KR20150034090A - Pharmaceutical composition for treating CNS injury-related diseases comprising CCL2 or CCL2 agonist - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating central nervous system injury-related diseases, wherein a CCL2 or CCL2 agonist is contained as an active ingredient. The CCL2 exhibits an effect in promoting the regeneration of the axon caused by conditioning injury (CI) by mediating an interaction between the neuron and the macrophagocyte, thereby effectively treating central nervous system injury diseases such as cerebral apoplexy and spinal cord injury. A candidate medicine for promoting the expression of activation of CCL2 is selected, thereby screening a medicine for treating central nervous system injury-related diseases.

Description

[0001] The present invention relates to a pharmaceutical composition for treating CNS injury-related diseases comprising CCL2 or CCL2 agonist as an active ingredient,

The present invention relates to a pharmaceutical composition for the treatment of central nervous system damage-related diseases containing CCL2 or CCL2 agonist as an active ingredient.

Adult mammals The central nervous system (CNS) axons of neurons are known to regenerate axons of the peripheral nervous system (PNS) rapidly while their ability to regenerate after injury is very limited. The limited regenerative capacity of CNS neurons is partly a characteristic of the CNS axons, but is also due to an unacceptable environment. The CNS mycelium is not the sole cause of the inhibitory function of neurite outgrowth, but it contains a number of inhibitory molecules that actively block axonal growth and thus constitute an important barrier to regeneration.

Stroke is a cerebrovascular disease that occurs when normal blood flow to the brain is compromised and the brain receives too much or too little blood, and functional recovery after stroke is associated with remodeling of the damaged brain, including cerebral angiogenesis and neurogenesis . Sudden obstruction of the cerebral artery leads to severe neurological impairment, while demyelination of the axon from multiple sclerosis causes gradual spread of neurological function and painful disturbances. Therefore, it is very important to induce axon formation and glial cell formation in cerebral apoplexy and to promote functional recovery after nerve injury.

Spinal cord injury is mainly caused by the compression of the spinal cord due to the displacement of the spine due to trauma. In addition to mechanical primary damage, immediate necrosis of the damaged cells leads to apoptosis which leads to apoptosis of neurons and white matter cells in the gray matter and demyelination of axons ), Which ultimately leads to permanent dysfunction. The pathophysiologic analysis showed that glutamate induced glutamate induced oxidative stress, ATP depletion, and anaerobic condition (ROS), which are initially liberated in the damaged area after spinal cord injury, ) And inflammatory mediators such as inflammatory mediators such as proinflammatory cytokines such as TNF-α and IL-1β and iNOS are known to be the main causes of apoptosis. In particular, the inflammatory response lasts not only in the early lesion but also in the long term. In particular, the inflammatory response caused by subcapsular apoptosis or axon degeneration in epidemics is a common pathologic feature in most neurological disorders, Its regulation is known to be important in preventing the spread of neurological diseases.

Korean Patent Publication No. 2009-0082480 discloses the use of a PirB / LILRB antagonist in the manufacture of a medicament for the treatment of a disease or condition in which beneficial results can be obtained from the promotion of neurite outgrowth, neuron growth, .

It is an object of the present invention to provide a pharmaceutical composition containing, as an active ingredient, a CCL2 or CCL2 agonist which can be usefully used for the treatment of diseases related to central nervous system damage such as stroke or spinal cord injury.

It is another object of the present invention to provide a method for providing information necessary for diagnosis of central nervous system damage using the expression profile of CCL2.

It is still another object of the present invention to provide a method for screening a drug for treating a central nervous system damage-related disease, which comprises selecting a candidate drug that promotes CCL2 expression or activity.

In order to achieve the above object, the present invention provides a biomarker composition for detecting central nervous system damage comprising CCL2 (chemokine (C-C motif) ligand 2].

The central nervous system damage may be selected from stroke or spinal cord injury.

The present invention also provides a pharmaceutical composition for the treatment of central nervous system damage-related diseases, which comprises CCL2 or CCL2 agonist as an active ingredient.

The CCL2 or CCL2 agonist has an axon regeneration effect, and the central nervous system injury-related disease can be selected from stroke or spinal cord injury.

The CCL2 agonist may be selected from the group consisting of a polypeptide, an antibody, a recombinant protein, and a GPCR (Ggycoprotein coupled receptor) ligand.

The present invention also provides a method of detecting CCL2 comprising: detecting the expression profile of CCL2 in a biological sample obtained from a subject having a central nervous system injury risk; And comparing the expression profile with an expression profile of CCL2 in a normal control group. The present invention also provides a method for providing information necessary for diagnosis of central nervous system damage.

