PCNT as a target protein for the treatment or diagnosis of neurological diseases

Abstract

To provide a pharmaceutical composition for preventing or treating a central nervous system disease, a composition or kit for diagnosing a central nervous system disease, a method of analyzing information required for diagnosing a central nervous system disease, and a method of screening a candidate substance for a therapeutic agent to treat a central nervous system disease.SOLUTION: Provided is a pharmaceutical composition including, as an active ingredient, cord blood or cord blood-derived cells expressing or secreting pericentrin (PCNT).SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for preventing or treating central nervous system diseases (neurological diseases) comprising umbilical cord blood or umbilical cord blood-derived cells that express or secrete PCNT as an active ingredient, a composition or kit for diagnosing neurological diseases comprising an agent for measuring the level of PCNT protein, a method for analyzing information necessary for diagnosing neurological diseases using PCNT protein, and a method for screening candidate substances for treating neurological diseases. [Background technology]

[0002] The cranial nervous system refers to the body's regulatory system, which is composed of the brain, spinal cord, cranial nerves, spinal nerves, and autonomic nervous system. Cranial nervous system disorders include cerebral palsy, brain injury, traumatic brain injury, ischemic brain injury, cerebral concussion, cerebral infarction, cerebral hemorrhage, Parkinson's disease, Alzheimer's disease, Huntington's disease, dementia, Lou Gehrig's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, primary lateral sclerosis, degenerative ataxia, multiple sclerosis, nervous system dysfunction, memory loss, epilepsy, encephalitis, prion disease, and other neurological disorders. Brain injury refers to a condition in which abnormalities in the brain's nervous tissue, caused by various internal or external factors, result in behavioral or functional abnormalities. Brain injury can result from open head injury, closed head injury, deceleration injury, toxic exposure, oxygen deprivation, tumors, infections, and cerebrovascular diseases such as stroke.

[0003] Cerebral palsy is not a single disease, but a collective term for a group of symptoms with similar clinical characteristics. It refers to a group of clinical symptoms that show movement and posture disorders caused by non-progressive lesions or damage to the immature brain. Cerebral palsy is a disease that causes problems with muscle control and maintaining posture while walking due to damage to the brain during the developmental process. Brain damage occurs mainly before, during, or after birth, but can occur at any time during pregnancy and even in childhood.

[0004] Although the brain injury is non-progressive, the neuromotor disorder and musculoskeletal disorder change over time, so rehabilitation treatment that is most appropriate for the changed clinical condition must be provided through periodic examinations. Although other treatment methods are available, there is currently no revolutionary or fundamental treatment method that can be used through these methods.

[0005] It has been reported that stimulating the potential of brain plasticity may be a potential treatment for cerebral palsy, and it has shown positive effects in patients with brain injury. One method for stimulating the potential of brain plasticity is cell therapy.

[0006] Korean Patent Publication No. 14093336 (Patent Document 1) relates to the use of oligodendrocyte precursor cells as a cell therapy agent for central nervous system diseases, and discloses a composition for treating central nervous system diseases containing, as an active ingredient, oligodendrocyte precursor cells, which are stem cells transformed with an expression vector containing an Olig2 gene-encoding nucleic acid molecule.

[0007] On the other hand, direct transplantation of stem cells, including mesenchymal stem cells, can result in problems such as failure to differentiate and survive in vivo and difficulty in quantitative administration, making their use in cell therapy difficult. Therefore, there is a need to develop a cell therapy that does not induce rejection in vivo and has therapeutic effects on motor and cognitive development. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Korean Patent Registration No. 14093336 Summary of the Invention [Problem to be solved by the invention]

[0009] To this end, the inventors, while researching umbilical cord blood as a cell therapy agent for nervous system diseases, identified PCNT protein as a target protein for the treatment or diagnosis of nervous system diseases, and completed the present invention.

[0010] One aspect provides a pharmaceutical composition for preventing or treating a neurological disease, comprising umbilical cord blood or umbilical cord blood-derived cells that express or secrete pericentrin (PCNT) as an active ingredient.

[0011] Another aspect provides a composition or kit for diagnosing a central nervous system disease, which comprises an agent for measuring the level of PCNT protein or mRNA of its gene.

[0012] Another aspect provides a method for analyzing information necessary for diagnosing a nervous system disease, comprising measuring the expression or activity level of PCNT protein or its gene in a sample isolated from an individual, and comparing the measured expression or activity level with the expression or activity level of PCNT protein measured in a control sample isolated from a normal individual.

[0013] Another aspect provides a method for screening candidate substances for treating cranial nervous system diseases, comprising the steps of contacting cells expressing PCNT protein with a test substance in vitro, measuring the expression level or activity of PCNT protein in the cells, and comparing the measured expression level or activity of PCNT protein with the expression level or activity measured in a control group, and selecting a test substance that increases the expression level or activity compared to the control group as a candidate substance for treating cranial nervous system diseases. [Means for solving the problem]

[0014] One aspect provides a pharmaceutical composition for preventing or treating a neurological disease, comprising umbilical cord blood or umbilical cord blood-derived cells that express or secrete PCNT as an active ingredient.

[0015] Another aspect provides a pharmaceutical composition for preventing or treating a central nervous system disease, which comprises the PCNT protein or an active fragment thereof as an active ingredient. [Effects of the Invention]

[0016] The use of compositions containing umbilical cord blood or umbilical cord blood-derived cells expressing or secreting PCNT in a specific manner can effectively prevent or treat cranial nervous system diseases. Furthermore, PCNT protein can be used as a target for early diagnosis of cranial nervous system diseases and for developing therapeutic agents for cranial nervous system diseases. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1A shows the results of two-dimensional gel electrophoresis to confirm changes in plasma proteins before (Pre) and 10 days after (Post+10 days) cord blood treatment, representing the results of Experiment 1 (analysis of samples from patients 4, 9, and 10). Figure 1B shows the results of two-dimensional gel electrophoresis to confirm changes in plasma proteins before (Pre) and 10 days after (Post+10 days) cord blood treatment, representing the results of Experiment 2 (analysis of samples from patients 6, 7, and 8). [Figure 2] These are the results of Western blot analysis confirming PCNT expression in the plasma of 13 cerebral palsy patients before (Pre) and after (Post) umbilical cord blood transfusion. [Figure 3] This is a graph showing the change in PCNT expression levels before and after cord blood treatment, combining the results of all the patients shown in Figure 2. [Figure 4] 1 shows the results of measuring PCNT expression levels four times after administration of umbilical cord blood mononuclear cells (UCB) in a mouse model of induced cerebral palsy. A model administered with EPO was used as a comparison group. [Figure 5] This is a graph showing the change in PCNT expression in Figure 4 converted into % based on a sham model in which cerebral palsy was not induced and umbilical cord blood was not administered. [Figure 6]Figure 6A shows the expression levels of PCNT in umbilical cord blood (UCB) plasma, normal plasma, and cerebral palsy patient plasma samples. Figure 6B shows the relative expression levels (%) of PCNT in umbilical cord blood (UCB) plasma, normal plasma, and cerebral palsy patient plasma samples, based on umbilical cord blood sample No. 1. [Figure 7] 1 is a graph showing the expression levels (Log10, ppm) of PCNT in the following components present in umbilical cord blood: serum, plasma, monocytes, neutrophils, B-lymphocytes, T-lymphocytes, CD4 T cells, CD8 T cells, NK cells, peripheral blood mononuclear cells, platelets, lymph nodes, tonsils, bone marrow stromal cells, and bone marrow mesenchymal stem cells. [Figure 8] This is a graph showing the GMFCS evaluation results (total GMFCS (%)) for cerebral palsy patients Nos. 5, 11, and 13 before (GMFCS_pre) and after (GMFCS_post) umbilical cord blood treatment. [Figure 9] This is a graph showing the results of the behavioral development domain assessment (BSID II motor) of the Bayley Scale of Infant Development for cerebral palsy patients Nos. 5, 11, and 13 before (BSID_Motor_pre) and after (BSID_Motor_post) cord blood treatment. [Figure 10] 1 is a graph showing the results of the mental development domain assessment (BSID II mental) of the Bayley Scale of Infant Development for cerebral palsy patients Nos. 5, 11, and 13 before (BSID_Mental_pre) and after (BSID_Mental_post) cord blood treatment. DETAILED DESCRIPTION OF THE INVENTION

[0018] The term "pericentrin (PCNT)" refers to a protein that binds calmodulin, is expressed in centrosomes, and forms the centrosome together with centrioles. PCNT protein is known to play an important role in cell division (Liu Q et al., Aug. 2010, Cell Research. 20 (8): 948-62), and abnormalities in this protein have been reported to cause mental disorders (Unal S et al., Feb. 2014, Pediatric Blood & Cancer. 61 (2): 302-5). It was confirmed that PCNT protein is expressed at higher levels in umbilical cord blood compared to plasma from normal individuals, but at lower levels in cerebral palsy patients compared to plasma from normal individuals. Furthermore, it was confirmed that PCNT protein is expressed at higher levels in cerebral palsy patients who underwent umbilical cord blood cell therapy compared to before treatment. These results suggest that PCNT protein can be used as a diagnostic marker for diagnosing cranial nervous system diseases such as cerebral palsy, and also that PCNT protein is involved in the prevention or treatment of cranial nervous system diseases. Therefore, umbilical cord blood or umbilical cord blood-derived cells with high PCNT protein expression may be used to prevent or treat cranial nervous system diseases.

