PHARMACEUTICAL COMPOSITION COMPRISING CD9 ANTIBODY FOR TREATING OR PREVENTING CELLULAR AGING OR AGING-RELATED DISEASES

The present invention refers to CD9 a unit having a cell senescence or aging compositions for the biomarkers related disorders.

According to one embodiment of the present invention, CD9 protein in cells Young aged than have been is upregulated in cells. Such CD9 protein reduction in the expression in cells aged aged when improve cell growth, senescence activity SA-β-gal indicative of the activity of the reduced. While, in cells Young safely when of the CD9, , is reduced in the cell growth is promoted under aging can be it is confirmed that. Said result a polyolefin, controlling cell senescence protein CD9 constitution: a trunk lid lock 2001 a biomarker composition it was found.

Such CD9 protein cell membrane region, cytoplasmic region, which a extracellular region, CD9 extracellular region of which is exposed on a surface a cell, of CD9 antibodies of the present invention CD9 extracellular region of specifically binding to the CD9 by inhibiting the action, aging of cells cell growth and aging precursor and CaO precursor effect recovery, cell senescence associated with atherosclerotic vascular disease reduced lesion curing.

Therefore, the present invention refers to CD9 antibodies that specifically bind to diseases which contain as the active ingredient cell senescence or aging medicament for treatment of preventing or related disorders provides composition.

13 14 list or sequencing of the present invention CD9 antibodies sequence list can be represented by the amino acid sequence of, human (human), mouse (mouse), RAT (goat) from the group consisting of of and go (rat) can be selected. More specifically a corresponding fria CD9 monoclonal antibodies or human CD9 may be monoclonal antibodies, more preferably human CD9 can be monoclonal antibodies, are not limited to.

In the present invention those parts of ' antibodies' antigenic email widow, a web page or for a protein that is specific indicated. molecules. Antibody will be and it will grow antibodies of the present invention, monoclonal antibodies may include both, the production of the antibodies using techniques widely publicly known art can be produced.

Said front board by establishing an optical fiber, of the present invention CD9 antibodies that specifically bind to cell senescence or aging effectively related conditions are can be preventing or treating.

In the present invention cell senescence vascular endothelial cells or fibroblast cell aging may be aging or replication, a cell aging said vascular endothelial cells or fibroblast can be derived by adriamycin to but, are not limited to.

Curing atherosclerotic disorders age-related in addition in the present invention, skin aging, osteoporosis, rheumatoid and degenerative osteoarthritis made of a value can be selected, are not limited to.

Preventing or related disorders or aging cell senescence of the present invention a composition for treating number pharmaceutically acceptable excipients, carrier, diluent may include further such as. Protein include carrier available in the present invention, polypeptides, liposome, polysaccharide, polylactic acid, polyglycolic acid, polymeric amino acid, amino acid copolymer and pharmacokinetics of slowly such as inactivated viral particles can be macromolecules. For example, hydrochloride, hydrogel bromide, phosphate and harvesting an inorganic salts; acetate, propionate, dichlorobenzoyl malonate and benzo, polycarbonate, to such as salt of an organic acid such pharmaceutically acceptable salts; water, brine, glycerol and a liquid, such as ethanol; and hydrophilizers, emulsion buffer pH or number and an adjuvant, such as material, use can be made of, either biological material,.

Furthermore, said pharmaceutical composition according to method of typically encountered in field within a patient's body for multiple type of the present invention show a release unit dosage suitable for administration by, preferably protein pharmaceuticals preparation form useful in administering where a new file does not exist per the formulated in a dosage commonly used in orally using method, or intravenous, intramuscular, in artery, intramedullary, inserted into the inserting tube and water, in ventricular, waste, transdermal, avoid, , either intraperitoneally, intranasal, in an alimentary tract, topical, the bisulphate, amniotic fluid in the vagina or rectal path including parenteral administration can be degraded and is administered to the patient by path, limited to not.