The present invention provides a method for screening a drug for treating a central nervous system damage-related disease, which comprises selecting a candidate drug that promotes CCL2 expression or activity.

The candidate drug may have an axon regeneration effect.

According to the present invention, since CCL2 mediates neuron-macrophage interactions and promotes axon regeneration due to conditioning damage (CI), it can effectively treat central nervous system damage diseases such as stroke and spinal cord injury, Candidate drugs that promote CCL2 expression or activity can be selected to screen drugs for treating central nervous system injury-related diseases.

FIG. 1 is a schematic diagram showing a neuron-macrophage interaction in dorsal root ganglia (DRG) sensory neurons,
FIG. 2 shows the result of confirming that the molecule that connects the damaged neuron and the macrophage is CCL2 by performing the chemokine array in the DRG sample obtained after CI,
FIG. 3 shows changes in axon regeneration effect by CCL2 neutralizing antibody treatment in neuron-macrophage co-culture or macrophage condition medium (CM)
FIG. 4 shows changes in CI-induced neurite outgrowth and growth promoting effect upon CCL2 neutralizing antibody injection in the ganglion,
FIG. 5 shows the macrophage increase and axon regeneration effect according to the recombinant CCL2 (rCCL2) treatment,
Fig. 6 shows the confirmation of axon regeneration of fractalkine, another chemokine that attracts macrophages,
FIG. 7 shows axon regeneration effect according to AAV5-CCL2 injection in the ganglion,
FIG. 8 shows axon regeneration in the central nervous system after the injection of AAV5-CCL2 in the ganglia instead of CI.

Hereinafter, the present invention will be described in more detail based on experimental results of the accompanying drawings.

FIG. 1 is a schematic diagram showing neuron-macrophage interactions in dorsal root ganglia (DRG) sensory neurons. Originally, the central part of the DRG sensory nerve is not regenerated spontaneously, but before peripheral injury, injury, CI) leads to regeneration of the central branch. This regeneration effect almost completely disappeared by the macrocyclic deactivator minocycline. On the other hand, cAMP injection in the ganglion is similar to CI induced axon regeneration, and cAMP injection in the ganglion leads to increase of macrophages, and since minocycline treatment suppresses increased neurite outgrowth of cAMP-injected DGR, The cAMP effect on axon regeneration was confirmed to be mediated by macrophages. However, cAMP-treated macrophage conditioned medium (CM) does not exhibit neurite outgrowth activity, whereas when cAMP obtained from neuron-macrophage co-culture is treated with cAMP, it significantly promotes neurite outgrowth and growth, - axon regeneration by macrophage interactions.

In order to identify signals mediating neuron-macrophage interactions, a chemokine array was performed on the DRG samples obtained after CI. As a result, it was confirmed that the molecule connecting the damaged neurons and macrophages was CCL2 as shown in Fig. CCL2 expression was increased in impaired DRG neurons, and cAMP upregulated CCL2 expression in DRG neuron cultures.

As shown in FIG. 3, when the neuron-macrophage co-culture was neutralized with CCL2 neutralizing antibody, the regeneration effect of CM was decreased, while in the macrophage condition medium (CM) I did.

As shown in FIG. 4, when CCL2 neutralizing antibody was injected into the ganglion, CI induced neurite outgrowth and growth promoting effect were inhibited. Recombinant CCL2 (rCCL2) increased macrophages and promoted axon regeneration as shown in FIG.

As shown in Fig. 6, fractalkine, another chemokine that attracts macrophages, did not show axon regeneration effect unlike CCL2.

As shown in FIG. 7, when CCL2 recombinant rAAV5-CCL2 was injected into the ganglion to increase CCL2 expression in the DRG neuron, the axon growth of the DRG neuron was lower than that of the control rAAV5- Respectively.

As shown in FIG. 8, when rAAV5-CCL2 was injected into the ganglia instead of CI and the central nervous system injury (spinal cord injury) was given 7 days later, the DRG neuron-derived regenerated axons traced by the cholera toxin B subunit (CTB) It was confirmed that it passed through the receiving area and extended to the head. On the other hand, when the control rAAV5-GFP was injected, axonal growth was stopped at the tail side of the injured area, and the end of the axon formed a club shape to lose regenerative ability. In addition, when rAAV5-CCL2 was injected into the ganglion one day after spinal cord injury, CCL2 could be used therapeutically as the axon regeneration was confirmed.

Thus, the present inventors have found that CCL2 mediates neuron-macrophage interactions and plays an important role in promoting CI-induced axonal regeneration. Further, when CCL2 is treated in neurons, DRG The present inventors have completed the present invention by confirming that the sensory neurons are exaggerated in axon regeneration ability after central nerve injury.