[0019] The PCNT protein may be secreted from umbilical cord blood or umbilical cord blood-derived cells. The PCNT protein may be derived from a mammal, such as a human, monkey, or rodent. In one embodiment, the PCNT protein consists of the amino acid sequence of SEQ ID NO: 1 (GenBank Accession No. XP_005261181).

[0020] In one embodiment, the cranial nervous system disease is a cranial nervous system disease in which PCNT expression is reduced compared to normal. In another embodiment, the cranial nervous system disease includes any disease in which abnormalities or disorders occur in part or all of the components of the cranial nervous system, such as the brain, spinal cord, cranial nerves, spinal nerves, and autonomic nervous system. In another embodiment, the cranial nervous system disease is a brain injury disease. In another embodiment, the cranial nervous system disease is a nerve injury disease. In another embodiment, the cranial nervous system disease is selected from the group consisting of, but not limited to, cerebral palsy, brain injury, traumatic brain injury, ischemic brain injury, brain concussion, brain contusion, stroke, cerebral infarction, cerebral hemorrhage, Parkinson's disease, Alzheimer's disease, Huntington's disease, dementia, Lou Gehrig's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, primary lateral sclerosis, degenerative ataxia, multiple sclerosis, nervous system dysfunction, memory loss, epilepsy, encephalitis, prion disease, and neurological diseases. In another embodiment, the cranial nervous system disease is cerebral palsy.

[0021] The term "cerebral palsy" refers to a disease in which non-progressive lesions or damage occur in the immature brain during or after birth due to various causative factors, resulting in clinically significant impairments of movement and posture.

[0022] The term "cord blood" refers to blood in the placenta or umbilical cord. Umbilical cord blood contains a large amount of hematopoietic stem cells that produce blood components such as white blood cells, red blood cells, and platelets, as well as various stem cells that produce cartilage, bone, fat, muscle, nerves, and other components. The term "stem cells" refers to primitive cells that are undifferentiated cells that can be transformed into any organ. Types of stem cells include embryonic stem cells and adult stem cells. Umbilical cord blood-derived stem cells include adult stem cells such as mesenchymal stem cells (MSCs). Umbilical cord blood can be obtained by separating it from the umbilical cord after birth. Umbilical cord blood can be used as a white blood cell concentrate, which excludes red blood cells and plasma. The cord blood leukocyte concentrate may contain a variety of cord blood-derived cells, including hematopoietic stem cells, monocytes, neutrophils, B-lymphocytes, T-lymphocytes, CD4 T cells, CD8 T cells, NK cells, peripheral blood mononuclear cells (PBMCs), platelets, lymph node cells, tonsil cells, bone marrow stromal cells, and bone marrow mesenchymal stem cells.

[0023] In one embodiment, the umbilical cord blood may be autologous or allogeneic. The allogeneic cord blood may be from a family member, such as a sibling or parent.

[0024] In one embodiment, the cord blood-derived cells are hematopoietic stem cells. In another embodiment, the cord blood-derived cells are cells that express or secrete PCNT. In another embodiment, the cord blood-derived cells are selected from the group consisting of, but not limited to, monocytes, B-lymphocytes, CD4 T cells, CD8 T cells, NK cells, peripheral blood mononuclear cells, platelets, and lymph nodes. In another embodiment, the cord blood-derived cells are peripheral blood mononuclear cells. In another embodiment, the cord blood-derived cells are genetically engineered to express or secrete, or to enhance, PCNT expression or secretion.

[0025] The pharmaceutical composition may further contain erythropoietin (EPO), a hormone that regulates red blood cell production in the bone marrow. When erythropoietin is administered in combination with cord blood or cord blood-derived cells, the therapeutic effects of cord blood are enhanced, and the motor and cognitive functions of patients can be further improved.

[0026] The term "treatment" refers to or includes the alleviation, slowing of progression, or prevention of a disease, disorder, or condition, or one or more symptoms thereof, and "active ingredient" or "pharmaceutically effective amount" means any amount of a composition utilized in the course of practicing the inventions provided herein sufficient to alleviate, slow the progression, or prevent a disease, disorder, or condition, or one or more symptoms thereof.

[0027] The terms "administer," "inject," and "implant" are used interchangeably and refer to the placement of a composition according to an embodiment into an individual by a method or route that results in at least partial localization of the composition according to an embodiment at a desired site. Administration may be by any suitable route that delivers cells, at least a portion of cellular components, or cell-derived secreted components of a composition according to an embodiment to a desired location within a living individual. The survival period of the cells after administration to an individual may be as short as a few hours, e.g., 24 hours to a few days, or as long as several years.

[0028] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable carriers or diluents are also known in the art. The carrier or diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water (e.g., saline and sterile water), syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, Ringer's solution, buffer, maltodextrin solution, glycerol, ethanol, dextran, albumin, or any combination thereof. The pharmaceutical composition may further comprise a lubricant, wetting agent, sweetener, flavoring agent, emulsifier, suspending agent, or preservative.

[0029] The pharmaceutical composition may be formulated using pharmaceutically acceptable carriers and / or excipients by methods known to those skilled in the art and prepared into a unit-dose form or in a multi-volume container. The dosage form may be a solution, suspension, syrup, or emulsion in an oil or aqueous solvent, or an extract, powder, powder, granule, tablet, or capsule, and may additionally contain a dispersant or stabilizer. The aqueous solvent may include physiological saline or PBS. According to one embodiment, the pharmaceutical composition may be formulated for oral or parenteral administration, preferably parenteral administration. For intramuscular, intraperitoneal, subcutaneous, and intravenous administration, a sterile solution of the active ingredient may generally be prepared, and a buffer may be added to appropriately adjust the pH of the solution. For intravenous administration, an isotonic agent may be added to impart isotonicity to the formulation.

[0030] In one embodiment, the pharmaceutical composition contains 1 x 10 nucleated cord blood-derived cells. 7 / kg or more, specifically, 2x10 7 / kg or more, 3x10 7 / kg or more, or 3x10 7 / kg or 10x107 / kg, e.g., 3x10 7 / kg or 5x10 7 / kg.

[0031] According to one embodiment, the effective daily dose of the pharmaceutical composition is about 0.01 to 200 ml, specifically about 0.01 to 150 ml, about 0.01 to 100 ml, about 0.1 to 150 ml, about 0.1 to 100 ml, about 1 to 100 ml, about 1 to 50 ml, about 5 to 50 ml, or about 10 to 40 ml, for example, 15 to 30 ml. If necessary, the umbilical cord blood can be appropriately concentrated before administration to reduce the total dose. However, the dose may vary depending on factors such as the formulation method, administration method, the patient's age, weight, sex, and condition, diet, administration time, administration route, excretion rate, and sensitivity, and those skilled in the art can appropriately adjust the dose taking these factors into consideration. The administration frequency can be once daily or two or more times per day within the range of clinically acceptable side effects. Administration can be at one or more sites, daily or at intervals of 2 to 5 days, with the total number of days of administration per treatment ranging from 1 to 30 days. If necessary, the same treatment can be repeated at an appropriate time. For non-human animals, the same dose per kg as for humans can be administered, or the dose can be converted based on, for example, the volume ratio (e.g., average) of organs (e.g., heart) between the target animal and humans. Possible administration routes include parenteral (e.g., subcutaneous, intramuscular, intra-arterial, intraperitoneal, intradural, or intravenous), topical (including transdermal), and injection or implantable device or substance insertion. In one embodiment, target animals for treatment include humans and other mammals, including humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, horses, and pigs.