Formulations may be in the form of a suited for this purpose is purification, bolus, confectionery (dragee), masked powders that are to be, capsule number, number syrup, solution number, gel system, number suspension, emulsion, microemulsion variety of orally dosable preparation and scanning for ampoule such as injection, injection number, and pos framer number spray such as (hypospray) such as. preferred agents for parenteral administration. When the scanning or infusion preparations for, suspension, solution or emulsion and may take the form of a such as, point that serves as a suspending agent, preservative, stabilizer agents such as anti-dispersant and/or may comprise an agent. Furthermore, prior to use a antibody molecules said sterile suitable the subtracter subtracts the liquid which can be used in a dried form may be formulated into.

Composition or pharmaceutical compositions of the present invention as the active ingredient including a person said antibodies against mammal 0.01 to 50 mg/kg weight per day, preferably 0.1 to 20 mg/kg weight 1 administration times once or can be divided into. However, for effective ingredient actual dose constitutes a preventing or treatment of diseases, the severity of diseases, route, , the body mass of the subject, age and sex, pharmaceutical combinations of, reaction sensitivity and an resistance/reactions, and so forth for the treatment a variety of associated factor falling on a detector a to be determined must be understood, thus, any dose constitutes said manner, surface of the present invention. are not limited to range.

In addition the present invention refers to CD9 antibodies that specifically bind to diseases which contain as the active ingredient cell senescence or aging related disorders provides foodstuffs composition health for treatment or prevention.

13 14 list or sequencing of the present invention CD9 antibodies sequence list can be represented by the amino acid sequence of, human (human), mouse (mouse), RAT (goat) from the group consisting of of and go (rat) can be selected. More specifically a corresponding fria CD9 monoclonal antibodies or human CD9 may be monoclonal antibodies, more preferably human CD9 can be monoclonal antibodies, are not limited to.

In the present invention cell senescence vascular endothelial cells or fibroblast cell aging may be aging or replication, a cell aging said vascular endothelial cells or fibroblast can be derived by adriamycin to but, are not limited to.

Curing atherosclerotic disorders age-related in addition in the present invention, skin aging, osteoporosis, rheumatoid and degenerative osteoarthritis made of a value can be selected, are not limited to.

Said post-treatment system powder, granules, purification, capsule, syrup or beverage and may be ground to provide the in the form of, as an active ingredient, an said post-treatment system the present invention according to CD9 antibodies that specifically bind to or moderate the sharp taste and smell, and for preparing foods or food-which is used in conjunction with, conventional method suitably according to can be used. For effective ingredient mixing thereof tilted prevented for example a use purpose, according to health or therapeutic treatment of can be determined for.

Said health food contained in specifically CD9 the capacitance of an antibody that binds said pharmaceutical composition effective to the effective capacity the height, health and sanitary pad intended to a long is intended to control health or in the case of ingestion of said range may be hereinafter, active ingredient with a fixing member of the back in the aspect of safety because of the absence of said range can be used also in an amount of from at least. secure.

Said type of health food and free from are restrictions with specific, examples meat, sausage, bread, chocolate, candy current, [...], confectionery, pizza, ramyon, other noodles, gum type, ice cream containing a dairy product, various sprocket, beverage, difference, abundant, complex and vitamin alcoholic beverage as to the aromatic hydrocarbon.

In addition the present invention refers to CD9 drug candidate for inhibiting the expression of screening to aging-related therapeutic agent for a disease characterized by including step provides screening method.

The atherosclerotic curing said screening method, skin aging, osteoporosis, rheumatoid and of degenerative osteoarthritis screening therapeutic agents are provided, comprised of salts of age-related diseases method ID:2, seq ID :.

Hereinafter, embodiment to assist in the understanding of the present invention rapidly and to reduce a memory example thereby, the cold air flows to the. Just of the following embodiment relate CDK exemplified content of the present invention of the present invention embodiment a range not limited aspect. Of the present invention embodiment of the chair are a mean water level in the art for relate user with the present invention to illustrate the an entire surface is to be provided for.