Accordingly, the present invention provides a biomarker composition for detecting central nervous system damage including CCL2.

The detection of the biomarker may be carried out by directly detecting the presence of the biomarker protein from human tissue or body fluid by two-dimensional electrophoresis, or by contacting the human tissue or body fluid with the antibody of the present invention to detect the presence of the biomarker protein This can be done by indirect verification. As the antigen-antibody reaction, immunoassay includes enzyme immunoassay (ELISA, Coated tube), magnetic particle method using antibody-bonded magnetic particles, and latex particle method using antibody-bonded latex.

The present invention also provides a diagnostic kit for central nervous system damage comprising a molecule specifically binding to CCL2.

The molecule may be a monoclonal antibody, a polyclonal antibody, a substrate, a ligand or a cofactor, preferably a polyclonal or monoclonal antibody, more preferably a monoclonal antibody.

The polyclonal antibody may be prepared by injecting an immunogen-causing biomarker protein or fragment thereof into an external host according to methods known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, and rabbits. The immunogen is injected intraperitoneally, intraperitoneally or subcutaneously, and is generally administered with an adjuvant to increase the antigenicity. Serum is collected periodically from an external host to collect the sera showing improved titer and specificity for the antigen or isolate and purify the antibody therefrom.

Such monoclonal antibodies can be produced by immortalized cell line generation techniques by fusion as known to those of skill in the art. For example, a biomarker protein is immunized in mice or a peptide is synthesized and bound to bovine serum albumin to be immunized in mice. Generation of immortalized hybridomas by fusing antigen-producing B lymphocytes isolated from mice with myeloma of human or mouse, and the generation of monoclonal antibodies using an indirect ELISA method with the hybridoma cells was examined A positive clone may be selected and cultured, and the antibody may be isolated or purified, or may be injected into the abdominal cavity of a rat, and then a plurality of the antibody may be harvested to prepare a monoclonal antibody.

The present invention also provides a pharmaceutical composition for the treatment of central nervous system damage-related diseases, which comprises CCL2 or CCL2 agonist as an active ingredient.

The CCL2 or CCL2 agonist has an axon regeneration effect, and the central nervous system injury-related disease can be selected from stroke or spinal cord injury.

The CCL2 agonist may be selected from the group consisting of a polypeptide, an antibody, a recombinant protein, and a GPCR (Ggycoprotein coupled receptor) ligand.

The recombinant protein may be, but is not limited to rAAV5-CCL2.

The pharmaceutical compositions according to the present invention may further comprise suitable carriers, excipients or diluents conventionally used in the production of pharmaceutical compositions.

Examples of the carrier, excipient or diluent which can be used in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to conventional methods.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose sucrose), lactose, gelatin, and the like.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The amount of the active ingredient to be used in the pharmaceutical composition may vary depending on the patient's age, sex, weight, route of administration, disease severity, disease type, and the like, and may be administered once to several times per day. Accordingly, such dosages do not in any way limit the scope of the invention.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention also provides a method of detecting CCL2 comprising: detecting the expression profile of CCL2 in a biological sample obtained from a subject having a central nervous system injury risk; And comparing the expression profile with an expression profile of CCL2 in a normal control group. The present invention also provides a method for providing information necessary for diagnosis of central nervous system damage.

The present invention provides a method for screening a drug for treating a central nervous system damage-related disease, which comprises selecting a candidate drug that promotes CCL2 expression or activity.

The candidate drug may have an axon regeneration effect.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

< Example  1>

1. Animals and surgical procedures

Sprague Dawley rats (250-300 g) were used, and all animal experiments were conducted according to the rules set by the Committee on Laboratory Animal Research. All operations were performed using chloral hydrate (400 mg / kg, ip) . The sciatic nerve injury (SNI) was induced by cutting the right sciatic nerve on days 1, 3 and 7 days before fixation for immunostaining and 7 days before the removal of DRGs for axon formation analysis. Nerve injury was induced seven days before the unilateral cleft of the spinal cord.

First, the skin covering the right hindlimb femur was cut longitudinally to expose the thigh muscles, and surgical scissors with blunt tips were inserted between the muscles to expose the sciatic nerve. The three-pronged sciatic nerve was tied near the center of the body and cut underneath the section bounded by microscopic surgical scissors.