[0032] Another aspect provides a composition or kit for diagnosing a central nervous system disease, which comprises an agent for measuring the level of PCNT protein or mRNA of its gene.

[0033] PCNT protein and cranial nervous system diseases are as described above.

[0034] In one example, it was confirmed that the expression level of PCNT protein is lower in plasma from cerebral palsy patients compared to that of normal individuals. Therefore, by measuring the expression and activity level of PCNT protein and comparing it with a sample from a normal individual, it is possible to diagnose the onset or progression of cranial nervous system diseases such as cerebral palsy. Therefore, PCNT protein is also a diagnostic marker for cranial nervous system diseases, such as cerebral palsy.

[0035] The term "diagnosis" means to confirm the existence or characteristics of a pathological condition. Accordingly, the term "diagnosis of a cranial nervous system disease" means to confirm whether or not a cranial nervous system disease has developed or the possibility of developing the disease, or to predict the risk of developing the disease.

[0036] The term "diagnosis marker" refers to a substance that can diagnose whether or not the above-mentioned cranial nervous system diseases have developed or the likelihood of developing the diseases.

[0037] The agent for measuring the protein may also be an antibody that specifically binds to the PCNT protein or a fragment thereof.

[0038] The agent for measuring the mRNA level of the gene may comprise a primer or probe that specifically binds to a nucleic acid encoding the PCNT protein or a fragment thereof.

[0039] Those skilled in the art can easily prepare antibodies that specifically bind to PCNT protein having a known amino acid sequence using known methods. Antibodies that specifically bind to PCNT protein or fragments thereof may include monoclonal antibodies, polyclonal antibodies, and recombinant antibodies, and may include not only complete forms having the full length of two heavy chains and two light chains, but also functional fragments such as Fab, F(ab'), F(ab')2, and Fv that retain at least the antigen-binding function.

[0040] Furthermore, those skilled in the art can design primers or probes that specifically amplify or recognize specific regions from the known PCNT gene sequence.

[0041] The term "primer" refers to a nucleic acid sequence of 7 to 50 strands with a short free 3' hydroxyl group that can base pair with a complementary template and serve as a starting point for template strand replication. While primers are usually synthesized, they can also be derived from naturally occurring nucleic acids. The primer sequence does not necessarily have to be exactly the same as that of the template; it only needs to be sufficiently complementary to hybridize with the template. The primer can initiate DNA synthesis in the presence of a polymerization reaction (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer solution and temperature. The PCR conditions and the lengths of the sense and antisense primers can be varied based on knowledge in the art. Therefore, PCR amplification can be performed using sense and antisense primers of the nucleotide sequence of the PCNT gene to diagnose nervous system diseases.

[0042] The term "probe" refers to a nucleic acid fragment, such as RNA or DNA, as short as a few bases or as long as several hundred bases, capable of specifically binding to mRNA and capable of detecting the presence or absence of a specific mRNA. Such probes may be prepared in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, RNA probes, etc. The selection of an appropriate probe and hybridization conditions may vary based on techniques known in the art. Therefore, hybridization can be performed using a probe complementary to the nucleotide sequence of the PCNT gene, and nervous system diseases can be diagnosed based on the degree of hybridization.

[0043] The primers or probes described above can be chemically synthesized using phosphoramidite solid support methods or other widely known methods. Such nucleic acid sequences can incorporate additional features that do not alter their basic properties. Examples of additional features that can be incorporated include, but are not limited to, methylation, capping, substitution of one or more nucleic acids with homologs, and variations between nucleic acids.

[0044] The kit may also be an RT-PCR kit, a microarray chip kit or a protein chip kit.

[0045] The kit may further include one or more other components suitable for the analytical method. When the analytical method is RT-PCR, the kit may further include necessary containers, reaction buffer, deoxynucleotides (dNTPs), PCR DNA polymerase, reverse transcriptase, etc. in addition to the primer set specific for PCNT protein. When the analytical method is ELISA, the kit may further include reagents for detecting the bound antibody, such as a secondary antibody, a chromophore, an enzyme, and its substrate.

[0046] Another aspect provides a method for analyzing information necessary for diagnosing a nervous system disease, comprising measuring the expression or activity level of PCNT protein or its gene in a sample isolated from an individual, and comparing the measured expression or activity level with the expression or activity level of PCNT protein measured in a control sample isolated from a normal individual.

[0047] PCNT protein and cranial nervous system diseases are as described above.

[0048] The method comprises measuring the expression or activity level of PCNT protein or its gene in a sample isolated from an individual.

[0049] The term "individual" refers to an individual for whom it is desired to determine whether or not a cranial nervous system disease has developed or to predict the risk of developing a disease. The individual is not limited to any species as long as it is an animal that can develop a cranial nervous system disease, and specifically includes mammals, such as humans (Homo sapiens).

[0050] The sample may include, but is not limited to, tissues isolated from an individual's body, cells, samples such as whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, urine, etc. In one embodiment, the sample is plasma.

[0051] The step of measuring the expression level of the PCNT protein or its gene can also be carried out by measuring the amount of mRNA of the PCNT gene or the PCNT protein.

[0052] The step of measuring the expression level of the PCNT protein, i.e., mRNA level, can also be performed by methods such as RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting, DNA chip, etc., but is not limited to these.

[0053] The step of measuring the activity level of the PCNT protein may also be performed by methods such as, but not limited to, Western blotting, magnetic bead-antibody immunoprecipitation, ELISA, mass spectrometry, radioimmunoassay, and immunoprecipitation.

[0054] The method includes a step of comparing the expression or activity level measured in the sample with the expression or activity level of PCNT protein measured in a control sample isolated from a normal individual. If the expression or activity level of PCNT protein in the sample is lower than that of the control sample isolated from a normal individual, it can be determined that the sample has a high probability of developing or progressing a cranial nervous system disease. Therefore, the method may include determining that a cranial nervous system disease is present if the measured expression or activity level is lower than that measured in the control sample.

[0055] Another aspect provides a method for screening candidate substances for treating cranial nervous system diseases, comprising the steps of contacting cells expressing PCNT protein with a test substance in vitro, measuring the expression level or activity of PCNT protein in the cells, and comparing the measured expression level or activity of PCNT protein with the expression level or activity measured in a control group, and selecting a test substance that increases the expression level or activity compared to the control group as a candidate substance for treating cranial nervous system diseases.

[0056] PCNT protein and cranial nervous system diseases are as described above.

[0057] We confirmed that PCNT protein expression levels were elevated in cerebral palsy patients who received umbilical cord blood cell therapy compared to before treatment, thereby confirming the positive effect on cerebral palsy. Therefore, substances that specifically increase the expression or activity of PCNT protein may be effective in treating cranial nervous system disorders such as cerebral palsy. Such effects indicate that PCNT protein or its gene is a target for the treatment of cranial nervous system disorders such as cerebral palsy.

[0058] The method comprises contacting a test substance in vitro with cells that express PCNT protein.

[0059] The cells expressing the PCNT protein include, but are not limited to, blood cells, brain cells, and the like.

[0060] The contact with the test substance may be by transfection, transformation or injection of the test substance.

[0061] The contact with the test substance is carried out in a medium capable of supporting the growth of the cells.

[0062] The method includes measuring the expression level or activity of PCNT protein in a cell.

[0063] The measurement can also be carried out using an antibody that specifically recognizes the PCNT protein or a fragment thereof, or a primer or probe that specifically recognizes a nucleic acid encoding the PCNT protein or a fragment thereof.

[0064] The expression level can also be measured by measuring the amount of PCNT gene mRNA or PCNT protein.

[0065] The amount of the mRNA can be measured by, but is not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting, or DNA chip.

[0066] The amount of said protein can be measured by, but is not limited to, Western blotting, magnetic bead-antibody immunoprecipitation, ELISA, mass spectrometry radioimmunoassay or immunoprecipitation.

[0067] The method includes the step of comparing the measured expression level or activity of PCNT protein with the expression level or activity measured in a control group, and selecting a test substance that increases the expression level or activity compared to the control group as a candidate substance for treating a nervous system disease.

[0068] If the expression or activity level of PCNT protein is further increased after treatment with the test substance, the test substance is also selected as a substance that increases the expression or activity of PCNT protein and can be used to treat nervous system diseases.

[0069] The control group is cells under identical conditions except for the step of contacting with the test substance.