<1aodirenc e.g. experiment > cell culture

Human umbilical cord vascular endothelial cells (human umbilical vascular endothelial cells, HUVECs) and human fibroblast (human dermal fibroblasts, HDFs) a LONZA Inc. The purchased in HUVECs (Walkersville, MD) cells EGM-2 the culture medium to which, Dulbecco HDFs cells' each the culture medium to which s Modified Eagle Medium (DMEM) 2×10⁵ cells the frequency divides the oven and entered into a cavity 100 mm culture 37 °C, 5% CO₂ good in incubator. Cell degree of dish culture 80-90% trypsin the presence of the processing (trypsin) isolated from the culture dish cells such as 1-5 wt % in an cell growth (population doubling; PDs) single house it is found out that to hydrolysis.

PD=log₂ F/log₂ I (F= finally collected in the reservoir the number of cells, I = the number of cells a divider initially)

After confirmation, <28 의 세포를 젊은 세포, PD>50 in cells PD aging cell at night.

<2aodirenc e.g. experiment > RNAextraction andreverse transcriptasepolymerase chain reaction(RT-PCR)

Human umbilical cord vascular endothelial cells and human fibroblast each 100 mm culture oven and entered into a cavity 4×10⁵ was frequency divides the sample with cells of the. Each cell (Trizol) sol dryer and control method thereof in the reagent extracted RNA. Separated RNA 1 micro g dNTP to, RNase inhibitor, AMV RTase oligo-d (T) and the reactor after the mixture obtained a cDNA. CDNA, table 1 primer (primer) and each such as using Taq polymerase (Taq polymerase) to (thermocycler) amplifier gene 25-30 cycle (cycle) after performing a PCR product is obtained. 1% agarose gel (agarose gel) electrophoretic in osteoclast maturation has been identified. The control group in the same using GAPDH PCR under conditions on electrophoretic osteoclast maturation has been identified.

<3aodirenc e.g. experiment > confirming cell growth between embodiment

Cells to 96-well 1×10³ after grown on time 24 and the in, CD9 adenovirus for processing to the lane width or CD9 siRNA (transfection) injection for transforming a good 24 and additional time. Culture after 2 time intervals and time program interfaces 96 Leica ASMDW confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) of cells in a photographing the. The cell growth cells photographing time intervals and 6 directly photographic Image or fixed human power by operating all the cell count.

<4aodirenc e.g. experiment > embodiment time PCR (real-timequantitativePCRanalysis)

SYBR Green (Applied Biosystems, USA) mRNA expression using sterile distilled l micro. 2 ×DNA master (SYBR Green) 10 it was determined that 6 micro l, l micro cDNA 2,2 µm CD9 (seq ID no:1) or GAPDH (sequence list 9) forward primer 2 µm CD9 and (forward primer) (seq ID no:2) or GAPDH (sequence list 10) reverse primer (reverse primer) at each 1 micro l capacity whole reaction doesn't have any error frames, 20 micro l on the experiment was carried out to. 7500 Real Time PCR System the PCR by cylinder to have (Applied Biosystems, USA). Process the 50 °C 2 ingredient, then reactions 94 °C 10,95 °C 15 seconds, the entire ingredient 54 °C 2 38 is relaxed by using at least corresponding advertisement based on the shown list, result to the computer of the LightCycler program was analyzing (Applied Biosystems, USA).

<5aodirenc e.g. experiment > protein extract and blotsblot analysis

Oven and entered into a cavity 100 mm culture 4×10⁵ cells cold and frequency divides the and, after washing the using PBS, 100 micro l RIPA buffer [25 mm Tris-HCl, pH 7.4,150 mm KCl, 5 mm EDTA, 1% NP-40,0.5% sodium deoxy [...] (sodium deoxycholate), 0.1% SDS, 1 mm Na₃ VO₄, 5 mm NaF, 1 mm phenylmethyl sulfoalkyl (phenylmethyl sulfonylfluoride) [...] ] adding a the dissolved of cells. 12,000g to 10 minutes then concentration of high protein centrifugation factor have been measured by bicinchoninic acid (BCA) method. After 10% SDS-PAGE for g micro 30 protein, nitrocellulose nitro moved to film (nitrocellulose). Film skim milk and miunutes 30 (skim milk) to 5%, 4 to the first antibody and, in its. 1 ×TBS reacted or more time using and, after washing the 30 minutes, (horseradish peroxidase) good earth[...]the lung rock it is sour, Oh sacrifice with secondary antibodies to 90 minutes and, after washing the whereby reactions can take place, ECL solution is confirmed LAS-3000 responsive to 2 minutes. Protein the amount and compared the GAPDH antibody.