To make the lesion of the dorsal column, the thoracic spinal cord was exposed to T9, and the laminectomy was performed. The dura was opened by an intermittent incision and the dorsal column paired adjacent to the side column was immersed 1.5 mm deep using the scissors for iris incision I inserted it and cut it out. In order to achieve perfect cutting of the dorsal column, a vacuum was inhaled to remove residual tissue and create an empty space between the proximal and distal lesions. Then, using a small piece of gel foam gauze, immediate hemorrhage of the lesion was blocked, the muscle and fascia layers were stitched together, and the skin was stapled.

Three days before sacrifice, 2 μl (0.1% in PBS) noncomplexed cholera toxin B subunit (CTB; List Biological Laboratories, Grand Hyatt, San Francisco, Calif., USA) was sacrificed to observe axonal regeneration of dorsal column After the incision, the remaining proximal sciatic nerve was injected.

1) cyclic AMP derivative dibutyryl-cAMP (db-cAMP; Calbiochem, Darmstadt, Germany; 100 mM in PBS), 2) recombinant CCL2 (250 ug / ml; R & D, Minneapolis, Minn. ), 3) anti-CCL2 neutralizing antibody or American hamster IgG (250 ㎍ / ml; eBioscience, Sandiego, CA, USA) and 4) recombinant fectalkain EGFP (10E12, UNC vector core service: The University of North Carolina at Chapel Hill) at a rate of 0.5 μl per minute And injected using a Hamilton syringe composed of a micropipette.

2. Management of minocycline in vivo

For the management of minocycline, the Alzette osmotic mini-pump was inserted into the epidural space for sciatic injury, the polyethylene tube (PE-10) was passed to the lower right beneath the vertebra of the L6 / S1 area, , The catheter was tied to the bones of the L6 / S1 portion to be securely positioned. In addition, the osmotic minipump was attached to the muscle adjacent to the vertebrae. Pumps were delivered at a rate of 1 μl per minute for 7 days with minocycline (Sigma, 50 μg / μl; Saint Louis, MO, USA) or PBS (control of catheter-related variables) It has been shown that minocycline effectively blocks macrophage infiltration without toxicity.

In another experiment, macrophages were inactive from 21 to 28 days after SNI and minocycline pumps could be installed up to 21 days after SNI.

3. Tissue processing and immunohistochemical analysis

The rats were anesthetized with an excess of chloral hydrate, perfused with heparinized PBS, perfused with 4% paraformaldehyde in 0.2 M phosphate buffer, and DRGs containing lesions and spinal cord tissues were dissected, % PFA. DRGs were then frozen to 20 μm thickness with a number of cruciform solutions and spinal cord tissues were frozen sections (20 μm thick) at a ratio of 1:10 to the sagittal plane and tissues were stained with Super Frost Plus slide (Fisher Scientific, Pittsburgh, PA, USA) and stored at -20 ° C until use.

Immunohistochemistry was performed by treating the tissues with 10% normal goat serum and 0.3% Triton X-100 for 1 hour, then dissolving the primary antibody in the blocking solution and treating at 4 ° C overnight. The primary antibodies were mouse anti-ED1 (1: 500; Serotec, Kidlington, UK), rabbit anti-Iba (1: 500; Wako, LA, CA, USA), rabbit anti- (1: 10000; List Biological Laboratories), rabbit anti-CCL2 (1: 1000, 1: 1000, ; Millipore, Danvers, MA, USA) and rabbit anti-GFAP (1: 500; Dako, Carpinteria, CA, USA).

The tissue sections were washed and incubated for 1 hour at room temperature with a secondary antibody (1: 500; Molecular Probes, Eugene, Oreg., USA) suitable for each primary antibody to which Alexa Fluor 488 and 594 were tagged.

To observe CTB tracking signals, biotinyl-conjugated anti-goto IgGs (Vector; Burlington, CA, USA) were used after primary antibody incubation and Alexa 594 streptavidin conjugate (Molecular Probes) Lt; / RTI &gt;

The cover slips were then placed on a slide using a glycerol-based mounting solution (Biomeda, Foster City, CA, USA) and imaged using an FV 300 confocal microscope (Olympus, Tokyo, Japan).

4. Quantitative Reverse Transcription Polymerase Chain Reaction

Total RNAs were extracted from DRGs using triazole (Gibco, Gland Island, CA, USA). The total amount of RNA was determined using spectroscopy at 260 nm, and 2 μg of RNA was reverse-transcribed with cDNA using standard RT protocol.

10 μM of complementary primer pair and PCR reaction pre-mixture (GenDEPOT, Barker, TX, USA) were added to 1 μl of cDNA.

At this time, the following primers were used.