[0070] The present invention will be described in more detail below through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples. [Example]

[0071] Example 1: Screening of proteins whose expression levels change after cord blood treatment Blood samples were collected from a child with cerebral palsy who underwent umbilical cord blood cell therapy before and after the treatment, and changes in specific proteins present in the plasma were observed. For this umbilical cord blood cell therapy, red blood cells and plasma were removed from the umbilical cord blood to prepare a leukocyte concentrate, which was then frozen and stored. The frozen leukocyte concentrate was thawed in a 37°C water bath before treatment and washed with a solution prepared by diluting equal amounts of 10% dextran 40 (100 g dextran 40 / 1 L water) and 5% albumin (50 g albumin / 1 L water). After washing, the leukocyte concentrate was diluted with 10 ml of a mixture of equal amounts of 10% dextran 40 and 5% albumin until a minimum total nucleated cell count of 3 x 10 7 The suspension was administered intravenously at a dose of 15 to 30 ml / kg.

[0072] 1-1. Preparation of plasma samples for screening Table 1 shows the diagnoses and cord blood treatments of cerebral palsy patients selected for plasma protein screening. Approximately 1-3 cc of blood from the cerebral palsy patients listed in Table 1 was collected in an EDTA tube and centrifuged at 2,000 rpm. The blood was separated into layers: red blood cells at the bottom, mononuclear cells in the middle, and plasma at the top. The plasma was then separated and stored in a -80°C ultra-low temperature freezer.

[0073] [Table 1]

[0074] 1-2. Screening of plasma proteins before and after cord blood cell therapy Two-dimensional gel electrophoresis (2DE) was used to confirm changes in specific proteins present in the plasma separated in Example 1-1. 2DE, image analysis, and qualitative analysis of the prepared samples were performed by the Yonsei Proteome Research Institute at Yonsei University.

[0075] Of the 10 cerebral palsy patients in Table 1, six showed improvement after receiving cord blood therapy, namely patients 4, 6, 7, 8, 9 and 10, and these were selected for the experiment.

[0076] Table 2 shows information about the experimental samples used to screen plasma proteins from six patients who showed improvement after receiving cord blood therapy. In Experiment 1, plasma from patients 4, 9, and 10 was mixed before cord blood therapy, and plasma from patient 4 was used as the sample for analysis 10 days after cord blood therapy. In Experiment 2, plasma from patients 6, 7, and 8 was mixed before cord blood therapy, and plasma from patient 6 was used as the sample for analysis 10 days after cord blood therapy.

[0077] [Table 2]

[0078] Figure 1A shows the results of two-dimensional gel electrophoresis to confirm changes in plasma proteins before cord blood treatment (Pre) and 10 days after treatment (Post+10 days), representing the results of Experiment 1 (analysis of samples from patients Nos. 4, 9, and 10). Figure 1B shows the results of two-dimensional gel electrophoresis to confirm changes in plasma proteins before cord blood treatment (Pre) and 10 days after treatment (Post+10 days), representing the results of Experiment 2 (analysis of samples from patients Nos. 6, 7, and 8). The horizontal axis represents pH, and the vertical axis represents protein size.

[0079] Table 3 shows the proteins that were found to increase more than three-fold in the plasma of patients who showed improvement after cord blood transfusion, based on the qualitative analysis of each protein using 2DE. The alpha-2-macroglobulin isoform is shown by two dots, suggesting that it is not completely filtered out of the sample when conducting experiments with proteins generally present in plasma. The protein with 626 dots is thought to be a fibrinogen-binding fragment, and this result also suggests that fibrinogen was not removed from the plasma. Therefore, pericentrin (PCNT) was confirmed to be the specific protein that increased in plasma after cord blood transfusion.

[0080] [Table 3]

[0081] Example 2: Confirmation of PCNT expression in cerebral palsy patients receiving umbilical cord blood therapy 2-1. Preparation of plasma samples from children with cerebral palsy Table 4 shows the list of clinical cerebral palsy patients, the type of umbilical cord blood administered, and information regarding erythropoietin (EPO) infusion. Allogeneic umbilical cord blood was obtained from siblings or family members. Blood was separated from the 13 cerebral palsy patients listed in Table 4, and plasma was collected. The collected plasma was diluted 5:1 with phosphate-buffered saline (PBS) and quantified using the Bradford assay. Quantified protein was prepared by mixing 30 μg with sample buffer, boiling for 7 minutes, and then spinning down.

[0082] [Table 4]

[0083] 2-2. Confirmation of PCNT protein expression after cord blood treatment After the cord blood treatment, the expression of PCNT protein was confirmed by Western blot technique.

[0084] First, the plasma sample prepared in Example 2-1 was dispensed onto an 8% SDS-polyacrylamide gel in the order of before and after the cord blood treatment. The proteins dispensed onto the gel were transferred to a nitrocellulose (NC) membrane and reacted with PCNT antibody for 16 hours or more. The expression levels of PCNT before and after the cord blood treatment were confirmed by band analysis.

[0085] Figure 2 shows the results of Western blot analysis confirming PCNT expression in the plasma of 13 cerebral palsy patients before (Pre) and after (Post) cord blood treatment.

[0086] As shown in Figure 2, although there were differences in the dates before and after treatment for each sample, it was confirmed that the amount of PCNT generally increased after cord blood treatment, regardless of the type of cord blood.

[0087] Figure 3 is a graph showing the changes in PCNT expression levels before and after cord blood treatment, combining the results of all the patients shown in Figure 2.

[0088] As shown in Figure 3, it was confirmed that the expression level of PCNT increased 3 or 10 days after the cord blood treatment, and the results were significant.

[0089] Therefore, it was confirmed that when umbilical cord blood therapy was administered to cerebral palsy patients, the expression level of PCNT increased significantly.

[0090] Example 3: Confirmation of PCNT changes in animal models of cranial nervous system diseases 3-1. Creation of a rat model of induced cerebral palsy A hypoxic ischemia (HI) model, a cerebral palsy-induced rat model, was created using 7-day-old ICR mice. The right common carotid artery of 7-day-old ICR mice was ligated using 5-0 blue nylon and placed in a sealed special container maintained at 8% O2 and 92% N2. The mice were kept at 37°C and hypoxia-cerebral ischemia was induced for 1 hour. Six days after creating the HI model, the extent of brain tissue damage was visually inspected, and models with damage of 50% or more were excluded.

[0091] 3-2. Umbilical cord blood administration to a rat model of cerebral palsy The umbilical cord blood used was donated by Icord, the cord blood bank at Cha Hospital, for research purposes. The cord blood was centrifuged at 2,200 rpm to separate red blood cells, mononuclear cell layers, and plasma. The plasma was stored separately, and the mononuclear cell layer was isolated using Ficoll (Pharmacia Corp., USA). Specifically, the plasma-free red blood cells and mononuclear cell layer were diluted with PBS and placed on top of the Ficoll, followed by centrifugation at 2,200 rpm for 20 minutes. Due to the Ficoll principle, red blood cells, which are heavier than the Ficoll, sink to the bottom, leaving the mononuclear cell layer directly above the Ficoll. The isolated mononuclear cell layer was then transferred separately and spun down using 4x10 5 The cells / 10 g concentration were dissolved in 100 μl of saline. The prepared umbilical cord blood mononuclear cells were intraperitoneally administered randomly to rat models of cerebral palsy induced on day 7.

[0092] 3-3. Confirmation of changes in PCNT expression in brain tissue One week after administration of umbilical cord blood mononuclear cells, the cerebral palsy-induced rat model was killed by removing blood and then the brain tissue was removed. The removed brain tissue was homogenized using a lysis buffer and centrifuged to separate the protein. The altered expression level of PCNT was confirmed using Western blot analysis of 30 μg of the separated protein.

[0093] Figure 4 shows the results of measuring PCNT expression levels four times after administration of umbilical cord blood mononuclear cells (UCB) in a mouse model of cerebral palsy. A model administered with EPO was used as a comparison group.

[0094] As shown in Figure 4, we confirmed that the expression level of PCNT increased in a mouse model of cerebral palsy induced by administration of umbilical cord blood. We also confirmed that the expression level of PCNT increased in umbilical cord blood when compared with a model administered EPO as a comparison group.

[0095] Figure 5 is a graph showing the change in PCNT expression in Figure 4 converted into a percentage, based on the level when umbilical cord blood was not administered to a sham model in which cerebral palsy was not induced.