<6aodirenc e.g. experiment > CD9adenovirusoverexpressing manufacturing and

PAdEasy-1 adenovirus vector (adenoviral vector) using constructed with a adenovirus overexpressing CD9. CD9 cDNA the pSM2/CD9 a massage cream, an essence, obtained by performing the cascade polymerization from, the CD9 cDNA bio-sequence listing dideoxy sequencing is confirmed. CD9 cDNA was inserted into a pShuttle vector. Recombinant vector pShuttle/CD9 Pme I restriction endonuclease and alkaline phosphatase was the and ethanol for processing. BJ5183 vector pAdEasy and a DNA transformed cells after, pAd/CD9 constructed with a recombinant vector. PAd/CD9 Pac I followed by a recombinant vector, AD293 the transformed cells. titer of it was determined that using pAdEasy titer kit.

<7aodirenc e.g. experiment > associated - senescence in cells and tissues β-galactosidase(gal -β- SA)confirming dyeing

Cells and refrigeration tissue fragments, and then being washed with 1 ×PBS, 3.7%(v/v) including aldehyde for controlling piezoelectric vibration was PBS is formed in the coupling space. Then 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D seed-galactosidase (5-bromo-4-chloro-3-indolyl-β-D-galactoside), 40 mm sodium-phosphate citric (citric acid-sodium phosphate; pH 6.0), 5 mm potassium cyanide ferrimagnetic (potassium ferricyanide), 150 mm NaCl, 2 mm MgCl₂ 17 time 30 minutes in a 37 °C reacting a metal salt of using optical microscope to check upper area of blue dyed to cells identified.

<8aodirenc e.g. experiment > human tissue specimens lesions curing atherosclerotic sample and vascular tissue operation errors of the device

The vascular tissue normal by age years 1995 doctor a favorite southeastern Korea 2012 ablation surgically until years from a spleen and testis in normal tissue around less than 10 cell sample 20,20 11-20 cell sample, sample 20 cell 21-30,31-40 cell sample 20,20 41-50 cell sample, sample 20 cell 51-60,61-70 cell sample 20, the consumer selects a desired object [...] sample 20 or more cell 71, the vascular tissue atherosclerosis 2012 from years 2000 doctor a favorite southeastern Korea until years after ablation (carotid artery) MAC the breeder bedded a diagnosed with atherosclerosis is a subject sample 6. MAC the breeder bedded and operation errors of the device made on 10% neutral formalin-fixed tissue after gun every grudge to paraffin and misdiagnosis - [...] (hematoxylin-eosin) has been observed on the dyeing. [...] - misdiagnosis dyeing slide in histological clients ask region representative opening in a block paraffin of the beam on him a portion corresponding. Tissue micro arrangement pulverized mechanism is used to the predetermined 5 mm diameter after punch sized portion of 5 mm of total row 5 heat 4 potted block recipient 20, total 8-up block includes the second was produced.

<9aodirenc e.g. experiment > oil-redO(red - Oil O) (Hematoxylin-eosin) dyeing texturing- [...] and

Oil-red O (Oil-red O) 3% 2-propanol (propanol) senses a rotation velocity of the disk to a to, 0.45 µm syringe filter for removing deposits the ratio 6:4 . 3% Oil-red O and a, the third to eo dyeing tissue plants. In addition the dyeing (Hematoxylin-eosin) texturing- [...][...] tissue specimens (Hematoxylin) solution soaked into minutes 7, tap water, to the 2 to 3 minutes to 1% HCl which washes the soaked into times, to tap water, to the cleaning was 3 minutes.

Then 1% ammonia to 3 minutes to tap water, to the neutralizing texturing (eosin) solution and, after washing the stining the 2 minutes, the dehydration alcohol.