18S rRNA, 5'-CGGCTACCACATCCAAGGAA-3 '(forward, SEQ ID NO: 1), 5'-TGCTGGCACCAGACTTGCCCTC-3' (reverse, SEQ ID NO: 2);

IL-6, 5'-ATATGTTCTCAGGGAGATCTTGGAA-3 '(forward, SEQ ID NO: 3), 5'-GTGCATCATCGCTGTTCATACA-3' (reverse, SEQ ID NO: 4);

IL-1 ?, 5'-TCTGTGACTCGTGGGATGAT-3 '(forward, SEQ ID NO: 5), 5'-GGCAGCCTTGTCCCTTGA-3' (reverse, SEQ ID NO: 6);

TNF- ?, 5'-GCCACCACGCTCTTCTGT-3 '(forward, SEQ ID NO: 7), 5'-GGCAGCCTTGTCCCTTGA-3' (reverse, SEQ ID NO: 8);

LIF, 5'-TCAACTGGCTCAACTCAACG-3 '(forward, SEQ ID NO: 9), 5'-ACCATCCGATACAGCTCGAC-3' (reverse, SEQ ID NO: 10);

CNTF, 5'-CACCCCAACTGAAGGTGACT-3 '(forward, SEQ ID NO: 11), 5'-ACCTTCAAGCCCCATAGCTT-3' (reverse direction, SEQ ID NO: 12);

oncodulin, 5'-AGGCGTGACTGCAGAAGAAT-3 '(forward, SEQ ID NO: 13), 5'-ACTTGGCTGGCAGACATCTT-3' (reverse, SEQ ID NO: 14);

BDNF, 5'-GGGTGAAACAAAGTGGCTGT-3 '(forward, SEQ ID NO: 15), 5'-ATGTTGTCAAACGGCACAAA-3' (reverse, SEQ ID NO: 16);

NT-3, 5'-GATCCAGGCGGATATCTTGA-3 '(forward, SEQ ID NO: 17), 5'-AGCGTCTCTGTTGCCGTAGT-3' (reverse direction, SEQ ID NO: 18);

NGF, 5'-CCTGCCAGAGTCCTTTTCTG-3 '(forward, SEQ ID NO: 19), 5'-GGTTCAGGCCACAAAGTGTT-3' (reverse, SEQ ID NO: 20);

CCL2, 5'-TCTCTTCCTCCACCACTATGCA-3 '(forward, SEQ ID NO: 21), 5'-TGCTACAGGCAGCAACTGTGA-3' (reverse, SEQ ID NO: 22).

qRT-PCR was performed using an Applied Biosystem SYBR Green PCR ^ㄾ according to the kit protocol 7500 Real-time PCR system (Applied Biosystems, Foster City, CA, USA).

Amplification was carried out at 94 ° C for 30 seconds, at 55-64 ° C for 31 seconds and at 72 ° C for 60 seconds for 34 cycles. Melting curves were generated in the final amplification step and CT values were determined using Applied Biosystem 7500 software &Lt; / RTI &gt; Standardization was performed using 18s ribosomal RNA as an internal control.

5. Analysis of magnetic-activated cell sorting (MACS) for macrophage isolation

Seven days after the sciatic nerve injury, the rat was anesthetized with chloral hydrate, cold PBS was first injected into the heart of the animal, perfused and dissociated with DMEM (Hyclone, Logan, UT, USA) in the L4 and L5 DRGs.

The cells were centrifuged at 1500 rpm for 3 minutes at 4 ° C and then washed with MACS buffer (0.5% BSA, 2 mM EDTA without PBS, pH 7.2).

Then, the cell suspension was incubated on ice for 15 minutes with a biotin-conjugated CD68 primary antibody (1: 100; Abcam, Cambridge, UK) and cytoplasmic granules of microwave- Biotec, Bergisch Gladbach, Germany) for 10 minutes on ice.

CD68 negative cells were then collected by passing the cell suspension through a mini column of MACS ^ㄾ Cell Separation kit (Miltenyi Biotec), and the column was washed once again with MACS buffer to collect the remaining negative cells.

CD68-positive cells adsorbed on the mini-column were separated using 500 el of elution buffer. CD68 positive cells collected by the second elution were labeled as the second positive group, and the collected secondary positive cells and negative cells were used as controls to compare the effect of MACS separation.

Total RNA was also extracted from each cell group.

6. ELISA

L4 and L5 DRGs were dissected and cAMP assay was performed immediately using cAMP enzyme immunoassay kit (Assay Designs, Ann Arbor, MI, USA).

Each well was filled with 100 μl of DRG solution at a concentration of 20 μg / μl, and each experiment was repeated three times in triplicate.

Concentrations of oncodomaine for addition to the culture were determined according to the manufacturer's protocol using an ELISA kit with a Rat Oncodomain (catalog number MBS908312; Mybiosource, San Diego, Calif., USA).