[0096] As shown in Figure 5, when umbilical cord blood was administered to the cerebral palsy-induced mouse model, the expression level of PCNT was even higher than when compared to the sham model, which is a normal mouse that did not receive umbilical cord blood.

[0097] Example 4: Verification of differences in PCNT levels present in umbilical cord blood, plasma of normal individuals, and plasma of children with cerebral palsy 4-1. Plasma separation in umbilical cord blood, normal human blood, and blood from cerebral palsy patients For the umbilical cord blood, research cord blood donated by iCord was centrifuged to separate the plasma, which was then frozen and stored in a -80°C ultra-low temperature freezer. For the normal blood, 3cc of blood from a woman and a man in their 20s and 30s was centrifuged to separate the plasma. For the cerebral palsy blood, blood from hospitalized cerebral palsy patients was centrifuged before cord blood treatment to separate the plasma. The two plasma samples were frozen and stored. Table 5 shows information on normal subjects and cerebral palsy patients, who were selected as comparison groups to confirm PCNT expression in umbilical cord blood.

[0098] [Table 5]

[0099] 4-2. Confirmation of PCNT Expression Levels in Umbilical Cord Blood, Normal Human Plasma, and Cerebral Palsy Patient Plasma The plasma stored in Example 4-1 was slowly thawed on ice and then diluted 5:1 with PBS. The diluted sample was quantified using Bradford reagent, and 30 μg of the sample was subjected to Western blot analysis.

[0100] Figure 6A shows the expression levels of PCNT in umbilical cord blood (UCB) plasma, normal plasma, and cerebral palsy patient plasma samples. Figure 6B shows the relative expression levels (%) of PCNT in umbilical cord blood (UCB) plasma, normal plasma, and cerebral palsy patient plasma samples, based on umbilical cord blood sample No. 1.

[0101] As shown in Figures 6A and 6B, the expression level of PCNT was significantly lower in the plasma of cerebral palsy patients, followed by that of normal individuals, and the highest level of PCNT was found in umbilical cord blood. Since there is no standard protein in plasma, the band stained with Ponceau was used as the standard, and the PCNT expression level was converted to a percentage, confirming that PCNT was most highly expressed in umbilical cord blood.

[0102] Example 5: Identification of cell types in umbilical cord blood that express PCNT Umbilical cord blood is blood found in the tissues of the umbilical cord and contains a large number of hematopoietic stem cells (HSCs). These stem cells are the main components of blood and have the potential to differentiate into monocytes, macrophages, and platelets produced in the bone marrow, as well as lymphoid cells such as T cells, B cells, and NK cells. Therefore, we identified the types of cells present in umbilical cord blood by determining whether they expressed PCNT. Information on PCNT-expressing cells was found on the GeneCards website (http: / / www.genecards.org / ).

[0103] Figure 7 shows the PCNT expression levels (Log 1) of the following components present in cord blood: serum, plasma, monocytes, neutrophils, B-lymphocytes, T-lymphocytes, CD4 T cells, CD8 T cells, NK cells, peripheral blood mononuclear cells (PBMCs), platelets, lymph nodes, tonsils, bone marrow stromal cells, and bone marrow mesenchymal stem cells. 10 , ppm).

[0104] As shown in Figure 7, PCNT was secreted by monocytes, B-lymphocytes, CD4 T cells, CD8 T cells, NK cells, peripheral blood mononuclear cells, platelets, and lymph nodes in the cells present in umbilical cord blood, and among these, peripheral blood mononuclear cells (PBMCs) showed the highest expression of PCNT.

[0105] Example 6: Confirmation of the therapeutic effect of umbilical cord blood on cranial nervous system diseases To confirm whether cord blood therapy is actually effective in treating neurological disorders in patients with neurological disorders, autologous cord blood therapy was performed on children with cerebral palsy.

[0106] In this cord blood cell therapy, cord blood was used after removing red blood cells and plasma to prepare a leukocyte concentrate, which was then frozen and stored. Before the procedure, the frozen leukocyte concentrate was thawed in a 37°C water bath and washed with a solution prepared by diluting equal amounts of 10% dextran 40 (100 g dextran 40 / 1 L water) and 5% albumin (50 g albumin / 1 L water). After washing, the leukocyte concentrate was diluted with 10 ml of a mixture of equal amounts of 10% dextran 40 and 5% albumin until a minimum total nucleated cell count of 3 x 10 was reached. 7The suspension was administered at a dose of between 15 ml and 30 ml / kg by intravenous injection.

[0107] Table 6 shows the gender, diagnosis, and functional evaluation results after cord blood treatment of 13 children with cerebral palsy who were selected to confirm the therapeutic effects of cord blood.

[0108] As shown in Table 6, it was confirmed that 13 cerebral palsy patients showed an improvement in overall function after cord blood treatment.

[0109] [Table 6]

[0110] In addition, 2 to 4 months after the cord blood treatment, patients 5, 11, and 13 were evaluated using the gross motor function classification system (GMFCS), Bayley Scales of Infant Development II motor (BSID II motor), and Bayley Scales of Infant Development II mental (BSID II mental).

[0111] FIG. 8 is a graph showing the GMFCS evaluation results (total GMFCS (%)) for cerebral palsy patients Nos. 5, 11, and 13 before (GMFCS_pre) and after (GMFCS_post) cord blood treatment.

[0112] FIG. 9 is a graph showing the results of the behavioral development domain assessment (BSID II motor) of the Bayley Scale of Infant Development for cerebral palsy patients Nos. 5, 11, and 13 before (BSID_Motor_pre) and after (BSID_Motor_post) cord blood treatment.

[0113] FIG. 10 is a graph showing the results of the mental development domain assessment (BSID II mental) of the Bayley Scale of Infant Development for cerebral palsy patients Nos. 5, 11, and 13 before (BSID_Mental_pre) and after (BSID_Mental_post) cord blood treatment.

[0114] As shown in Figures 8 to 10, the GMFCS, BSID II motor, and BSID II mental tests were conducted on cerebral palsy patients before and after cord blood therapy, and improvements were observed after cord blood therapy. [Industrial Applicability]

[0115] We have identified PCNT protein as a target protein for the treatment or diagnosis of nervous system diseases, and PCNT protein can be used as a target for diagnosing nervous system diseases and developing therapeutic agents for nervous system diseases in the early stages.

[0116] Therefore, by using a composition containing umbilical cord blood or umbilical cord blood-derived cells that express or secrete PCNT, diseases of the central nervous system can be effectively prevented or treated.

[0117] Furthermore, nervous system diseases can be diagnosed using a composition or kit containing an agent that measures the level of PCNT protein or the mRNA of its gene.

[0118] Furthermore, by measuring the expression level or activity level of the PCNT protein or its gene, it is possible to analyze information necessary for diagnosing cranial nervous system diseases.

[0119] Furthermore, candidate substances for treating central nervous system diseases can be screened by contacting cells expressing PCNT protein with a test substance and then measuring the expression level or activity of PCNT protein. The present disclosure includes the following embodiments. [1] A pharmaceutical composition for preventing or treating a central nervous system disease, comprising umbilical cord blood or umbilical cord blood-derived cells that express or secrete pericentrin (PCNT) as an active ingredient. [2] The pharmaceutical composition described in embodiment 1, characterized in that the PCNT has the amino acid sequence of SEQ ID NO: 1. [3] The pharmaceutical composition according to embodiment 1, characterized in that it is for preventing or treating a central nervous system disease in which the PCNT expression level is reduced compared to that of a normal individual. [4] The pharmaceutical composition of embodiment 1, wherein the central nervous system disease is selected from the group consisting of cerebral palsy, brain injury, traumatic brain injury, ischemic brain injury, cerebral concussion, cerebral infarction, cerebral hemorrhage, Parkinson's disease, Alzheimer's disease, Huntington's disease, stroke, dementia, Lou Gehrig's disease, Pick's disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, primary lateral sclerosis, degenerative ataxia, multiple sclerosis, nervous system dysfunction, memory loss, epilepsy, encephalitis, prion disease, and neurological diseases. [5] The pharmaceutical composition of embodiment 1, wherein the umbilical cord blood is autologous or allogeneic umbilical cord blood. [6] The pharmaceutical composition described in embodiment 1, characterized in that the umbilical cord blood-derived cells are hematopoietic stem cells. [7] The pharmaceutical composition of embodiment 1, wherein the cord blood-derived cells are selected from the group consisting of mononuclear cells, B-lymphocytes, CD4 T cells, CD8 T cells, NK cells, peripheral blood mononuclear cells, platelets, and lymph nodes. [8] The pharmaceutical composition of embodiment 1, further comprising erythropoietin (EPO). [9] A composition for diagnosing a central nervous system disease, comprising an agent for measuring the level of pericentrin (PCNT) protein or mRNA of its gene.