< 실시예 1>Confirming expression CD9 in aging cell

Cell senescence degree according to CD9 expression of antigen to make sure that, aging of replicate through sebocytes are excellently derived adriamycin by HUVEC and HDF cells derived is aging RT-PCR in HUVEC and HDF, between embodiment (realtime) PCR, through automatic analysis (Western blot) blot blots expression of CD9 antigen degree identified.

Sebocytes are excellently an experiment using a computer to replicate through senescence inducing cells 1 e.g. such as a cell senescence by culturing of cells in method derived between embodiment (Western blot) blot blots and after (realtime) PCR expression of CD9 analyzer analyze a spectrum and a phase degree identified.

In addition a cell senescence induced by oh it will give oven and entered into a cavity 60 mm culture 2×10⁵ 24 and frequency divides the each cell to good time. Dulbecco ' using s Modified Eagle Medium (DMEM) adriamycin and, after washing the burn 3 (adriamycin) 500 nm for 4 time was filtered cells. Adriamycin after removing contaminants and metal oxides in cleaning burn 3 in DMEM to either a human umbilical cord endothelial cells are the vein 10% fetal small serum (fetal bovine serum), (penicillin) 100 U/ml penicillin, 100 ug/ml (streptomycin) improves EGM-2 with Streptomyces erythromycin, human fibroblast cells (fetal bovine serum) 10% fetal small serum, (penicillin) 100 U/ml penicillin, 100 ug/ml DMEM with Streptomyces erythromycin (streptomycin) grown on during 4 improves by extracting the RNA from each cell after RT-PCR CD9, p53 to expression of has been confirmed.

As a result, also 1, such as adriamycin or sebocytes are excellently each is derived aging by HUVEC and HDF increased expression of CD9 antigen in cells capable of confirming the was.

< 실시예 2>Confirming change cells according to CD9 expression

In the embodiment 1 and the goal such as, in cells than in Young aging cell with a selective ratio relative to the expression of CD9 by identified, CD9 cell it is found out that whether involved in controlling senescence.

1. CD9 confirming increase cell senescence by overexpression

Young cells process adenovirus CD9, increasing the expression of CD9 it is found out that OSC of a cell.

100 mm culture oven and entered into a cavity each cells Young HDF and HUVEC 4×10⁵ supplied to the light and the 24 time 37 °C, 5% CO₂ good in incubator. CD9 or overexpression of the tnf-to derive then, are protruded from both sides of the and 6 e.g. experiment process adenovirus CD9 24 time after incubating the culture fluid having between 6 and 4 replacing a additional culturing the cell line in the cells was cutting device.

CD9 is overexpression in cells and order to ascertain if the aging proceeds, such as 7 e.g. experiment SA-β-gal dyeing method in modulating cells via aging degree identified.

As a result, also 2A, overexpression is CD9 such as a 2C and 2B UVEC and HDF p53, p21 in cells, the base station can check the increase of expression of IL-1 β and IL-6 been, also 2C 2D and in reduction of growth expression and cell pRb were identified. While, also 2C reference to an surface, an important role to damage DNA ATM didn't indicative of a variation in the activity.

Said result in cells Young surface is upregulated of CD9, it is confirmed that the vacuum annealing equipment partly cell senescence can be.

2. Confirming inhibitors cell senescence by reducing aCD9

Expression of CD9 in aging cell reduction process is performed to reduce a cell senescence degree identified.

In aging cell to reduce expression of CD9, Life Technologies yarn Stealth^TM CD9 siRNA [sense, 5 '-AAGGUUUCGAGUACGUCCUUCUUGG-3' (sequence list 11); and antisense, 5 '-CCAAGAAGGACGUACUCGAAACCUU-3' (sequence list 12)] the first voice portion out of an. Aging cells oven and entered into a cavity 100 mm culture 4×10⁵ supplied to the light and the 37 °C, 5% CO₂[...] 24 in incubator (Lipofectamine) 2000 and the reformer provides the culture time using the control group and a CD9 siRNA (transfection) and injection for transforming a siRNA, good during 4. Culture nor post-incubation CD9 corresponding advertisement based on the shown list identified as (Western blot) blot blots degree of expression, degree of aging via telemetry and cell growing dyeing active SA-β-gal it is found out that.