100 μl of each culture was added to each well and cultured under the respective conditions.

7. Premature culture of dissociated adult DRG neurons

The adult DRGs of the lumbar spine 4 and 5 were dissected and treated with 125 U / ml type XI collagenase (Sigma) and dissolved in DMEM (Hyclone, Logan, UT, USA) And washed 5 times with DMEM.

Cells were dissociated using a pipette tip and centrifuged at 1,500 rpm for 3 min.

The cell pellet was resuspended in Gibco containing B-27 (Gibco) and resuspended in 0.01% poly-D-lysine (Sigma) and 3 ug / ml laminin (Invitrogen, Grand Island, (BD Bioscience, San Jose, Calif., USA).

The incubation period was strictly limited to 15 hours, which was the minimum axonal production of the injured control group. To confirm axon production, mouse anti-beta III tubulin (1: 1000; Promega, Madison, Wis., USA) and Alexa 594 Immunostaining was performed using the combined anti-mouse secondary antibody.

8. Premulture microwave incubation and co-culture of neuron-microwave paper

Adult female rats (Sprague Dawley rats, 250-300 g) were anesthetized with an excess of chloral hydrate. Immediately after the uterine transposition animal was euthanized, the abdominal cavity was washed with 50 ml of cold PBS to harvest macrophages in the abdominal cavity.

The washed solution was centrifuged at 1500 rpm for 10 minutes at 4 ° C to obtain cell pellet. Cell pellets were resuspended in 5 ml RBC lysis buffer (Qiagen, Valencia, CA, USA) for 3 minutes at room temperature and washed three times with PBS And washed.

After the last wash, the cells were resuspended in RPMI-1640 (Hyclone) supplemented with 10% fetal bovine serum (FBS) and dispensed into a 100 mm culture dish (BD Bioscience) coated with 0.01% poly-D- Lt; 0 &gt; C, 5% CO ₂ incubator.

After 4 hours, the cultured microwave was treated with db-cAMP (100 μM) or zymosan (12.5 μg / ml) and minocycline (10 μg / μl) Was replaced with fresh DMEM containing B-27 (Gibco). After 72 hours of culture without culture medium replacement, the medium was centrifuged at 1500 rpm for 5 minutes, and the culture was passed through a 0.2 μm filter (BD Bioscience) to remove residual cell debris. Primary cultures of the dissolved DRG neurons were carried out in the manner described above, maintained for 2 hours to settle in the culture dishes, and replaced with media of each condition and incubated for 15 hours.

To confirm axonal formation in fixed cells, neurons were incubated in DMEM (Gibco) containing B-27 (Gibco) for 4 hours for immunostaining with beta III tubulin and co-culture of neuron-microwave plaques. After incubation, intraperitoneal macrophages were divided into neurons at a ratio of 1: 5 (neurons to macrophages) and treated for 4 hours. Then, the medium was collected and co-cultured with neuron-macrophages using cell culture insert (BD Bioscience) First, neurons were injected into 6-wells of cell culture insertions and intraperitoneal macrophages were injected after 4 hours.

 IL-6 (20 μg / ml; R & D, Minneapolis, Minnesota, USA), LIF (20 μg / ml; R & D), and the like were used to inactivate the potential activity of IL-6, LIF and oncodomainin in the harvested culture medium. Inocomodulin (20 μg / ml, kindly provided by Dr. Benowitz at Harvard University) was added to the control and the same amount of ghoul or rabbit IgG (R & D) was added.

CCL2 neutralizing antibody (1 ug / ml; eBioscience, Sandiego, CA, USA) was treated to nullify the effect of CCL2 in co-culture of neuron-macrophages.

1) Neuron-macrophage co-cultivation was performed as described above, and after 4 hours of incubation, cultured neurons and microwaved db-cAMP (100 μM) or PBS and CCL2 neutralizing antibody or american hamster IgG were incubated with 24 After incubation for 24 hours, the culture medium was replaced with fresh DMEM containing B-27 (Gibco) for 24 hours. The medium was centrifuged at 1500 rpm for 5 minutes and the culture was passed through a 0.2 μm filter (BD Bioscience) To remove remaining cell debris.

2) CCL2 neutralizing antibody or American hamster IgG was added during the primary culture of dissociated adult DRG neurons using the culture medium of dc-cAMP or PBS-added neuron-macrophage co-culture performed through the above-described method.

9. Accumulation of axon during culture and quantification of vital macrophage count and axon regeneration

For axonal production analysis, the average neurite length per neuron was measured and the other experimental conditions and axon production were compared.