[10] The composition of embodiment 9, wherein the agent for measuring the protein is an antibody that specifically binds to the PCNT protein or a fragment thereof, or the agent for measuring the mRNA level of the gene comprises a primer or probe that specifically binds to a nucleic acid encoding the PCNT protein or a fragment thereof.

[11] The composition of embodiment 9, wherein the central nervous system disease is selected from the group consisting of cerebral palsy, brain injury, traumatic brain injury, ischemic brain injury, cerebral concussion, cerebral infarction, cerebral hemorrhage, Parkinson's disease, Alzheimer's disease, Huntington's disease, stroke, dementia, Lou Gehrig's disease, Pick's disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, primary lateral sclerosis, degenerative ataxia, multiple sclerosis, nervous system dysfunction, memory loss, epilepsy, encephalitis, prion disease, and neurological diseases.

[12] A kit for diagnosing a central nervous system disease, comprising an agent for measuring the level of pericentrin (PCNT) protein or mRNA of its gene.

[13] Measuring the expression or activity level of pericentrin (PCNT) protein or its gene in a sample isolated from an individual; A method for analyzing information necessary for diagnosing central nervous system diseases, comprising a step of comparing the measured expression or activity level with the expression or activity level of PCNT protein measured in a control sample isolated from a normal individual.

[14] The method of embodiment 13, wherein the sample is plasma.

[15] The method of embodiment 13, further comprising determining that there is a central nervous system disease if the measured expression or activity level is lower than the expression or activity level measured in the control sample.

[16] The method of embodiment 13, wherein the central nervous system disease is selected from the group consisting of cerebral palsy, brain injury, traumatic brain injury, ischemic brain injury, cerebral concussion, cerebral infarction, cerebral hemorrhage, Parkinson's disease, Alzheimer's disease, Huntington's disease, stroke, dementia, Lou Gehrig's disease, Pick's disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, primary lateral sclerosis, degenerative ataxia, multiple sclerosis, nervous system dysfunction, memory loss, epilepsy, encephalitis, prion disease, and neurological diseases.

[17] Contacting a test substance with cells expressing a pericentrin (PCNT) protein in vitro; measuring the expression level or activity of PCNT protein in the cells; and a step of comparing the measured expression level or activity of PCNT protein with the expression level or activity measured in a control group and selecting a test substance that increases the expression level or activity of PCNT protein as a candidate substance for treating central nervous system diseases.

[18] The method of embodiment 17, wherein the cells are contacted with the test substance in a medium capable of supporting the growth of the cells.

[19] The method described in embodiment 17, wherein the expression level is measured by measuring the amount of PCNT gene mRNA or PCNT protein.

[20] The method of embodiment 17, wherein the central nervous system disease is selected from the group consisting of cerebral palsy, brain injury, traumatic brain injury, ischemic brain injury, cerebral concussion, cerebral infarction, cerebral hemorrhage, Parkinson's disease, Alzheimer's disease, Huntington's disease, stroke, dementia, Lou Gehrig's disease, Pick's disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, primary lateral sclerosis, degenerative ataxia, multiple sclerosis, nervous system dysfunction, memory loss, epilepsy, encephalitis, prion disease, and neurological diseases.