As a result, from the outside during a CD9 expression, also 3A p21 and p53 such as a been reduced expression of proteins, also 3B been accelerated cell growth such as a. In addition also 3C SA-β-gal such as a the activity of the reduced.

CD9 said aging cell result in decreasing expression, cell senescence that it is inhibited from again a Young was capable of confirming the.

Therefore, cell CD9 important controlling senescence it was found to play a role.

< 실시예 3>Human angiogenic confirming expression amount according to CD9 age in tissues

CD9 expression cell senescence or increased as well as, actual tissue enhanced it is found out that whether.

First, angiogenic tissue vascular endothelial cells operation errors of the device of a person in cell 0 sample collecting and by age until cell 90 9 e.g. experiment method of manufacturing tissue specimens in immune tissue dyeing is performed for all the.

Human angiogenic active (peroxidase) tableted peroxidase activity of tissue to remove a dilution methanol 3% H₂ O₂ to CD9 antibodies reacting a metal salt of 10 minutes (Abcam, ab92726) in a 1:50 dilution in the 4 °C Dako Envision kit and age reacted night CD9 expression using the same thickness as that of the corresponding advertisement based on the shown list, nuclear the Mayer ' [...] s it is found out that the dyeing (hematoxylin).

Also 4 such as a result of a V age is into its original increasing the stiffness for dyeing CD9, each age in vascular tissue of tissue specimens of 20 by CD9 positive expression in cell 9 to 0 0%, 15% in cell 10 to 19,20 to 29 in cell 10%, 15% in cell 30 to 39,40 to 49 in cell 25%, 70% in cell 50 to 59,60 to 69 in cell 70%, 60% and 80 to 89 70 to 79 in cell in cell 40% expressed it has been confirmed.

Said result in vascular tissue a person's expression CD9 increases in thickness according to age, aging tissue of a blood vessel is or ester are capable of engaging in identifying improving.

< 실험예 4>Human CD9 confirming a cell senescence regulatory effects by means of bi-specific antibodies

CD9 from prior results of the experiments cell play a significant role to controlling senescence it is ascertained that the is, CD9 cell human antibodies identified an effect on controlling senescence.

1. CD9 manufacturing human antibodies

10E4 anti-CD9 monoclonal human antibodies (sequence list 13 14 list or sequencing) according to the fabrication of 25th which patent registration number number 10-1227971 been performed for production of. 10E4 cell master HEK293F cells suspensions FreeStyle^TM 293 expression medium (Life Technology, USA) to 1×10⁵ cells/ml thereby to 24 time culturing the cell line in the, PEI : (transfection) injection transgenic on the DNA ratio was 4:1. Between 6 37 °C, 8% CO₂ debris and cells following cultured in incubator suspension (debris) to 1,000 × g 5 minutes a centrifugal separator in precipitating the centrifugation, the harvesting culture the sterilization through 0.22 um top filter. Protein A resin purification thereof antibodies harvested from (GE Healthcare, USA) through corresponding advertisement based on the shown list for performing affinity column chromatography, 0.2M glycine buffer (glycine buffer; pH2.5) to antibodies was eluting fractions from the column (column). The, antibody pH of buffers included in order to increase the eluate to a 1/4 capacity (volume) added in PBS (pH 7.4). Fraction eluates, which are high in which fraction during antibodies Coomassie (Bradford) Protein Assay kit (Thermo, USA) an corresponding advertisement based on the shown list, these fraction combined both Centricon (MWCO 10 kDa, Millipore, USA) concentrated PBS dilution to wherein the shaft back to the storing final antibody in repeated 3-4 turn light with the PBS suspending solution. The (endotoxin level) level toxin of an antibody purified LAL assay kit (Lonza, USA) mg/1 EU corresponding advertisement based on the shown list of a digital signal, the third to eo antibody alone less than.

10E4 said anti-CD9 monoclonal human antibodies sequence of the a sequence list 14 and a equal 13 sequence list.