Neuron length was measured and quantified using a Neuron J plugin from image analysis software Image J (publicly available from http://rsbweb.nih.gov/ij/index.html). Each well was divided into 4 wells, and 200 × magnified images were obtained based on the center of each part.

All neuron nerves in each image were tracked and the number of DRG neurons per image was measured. As a result, 20 neurons per image were measured and 80 neurons per well were measured.

The average neurite length per neuron in each well was calculated by dividing the length of the entire neuron of about 80 neurons by the number of neurons.

Averaged over 4 wells for each culture condition, the average group value of neuron per neuron length included an average value of 3-4 independent cultures.

To quantify macrophage infiltration in DRGs, images of Iba1 immunostained tissue sections were magnified 200 × to quantify the number of macrophages in the DRGs.

The number of macrophages was counted in four images per animal. To obtain the number of macrophages per unit area, the total number of macrophages was divided into imaged DRGs.

Iba1-positive macrophages are located close to nerve fiber positive DRG neurons, and there is no notable background signal between these two cells, suggesting that macrophages are physically related to neurons.

 The number of macrophages representing the physical connection was divided by the total number of macrophages in the same image, including the percentage of macrophages associated with the DRG neurons.

 To quantify the growth of dorsal column axilla after injury, all 10th temporal homogeneous spinal cord segments (200 μm cross section) containing CBT tracking signals were analyzed. CTB-stained lesions were obtained from 5 mm to 5 mm from the anterior side of the lesion, and the lesion boundary was identified by GFAP staining. After calculating the calculation unit from the lower lesion boundary, a vertical line was drawn on the vertical axis at intervals of 100 μm. The name is shown as the shortest distance from the lesion border. For example, the number of axons between +100 and +200 was recorded as +100.

The number of axon protrusions was measured by measuring the number of CTB-positive axons between consecutive lines, and the number of axon protrusions was recorded in each unit.

Other axons were also recorded at distances beyond the lesion border.

10. Chemokine analysis

Animals were anesthetized with an excess of chloral hydrate and sacrificed, followed by perfusion with cold saline to remove blood components from the tissue.

Dorsal root ganglia tissues were rapidly dissected and total RNA was isolated using the RNase-free DNase set chemokine array tissue mini kit (Qiagen, Hilgen, Germany) according to the manufacturer's instructions.

For chemokine analysis, the concentration of the RNA sample was quantified using Nanodrop 1000 (Thermo Fisher Scientific, Wilmington, DE, USA) and 2 ug of RNA was reverse transcribed with cDNA using standard RT protocol.

^{RT 2 Profiler PCR Array kit (Qiagen} , Hilgen, Germany) was used on a cDNA of 1㎕ Quantitative Real-Time PCR is RT ² Profiler PCR according to the protocol provided by the Array kit 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) for 10 minutes at 90 ° C and 1 minute at 60 ° C for 40 cycles. In the last amplification step, a melting curve was observed and the CT values were quantified with Applied Biosystem 7500 software.

Expression of the target gene was normalized in relation to the expression of the acidic ribosomal protein 60S as an internal control.

11. Recombination AAV5 ( Recombinant adeno - associate virus serotype  5) Production

CCL2 cDNA was subcloned into the pAAV-CAG-GFP vector and then prepared as an AAV virus of serotype 5 (Viral vector core service, The University of North Carolina at Chapel Hill, USA). As a control group, rAAV5-eGFP with a fluorescent label attached to the same antigenic AAV5 was prepared.

12. Statistical Analysis

All values are expressed as mean ± SEM. Statistical comparisons of mean values were performed using one-way ANOVA followed by Tukey's posthoc test and repeated measurements of two-way ANOVA were used to compare the number of axons at a distance from the center Respectively.

Quantitative graphs were made using GraphPad Prism version 5.00 (GraphPad Software, San Diego, Calif., USA).

13. Experimental results

FIG. 1 is a schematic diagram showing neuron-macrophage interactions in dorsal root ganglia (DRG) sensory neurons. Originally, the central part of the DRG sensory nerve is not regenerated spontaneously, but before peripheral injury, injury, CI) leads to regeneration of the central branch. This regeneration effect almost completely disappeared by the macrocyclic deactivator minocycline. On the other hand, cAMP injection in the ganglion is similar to CI induced axon regeneration, and cAMP injection in the ganglion leads to increase of macrophages, and since minocycline treatment suppresses increased neurite outgrowth of cAMP-injected DGR, The cAMP effect on axon regeneration was confirmed to be mediated by macrophages. However, cAMP-treated macrophage conditioned medium (CM) does not exhibit neurite outgrowth activity, whereas when cAMP obtained from neuron-macrophage co-culture is treated with cAMP, it significantly promotes neurite outgrowth and growth, - axon regeneration by macrophage interactions.