[0120] The present disclosure includes the following sequence information: SEQUENCE LISTING <110> SUNGKWANG MEDICAL FOUNDATION <120> PCNT AS A TARGET PROTEIN FOR TREATMENT OR DIAGNOSIS OF Brain-nervous system diseases <130> PA23-455 <150> KR 10-2017-0004167 <151> 2017-01-11 <160> 1 <170> PatentIn version 3.2 <210> 1 <211> 3229 <212> PRT <213> Homo sapiens <400> 1 Met Phe Thr Val Ser Asp His Pro Pro Glu Gln His Gly Met Phe Thr 1 5 10 15 Val Gly Asp His Pro Pro Glu Gln Arg Gly Met Phe Thr Val Ser Asp 20 25 30 His Pro Pro Glu Gln His Gly Met Phe Thr Val Ser Asp His Pro Pro 35 40 45 Glu Gln Arg Gly Met Phe Thr Ile Ser Asp His Gln Pro Glu Gln Arg 50 55 60 Gly Met Phe Thr Val Ser Asp His Thr Pro Glu Gln Arg Gly Ile Phe 65 70 75 80 Thr Ile Ser Asp His Pro Ala Glu Gln Arg Gly Met Phe Thr Lys Glu 85 90 95 Cys Glu Gln Glu Cys Glu Leu Ala Ile Thr Asp Leu Glu Ser Gly Arg 100 105 110 Glu Asp Glu Ala Gly Leu His Gln Ser Gln Ala Val His Gly Leu Glu 115 120 125 Leu Glu Ala Leu Arg Leu Ser Leu Ser Asn Met His Thr Ala Gln Leu 130 135 140 Glu Leu Thr Gln Ala Asn Leu Gln Lys Glu Lys Glu Thr Ala Leu Thr 145 150 155 160 Glu Leu Arg Glu Met Leu Asn Ser Arg Arg Ala Gln Glu Leu Ala Leu 165 170 175 Leu Gln Ser Arg Gln Gln His Glu Leu Glu Leu Leu Arg Glu Gln His 180 185 190 Ala Arg Glu Lys Glu Glu Val Val Leu Arg Cys Gly Gln Glu Ala Ala 195 200 205 Glu Leu Lys Glu Lys Leu Gln Ser Glu Met Glu Lys Asn Ala Gln Ile 210 215 220 Val Lys Thr Leu Lys Glu Asp Trp Glu Ser Glu Lys Asp Leu Cys Leu 225 230 235 240 Glu Asn Leu Arg Lys Glu Leu Ser Ala Lys His Gln Ser Glu Met Glu 245 250 255 Asp Leu Gln Asn Gln Phe Gln Lys Glu Leu Ala Glu Gln Arg Ala Glu 260 265 270 Leu Glu Lys Ile Phe Gln Asp Lys Asn Gln Ala Glu Arg Ala Leu Arg 275 280 285 Asn Leu Glu Ser His His Gln Ala Ala Ile Glu Lys Leu Arg Glu Asp 290 295 300 Leu Gln Ser Glu His Gly Arg Cys Leu Glu Asp Leu Glu Phe Lys Phe 305 310 315 320 Lys Glu Ser Glu Lys Glu Lys Gln Leu Glu Leu Glu Asn Leu Gln Ala 325 330 335 Ser Tyr Glu Asp Leu Lys Ala Gln Ser Gln Glu Glu Ile Arg Arg Leu 340 345 350 Trp Ser Gln Leu Asp Ser Ala Arg Thr Ser Arg Gln Glu Leu Ser Glu 355 360 365 Leu His Glu Gln Leu Leu Ala Arg Thr Ser Arg Val Glu Asp Leu Glu 370 375 380 Gln Leu Lys Gln Arg Glu Lys Thr Gln His Glu Ser Glu Leu Glu Gln 385 390 395 400 Leu Arg Ile Tyr Phe Glu Lys Lys Leu Arg Asp Ala Glu Lys Thr Tyr 405 410 415 Gln Glu Asp Leu Thr Leu Leu Gln Gln Arg Leu Gln Gly Ala Arg Glu 420 425 430 Asp Ala Leu Leu Asp Ser Val Glu Val Gly Leu Ser Cys Val Gly Leu 435 440 445 Glu Glu Lys Pro Glu Lys Gly Arg Lys Asp His Val Asp Glu Leu Glu 450 455 460 Pro Glu Arg His Lys Glu Ser Leu Pro Arg Phe Gln Ala Glu Leu Glu 465 470 475 480 Glu Ser His Arg His Gln Leu Glu Ala Leu Glu Ser Pro Leu Cys Ile 485 490 495 Gln His Glu Gly His Val Ser Asp Arg Cys Cys Val Glu Thr Ser Ala 500 505 510 Leu Gly His Glu Trp Arg Leu Glu Pro Ser Glu Gly His Ser Gln Glu 515 520 525 Leu Pro Trp Val His Leu Gln Gly Val Gln Asp Gly Asp Leu Glu Ala 530 535 540 Asp Thr Glu Arg Ala Ala Arg Val Leu Gly Leu Glu Thr Glu His Lys 545 550 555 560 Val Gln Leu Ser Leu Leu Gln Thr Glu Leu Lys Glu Glu Ile Glu Leu 565 570 575 Leu Lys Ile Glu Asn Arg Asn Leu Tyr Gly Lys Leu Gln His Glu Thr 580 585 590 Arg Leu Lys Asp Asp Leu Glu Lys Val Lys His Asn Leu Ile Glu Asp 595 600 605 His Gln Lys Glu Leu Asn Asn Ala Lys Gln Lys Thr Glu Leu Met Lys 610 615 620 Gln Glu Phe Gln Arg Lys Glu Thr Asp Trp Lys Val Met Lys Glu Glu 625 630 635 640 Leu Gln Arg Glu Ala Glu Glu Lys Leu Thr Leu Met Leu Leu Glu Leu 645 650 655 Arg Glu Lys Ala Glu Ser Glu Lys Gln Thr Ile Ile Asn Lys Phe Glu 660 665 670 Leu Arg Glu Ala Glu Met Arg Gln Leu Gln Asp Gln Gln Ala Ala Gln 675 680 685 Ile Leu Asp Leu Glu Arg Ser Leu Thr Glu Gln Gln Gly Arg Leu Gln 690 695 700 Gln Leu Glu Gln Asp Leu Thr Ser Asp Asp Ala Leu His Cys Ser Gln 705 710 715 720 Cys Gly Arg Glu Pro Pro Thr Ala Gln Asp Gly Glu Leu Ala Ala Leu 725 730 735 His Val Lys Glu Asp Cys Ala Leu Gln Leu Met Leu Ala Arg Ser Arg 740 745 750 Phe Leu Glu Glu Arg Lys Glu Ile Thr Glu Lys Phe Ser Ala Glu Gln 755 760 765 Asp Ala Phe Leu Gln Glu Ala Gln Glu Gln His Ala Arg Glu Leu Gln 770 775 780 Leu Leu Gln Glu Arg His Gln Gln Gln Leu Leu Ser Val Thr Ala Glu 785 790 795 800 Leu Glu Ala Arg His Gln Ala Ala Leu Gly Glu Leu Thr Ala Ser Leu 805 810 815 Glu Ser Lys Gln Gly Ala Leu Leu Ala Ala Arg Val Ala Glu Leu Gln 820 825 830 Thr Lys His Ala Ala Asp Leu Gly Ala Leu Glu Thr Arg His Leu Ser 835 840 845 Ser Leu Asp Ser Leu Glu Ser Cys Tyr Leu Ser Glu Phe Gln Thr Ile 850 855 860 Arg Glu Glu His Arg Gln Ala Leu Glu Leu Leu Arg Ala Asp Phe Glu 865 870 875 880 Glu Gln Leu Trp Lys Lys Asp Ser Leu His Gln Thr Ile Leu Thr Gln 885 890 895 Glu Leu Glu Lys Leu Lys Arg Lys His Glu Gly Glu Leu Gln Ser Val 900 905 910 Arg Asp His Leu Arg Thr Glu Val Ser Thr Glu Leu Ala Gly Thr Val 915 920 925 Ala His Glu Leu Gln Gly Val His Gln Gly Glu Phe Gly Ser Glu Lys 930 935 940 Lys Thr Ala Leu His Glu Lys Glu Glu Thr Leu Arg Leu Gln Ser Ala 945 950 955 960 Gln Ala Gln Pro Phe His Gln Glu Glu Lys Glu Ser Leu Ser Leu Gln 965 970 975 Leu Gln Lys Lys Asn His Gln Val Gln Gln Leu Lys Asp Gln Val Leu 980 985 990 Ser Leu Ser His Glu Ile Glu Glu Cys Arg Ser Glu Leu Glu Val Leu 995 1000 1005 Gln Gln Arg Arg Glu Arg Glu Asn Arg Glu Gly Ala Asn Leu Leu 1010 1015 1020 Ser Met Leu Lys Ala Asp Val Asn Leu Ser His Ser Glu Arg Gly 1025 1030 1035 Ala Leu Gln Asp Ala Leu Arg Arg Leu Leu Gly Leu Phe Gly Glu 1040 1045 1050 Thr Leu Arg Ala Ala Val Thr Leu Arg Ser Arg Ile Gly Glu Arg 1055 1060 1065 Val Gly Leu Cys Leu Asp Asp Ala Gly Ala Gly Leu Ala Leu Ser 1070 1075 1080 Thr Ala Pro Ala Leu Glu Glu Thr Trp Ser Asp Val Ala Leu Pro 1085 1090 1095 Glu Leu Asp Arg Thr Leu Ser Glu Cys Ala Glu Met Ser Ser Val 1100 1105 1110 Ala Glu Ile Ser Ser His Met Arg Glu Ser Phe Leu Met Ser Pro 1115 1120 1125 Glu Ser Val Arg Glu Cys Glu Gln Pro Ile Arg Arg Val Phe Gln 1130 1135 1140 Ser Leu Ser Leu Ala Val Asp Gly Leu Met Glu Met Ala Leu Asp 1145 1150 1155 Ser Ser Arg Gln Leu Glu Glu Ala Arg Gln Ile His Ser Arg Phe 1160 1165 1170 Glu Lys Glu Phe Ser Phe Lys Asn Glu Glu Thr Ala Gln Val Val 1175 1180 1185 Arg Lys His Gln Glu Leu Leu Glu Cys Leu Lys Glu Glu Ser Ala 1190 1195 1200 Ala Lys Ala Glu Leu Ala Leu Glu Leu His Lys Thr Gln Gly Thr 1205 1210 1215 Leu Glu Gly Phe Lys Val Glu Thr Ala Asp Leu Lys Glu