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

2. Cell senescence regulatory effects confirming

Human umbilical cord vein endothelial cells (HUVEC) and human fibroblast (HDF) passaging each derived after aging of by culturing, oven and entered into a cavity 60 mm culture 1×10⁵ 24 and the cells of good time. CD9 made a IgG human soldiers/correlation human antibodies to g/ml and each handling a micro 1-10, 37 °C, 5% CO₂ incubator in CD9 culturing the cell line in the between 3 subjecting the film to a treatment by human antibodies overcome whether aged cell aging SA-β-gal active dyeing and cell growing is confirmed.

Immunisation to automate and the cell human antibodies in addition CD9 express using fluorescent staining is confirmed.

First, 5×10³ types of cells to slide the division and 24 time 37 °C, 5% CO₂ good in incubator. Cells which washes the PBS 3 times to 3.7%(v/v) (paraformaldehyde) aldehyde for controlling piezoelectric vibration including 15 minutes was secures the room temperature PBS to. 3% (goat serum) serum hypertriglyceridemia, with 45 minutes using PBS was filtered. Humanized anti-human combined fluorescence is then 3% a IgG (Fluorescein-conjugated anti-human IgG) 150 to the dilution back (goat serum) serum hypertriglyceridemia, 37 °C reacted in 90 minutes. Then to 100 ng/ml DAPI which washes the PBS 3 times for 10 minutes for cells following incubation at room temperature has been observed on in fluorescence microscopy.

As a result, also 5A CD9 such as a coupled and the cell human antibodies were identified.

In addition also 5B reference to an surface, treated with human soldiers contrast IgG treated with human antibodies CD9 than increased cell growth of confirming corresponding advertisement based on the shown list, also 5C such as a CD9 SA-β-gal of cells is processed human antibodies the significance of active the part recessed from the conductive surface.

Said result that bind to CD9 extracellular region of human CD9 antibody cell senescence, which compounds inhibit certain transmembrane it is found out that can be.

< 실시예 5>Atherosclerotic ApoE LDLR and mouse defect mouse defect confirming expression amount of CD9 in lesions curing

CD9 expression associated with aging exemplary angiogenesis tissue lesions curing atherosclerotic disease reference skin tones in order to identify whether enhanced, models of MICE cured atherosclerotic ApoE atherosclerotic of mouse defective LDLR and mouse defect CD9 expression in lesions curing immune dyeing is confirmed.

After sacrifice MICE each, while the heart and an aqueous solution of, 3.7%. 10% the solution (paraformaldehyde) aldehyde for controlling piezoelectric vibration (sucrose) sucrose, 20% sucrose 30% sucrose (sucrose) and reacting a metal salt of each solution (sucrose) at day, and gun whip to OCT compound, using rice cake making machine refrigeration LEICA CM1850 the refrigerated fragment was produced. After positioned in the front of left aortic oyster a tissue slice, CD9 expression in atherosclerotic lesions curing immune dyeing is confirmed.

Atherosclerotic of MICE made using tissue specimens paraffin lesions curing, active (peroxidase) tableted peroxidase activity of tissue to remove a dilution methanol 3% H₂ O₂ to CD9 antibodies reacting a metal salt of 10 minutes (Abcam, ab92726) in a 1:50 dilution in the 4 °C Dako Envision kit and age reacted night CD9 expression using the same thickness as that of the corresponding advertisement based on the shown list, nuclear the Mayer ' [...] s it is found out that the dyeing (hematoxylin).

In addition are protruded from both sides of the and 8 e.g. experiment human atherosclerotic lesions cured using tissue specimens CD9 expression in atherosclerotic lesions curing immune dyeing is confirmed.

As a result also 6A defect ApoE such as a mouse's age of a V atherosclerosis lesions corresponding advertisement based on the shown list as the degree of crosslinking increases, while for glyphosate having increased activity, SA-β-gal in tissues lesions, checks that the is upregulated CD9 can be. In addition also 6B even tissue lesions curing atherosclerotic of mouse defective LDLR of CD9 is upregulated of confirming the can be. In addition also even tissue lesions curing atherosclerotic human CD9 is upregulated 6C such as a can be of confirming the.