In order to identify signals mediating neuron-macrophage interactions, a chemokine array was performed on the DRG samples obtained after CI. As a result, it was confirmed that the molecule connecting the damaged neurons and macrophages was CCL2 as shown in Fig. CCL2 expression was increased in impaired DRG neurons, and cAMP upregulated CCL2 expression in DRG neuron cultures.

As shown in FIG. 3, when the neuron-macrophage co-culture was neutralized with CCL2 neutralizing antibody, the regeneration effect of CM was decreased, while in the macrophage condition medium (CM) I did.

As shown in FIG. 4, when CCL2 neutralizing antibody was injected into the ganglion, CI induced neurite outgrowth and growth promoting effect were inhibited. Recombinant CCL2 (rCCL2) increased macrophages and promoted axon regeneration as shown in FIG.

As shown in Fig. 6, fractalkine, another chemokine that attracts macrophages, did not show axon regeneration effect unlike CCL2.

As shown in FIG. 7, when CCL2 recombinant rAAV5-CCL2 was injected into the ganglion to increase CCL2 expression in the DRG neuron, the axon growth of the DRG neuron was lower than that of the control rAAV5- Respectively.

As shown in FIG. 8, when rAAV5-CCL2 was injected into the ganglia instead of CI and the central nervous system injury (spinal cord injury) was given 7 days later, the DRG neuron-derived regenerated axons traced by the cholera toxin B subunit (CTB) It was confirmed that it passed through the receiving area and extended to the head. On the other hand, when the control rAAV5-GFP was injected, axonal growth was stopped at the tail side of the injured area, and the end of the axon formed a club shape to lose regenerative ability. In addition, when rAAV5-CCL2 was injected into the ganglion one day after spinal cord injury, CCL2 could be used therapeutically as the axon regeneration was confirmed.

Thus, it has been found that CCL2 mediates neuron-macrophage interactions and plays an important role in promoting CI-induced axonal regeneration. In addition, treatment of CCL2 with neuronal cells showed that DRG sensory neurons were able to regenerate axon regeneration ability after CNS damage without CI.

 While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is to be understood that various modifications and changes may be made without departing from the scope of the appended claims.

<110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Pharmaceutical composition for treating CNS injury related          naturally occurring CCL2 or CCL2 agonist <130> ADP-2014-0397 <150> KR 10/20130113083 <151> 2013-09-24 <160> 22 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cggctaccac atccaaggaa 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tgctggcacc agacttgccc tc 22 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 atatgttctc agggagatct tggaa 25 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gtgcatcatc gctgttcata ca 22 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tctgtgactc gtgggatgat 20 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ggcagccttg tcccttga 18 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gccaccacgc tcttctgt 18 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ggcagccttg tcccttga 18 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tcaactggct caactcaacg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 accatccgat acagctcgac 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 caccccaact gaaggtgact 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 accttcaagc cccatagctt 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 aggcgtgact gcagaagaat 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 acttggctgg cagacatctt 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gggtgaaaca aagtggctgt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 atgttgtcaa acggcacaaa 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gatccaggcg gatatcttga 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 agcgtctctg ttgccgtagt 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 cctgccagag tccttttctg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 ggttcaggcc acaaagtgtt 20 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 tctcttcctc caccactatg ca 22 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 tgctacaggc agcaactgtg a 21

Claims (9)

CCL2 [chemokine (C-C motif) ligand 2]. The biomarker composition of claim 1, wherein the central nervous system injury is selected from stroke or spinal cord injury. A pharmaceutical composition for the treatment of a central nervous system damage-related disease containing as an active ingredient CCL2 or CCL2 agonist. [Claim 4] The pharmaceutical composition according to claim 3, wherein the CCL2 or CCL2 agonist has axon regeneration effect. [Claim 5] The pharmaceutical composition according to claim 3, wherein the central nervous system damage-related disease is selected from stroke or spinal cord injury. [Claim 5] The pharmaceutical composition according to claim 3, wherein the CCL2 agonist is selected from the group consisting of a polypeptide, an antibody, a recombinant protein, and a GPCR (Ggycoprotein coupled receptor) ligand. Detecting an expression profile of CCL2 in a biological sample obtained from a subject having a central nervous system injury risk; And comparing the expression profile with an expression profile of CCL2 in a normal control. &Lt; Desc / Clms Page number 19 &gt; Selecting a candidate drug that promotes CCL2 expression or activity. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; [Claim 9] The method of claim 8, wherein the candidate drug has axon regeneration effect.

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