gal staining was performed to measure the degree of aging, and Oil-red O staining was performed to identify atherosclerotic lesions.

To measure the atherosclerotic lesion at the aortic tunnel, the aortic tunnel was excised from the heart of the mouse and embedded in an OCT compound. Continuous frozen sections were fabricated with a thickness of 20 μm from the start of the coronary artery to the end of the aortic valve using a frozen tissue slicer. Oil-red O staining and Hematoxylin-eosin staining were performed on the prepared tissue section by the method of Experimental Example 11, and SA-β-gal staining was performed by the method of Experimental Example 7, and the volume of atherosclerotic lesions was determined by the total artery. The area stained with Oil-red O was measured using the Image J program (Wayne Rasband, National Institutes of Health, USA).

As a result, as shown in FIG. 7A, the body weight was decreased in the CD9 rat antibody-administered group (αmCD9) compared to the IgG-administered control group (mIgG), but there was no difference in food intake as shown in FIG. 7B. In addition, there was no difference in the concentration of serum total triglyceride and high-density lipoprotein in the CD9 rat antibody-administered group (αmCD9) than in the control group (mIgG) administered with IgG as shown in FIG. 7C, but the concentration of total cholesterol and low-density lipoprotein was decreased.

On the other hand, as a result of Oil-red O staining and SA-β-gal staining in the aorta, as shown in FIG. In the results of Oil-red O staining and SA-β-gal staining of atherosclerotic lesions in aortic tunnel tissue specimens, as shown in FIG. (mIgG) showed a statistically significant decrease.

From the above results, it was confirmed that the CD9 antibody has an effect of inhibiting the occurrence of atherosclerotic lesions.

As described above, a specific part of the present invention has been described in detail, and for those of ordinary skill in the art, it is obvious that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<110> Research Cooperation Foundation of Yeungnam University <120> Pharmaceutical composition for treating or preventing aging or age related diseases comprising CD9 antibody <130> ADP-2015-0269 <150> 2014/099,872 <151> 2014-08-04 <160> 14 <170> KopatentIn 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 catgatgctg gtgggcttcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ccaaaccaca gcagttcaac 20 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gcctgaggtt ggctctga 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gtggtgaggc tccccttt 18 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tgaggagact tgcctggtg 19 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tgcccagtgg acaggttt 18 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tctccacctc cagggaca 18 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ctgccagccc tagggatt 18 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 cgaccacttt gtcaagctca 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 cgaccacttt gtcaagctca 20 <210> 11 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 11 aagguuucga guacguccuu cuugg 25 <210> 12 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 12 ccaagaagga cguacucgaa accuu 25 <210> 13 <211> 121 <212> PRT <213> CD9 10E4 HC <400> 13 Met Ala Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp 20 25 30 Asp Phe Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Gly Ile Ser Trp Asn Ser Gly Asp Ile Arg Tyr Ala Asp 50 55 60 Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Ser Pro Val Gly Thr Thr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Ala Leu Ile Thr Val Ser Ser 115 120 <210> 14 <211> 107 <212> PRT <213> CD9 10E4 LC <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Glu Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ile Phe Pro Leu 85 90 95 Thr Phe Gly Gly Thr Lys Val Asp Ile Lys Arg 100 105

Claims (6)

A pharmaceutical composition for preventing or treating atherosclerosis comprising an antibody that specifically binds to CD9 as an active ingredient. The pharmaceutical composition for preventing or treating atherosclerosis according to claim 1, wherein the antibody is a CD9 human antibody having the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14. The pharmaceutical composition for preventing or treating atherosclerosis according to claim 1, wherein the atherosclerosis is caused by transcriptional aging of vascular endothelial cells or aging induced by adriamycin. A health food for preventing or ameliorating atherosclerosis comprising an antibody specifically binding to CD9 as an active ingredient. The health food for preventing or ameliorating atherosclerosis according to claim 4, wherein the antibody is a CD9 human antibody having an amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14. [Claim 5] The health food for preventing or ameliorating atherosclerosis according to claim 4, wherein the atherosclerosis is caused by aging of the vascular endothelial cells or aging induced by adriamycin.

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