Said result aging angiogenesis in a warm-CD9 expression animals and humans increased lesions curing atherosclerotic disease can be it is confirmed that.

< 실시예 6>A corresponding fria CD9 atherosclerotic ApoE a by means of bi-specific antibodies of mouse defect confirming reduced lesions curing

Antibody CD9 actual from result said lesion curing atherosclerotic ApoE whether the inhibitors it is found out that in mouse defect.

While ApoE [...] fat to mouse defect, a collection, contrary to a corresponding fria CD9 antibodies each IgG 15 in one interval 3.5 a scanning unit scans abdominal weeks after the serum triglyceride level are mouse sacrifice, total cholesterol, concentration of density 105,000xg for 30min 105,000xg for 30min and it was determined that. Serum cholesterol, neutral lipid-depleted, high density lipoprotein, Kyowa medex lipoprotein concentration (Tokyo, Japan) of total cholesterol assay kit (Cat No. 132614), neutral fat measuring kit (Cat No. 132691), high density lipoprotein assay kit (Cat No. 136148), lipoprotein assay kit (Cat No. 137520) it was determined that the.

Aortic atherosclerotic curing of lesions in oyster aortic and in addition change identified.

Aortic atherosclerotic lesions curing of surgical microscope to measure after putting a mouse, aortic heart, , carotid artery, fault artery, abdominal bandage fine while checking the microscope artery using surgical instrument was an aqueous solution of carefully for aortic. After removing the adipose tissue around aortic, for evaluating a degree of aging SA-β-gal corresponding advertisement based on the shown list performing dyeing, atherosclerosis lesions to make sure that dyeing is performed for all the Oil-red O.

Oyster aortic atherosclerotic lesions curing of mouse to measure the ablation sites oyster aortic from's heart, adaptation gun whip to OCT compound. Refrigeration organized rice cake using coronary artery is started up until site is terminated the aortic valve from site 20 µm thickness continuous refrigeration fragment was produced. 11 e.g. experiment to a tissue slice made in method of dyeing Oil-red O, corresponding advertisement based on the shown list performing dyeing Hematoxylin-eosin, such as 7 e.g. experiment method performs dyeing SA-β-gal in, area, artery entire volume of lesions curing atherosclerotic Oil-red O portion which are dyed with Image J program (Wayne Rasband, National Institutes of Health, USA) was by using.

As a result, also 7A such as a (α mCD9) CD9 IgG is applied to the back plate in a corresponding fria antibodies administration group compared to the control group (mIgG) but reduced weight, also 7B was no differentiation is made increased food-uptake such as a. Furthermore, also 7C such as a (mIgG) than the control group is applied to the back plate IgG a corresponding fria CD9 antibodies (α mCD9) group administration concentration of total neutral lipid-depleted serum in no differentiation is made but 105,000xg for 30min as well as, the concentration of 105,000xg for 30min and total cholesterol reduced.

While, in aortic Oil-red O dyeing and SA-β-gal dyeing result, also 7D such as a CD9 than the control group is applied to the back plate IgG (mIgG) administration group in a corresponding fria antibodies (α mCD9) curing atherosclerotic lesions area are statistically significantly reduce the corresponding advertisement based on the shown list, oyster aortic atherosclerotic curing of lesions in tissue specimens Oil-red O 7E also even dyeing and SA-β-gal dyeing result a corresponding fria CD9 such as a (α mCD9) group administration antibodies in the oyster aortic atherosclerotic curing of lesions volume than the control group is applied to the back plate IgG (mIgG) showed reduction in a significant statistically.

Said curing atherosclerotic antibody CD9 result that inhibit the development of lesions been found an effect.

The present invention at least a particular content detail the portion 25mbps and techniques, homogeneously distributed to be person with skill in the art, such a preferred embodiment only the specifically and the user makes a is aspect, of the present invention the not range is limited will the apparent. Thus substantial of the present invention by issuing an ranges are defined by claim and their equivalent will the pixels include.