Val Leu 1220 1225 1230 Ala Gly Lys Glu Asp Ser Glu His Arg Leu Val Leu Glu Leu Glu 1235 1240 1245 Ser Leu Arg Arg Gln Leu Gln Gln Ala Ala Gln Glu Gln Ala Ala 1250 1255 1260 Leu Arg Glu Glu Cys Thr Arg Leu Trp Ser Arg Gly Glu Ala Thr 1265 1270 1275 Path Thr Asp Path Glu Path Arg Glu Path Gly Thr Path Val Thr Path 1280 1285 1290 Path His Lys Asp Ser Path Leu Arg Lys Glu Val Glu Asp Leu Thr 1295 1300 1305 Lys Glu Gln Ser Glu Thr Arg Lys Gln Ala Glu Lys Asp Arg Ser 1310 1315 1320 Ala Leu Leu Ser Gln Met Lys Ile Leu Glu Ser Glu Leu Glu Glu 1325 1330 1335 Gln Leu Ser Gln His Arg Gly Cys Ala Lys Gln Ala Glu Ala Val 1340 1345 1350 Thr Ala Leu Glu Gln Gln Val Ala Ser Leu Asp Lys His Leu Arg 1355 1360 1365 Asn Gln Arg Gln Phe Met Asp Glu Gln Ala Ala Glu Arg Glu His 1370 1375 1380 Glu Arg Glu Glu Phe Gln Gln Glu Ile Gln Arg Leu Glu Gly Gln 1385 1390 1395 Leu Arg Gln Ala Ala Lys Pro Gln Pro Trp Gly Pro Arg Asp Ser 1400 1405 1410 Gln Gln Ala Pro Leu Asp Gly Glu Val Glu Leu Leu Gln Gln Lys 1415 1420 1425 Leu Arg Glu Lys Leu Asp Glu Phe Asn Glu Leu Ala Ile Gln Lys 1430 1435 1440 Glu Ser Ala Asp Arg Gln Val Leu Met Gln Glu Glu Glu Ile Lys 1445 1450 1455 Arg Leu Glu Glu Met Asn Ile Asn Ile Arg Lys Lys Val Ala Gln 1460 1465 1470 Leu Gln Glu Glu Val Glu Lys Gln Lys Asn Ile Val Lys Gly Leu 1475 1480 1485 Glu Gln Asp Lys Glu Val Leu Lys Lys Gln Gln Met Ser Ser Leu 1490 1495 1500 Leu Leu Ala Ser Thr Leu Gln Ser Thr Leu Asp Ala Gly Arg Cys 1505 1510 1515 Pro Glu Pro Pro Ser Gly Ser Pro Pro Glu Gly Pro Glu Ile Gln 1520 1525 1530 Leu Glu Val Thr Gln Arg Ala Leu Leu Arg Arg Glu Ser Glu Val 1535 1540 1545 Leu Asp Leu Lys Glu Gln Leu Glu Lys Met Lys Gly Asp Leu Glu 1550 1555 1560 Ser Lys Asn Glu Glu Ile Leu His Leu Asn Leu Lys Leu Asp Met 1565 1570 1575 Gln Asn Ser Gln Thr Ala Val Ser Leu Arg Glu Leu Glu Glu Glu 1580 1585 1590 Asn Thr Ser Leu Lys Val Ile Tyr Thr Arg Ser Ser Glu Ile Glu 1595 1600 1605 Glu Leu Lys Ala Thr Ile Glu Asn Leu Gln Glu Asn Gln Lys Arg 1610 1615 1620 Leu Gln Lys Glu Lys Ala Glu Glu Ile Glu Gln Leu His Glu Val 1625 1630 1635 Ile Glu Lys Leu Gln His Glu Leu Ser Leu Met Gly Pro Val Val 1640 1645 1650 His Glu Val Ser Asp Ser Gln Ala Gly Ser Leu Gln Ser Glu Leu 1655 1660 1665 Leu Cys Ser Gln Ala Gly Gly Pro Arg Gly Gln Ala Leu Gln Gly 1670 1675 1680 Glu Leu Glu Ala Ala Leu Glu Ala Lys Glu Ala Leu Ser Arg Leu 1685 1690 1695 Leu Ala Asp Gln Glu Arg Arg His Ser Gln Ala Leu Glu Ala Leu 1700 1705 1710 Gln Gln Arg Leu Gln Gly Ala Glu Glu Ala Ala Glu Leu Gln Leu 1715 1720 1725 Ala Glu Leu Glu Arg Asn Val Ala Leu Arg Glu Ala Glu Val Glu 1730 1735 1740 Asp Met Ala Ser Arg Ile Gln Glu Phe Glu Ala Ala Leu Lys Ala 1745 1750 1755 Lys Glu Ala Thr Ile Ala Glu Arg Asn Leu Glu Ile Asp Ala Leu 1760 1765 1770 Asn Gln Arg Lys Ala Ala His Ser Ala Glu Leu Glu Ala Val Leu 1775 1780 1785 Leu Ala Leu Ala Arg Ile Arg Arg Ala Leu Glu Gln Gln Pro Leu 1790 1795 1800 Ala Ala Gly Ala Ala Pro Pro Glu Leu Gln Trp Leu Arg Ala Gln 1805 1810 1815 Cys Ala Arg Leu Ser Arg Gln Leu Gln Val Leu His Gln Arg Phe 1820 1825 1830 Leu Arg Cys Gln Val Glu Leu Asp Arg Arg Gln Ala Arg Arg Ala 1835 1840 1845 Thr Ala His Thr Arg Val Pro Gly Ala His Pro Gln Pro Arg Met 1850 1855 1860 Asp Gly Gly Ala Lys Ala Gln Val Thr Gly Asp Val Glu Ala Ser 1865 1870 1875 His Asp Ala Ala Leu Glu Pro Val Val Pro Asp Pro Gln Gly Asp 1880 1885 1890 Leu Gln Pro Val Leu Val Thr Leu Lys Asp Ala Pro Leu Cys Lys 1895 1900 1905 Gln Glu Gly Val Met Ser Val Leu Thr Val Cys Gln Arg Gln Leu 1910 1915 1920 Gln Ser Glu Leu Leu Leu Val Lys Asn Glu Met Arg Leu Ser Leu 1925 1930 1935 Glu Asp Gly Gly Lys Gly Lys Glu Lys Val Leu Glu Asp Cys Gln 1940 1945 1950 Leu Pro Lys Val Asp Leu Val Ala Gln Val Lys Gln Leu Gln Glu 1955 1960 1965 Lys Leu Asn Arg Leu Leu Tyr Ser Met Thr Phe Gln Asn Val Asp 1970 1975 1980 Ala Ala Asp Thr Lys Ser Leu Trp Pro Met Ala Ser Ala His Leu 1985 1990 1995 Leu Glu Ser Ser Trp Ser Asp Asp Ser Cys Asp Gly Glu Glu Pro 2000 2005 2010 Asp Ile Ser Pro His Ile Asp Thr Cys Asp Ala Asn Thr Ala Thr 2015 2020 2025 Gly Gly Val Thr Asp Val Ile Lys Asn Gln Ala Ile Asp Ala Cys 2030 2035 2040 Asp Ala Asn Thr Thr Pro Gly Gly Val Thr Asp Val Ile Lys Asn 2045 2050 2055 Trp Asp Ser Leu Ile Pro Asp Glu Met Pro Asp Ser Pro Ile Gln 2060 2065 2070 Glu Lys Ser Glu Cys Gln Asp Met Ser Leu Ser Ser Pro Thr Ser 2075 2080 2085 Val Leu Gly Gly Ser Arg His Gln Ser His Thr Ala Glu Ala Gly 2090 2095 2100 Pro Arg Lys Ser Pro Val Gly Met Leu Asp Leu Ser Ser Trp Ser 2105 2110 2115 Ser Pro Glu Val Leu Arg Lys Asp Trp Thr Leu Glu Pro Trp Pro 2120 2125 2130 Ser Leu Pro Val Thr Pro His Ser Gly Ala Leu Ser Leu Cys Ser 2135 2140 2145 Ala Asp Thr Ser Leu Gly Asp Arg Ala Asp Thr Ser Leu Pro Gln 2150 2155 2160 Thr Gln Gly Pro Gly Leu Leu Cys Ser Pro Gly Val Ser Ala Ala 2165 2170 2175 Ala Leu Ala Leu Gln Trp Ala Glu Ser Pro Pro Ala Asp Asp His 2180 2185 2190 His Val Gln Arg Thr Ala Val Glu Lys Asp Val Glu Asp Phe Ile 2195 2200 2205 Thr Thr Ser Phe Asp Ser Gln Glu Thr Leu Ser Ser Pro Pro Pro 2210 2215 2220 Gly Leu Glu Gly Lys Ala Asp Arg Ser Glu Lys Ser Asp Gly Ser 2225 2230 2235 Gly Phe Gly Ala Arg Leu Ser Pro Gly Ser Gly Gly Pro Glu Ala 2240 2245 2250 Gln Thr Ala Gly Pro Val Thr Pro Ala Ser Ile Ser Gly Arg Phe 2255 2260 2265 Gln Pro Leu Pro Glu Ala Met Lys Glu Lys Glu Val Arg Pro Lys 2270 2275 2280 His Val Lys Ala Leu Leu Gln Met Val Arg Asp Glu Ser His Gln 2285 2290 2295 Ile Leu Ala Leu Ser Glu Gly Leu Ala Pro Pro Ser Gly Glu Pro 2300 2305 2310 His Pro Pro Arg Lys Glu Asp Glu Ile Gln Asp Ile Ser Leu His 2315 2320 2325 Gly Gly Lys Thr Gln Glu Val Pro Thr Ala Cys Pro Asp Trp Arg 2330 2335 2340 Gly Asp Leu Leu Gln Val Val Gln Glu Ala Phe Glu Lys Glu Gln 2345 2350 2355 Glu Met Gln Gly Val Glu Leu Gln Pro Arg Leu Ser Gly Ser Asp 2360 2365 2370 Leu Gly Gly His Ser Ser Leu Leu Glu Arg Leu Glu Lys Ile Ile 2375 2380 2385 Arg Glu Gln Gly Asp Leu Gln Glu Lys Ser Leu Glu His Leu Arg 2390 2395 2400 Leu Pro Asp Arg Ser Ser Leu Leu Ser Glu Ile Gln Ala Leu Arg 2405 2410 2415 Ala Gln Leu Arg Met Thr His Leu Gln Asn Gln Glu Lys Leu Gln 2420 2425 2430 His Leu Arg Thr Ala Leu Thr Ser Ala Glu Ala Arg Gly Ser Gln 2435 2440 2445 Gln Glu His Gln Leu Arg Arg Gln Val Glu Leu Leu Ala Tyr Lys 2450 2455 2460 Val Glu Gln Glu Lys Cys Ile Ala Gly Asp Leu Gln Lys Thr Leu 2465 2470 2475 Ser Glu Glu Gln Glu Lys Ala Asn Ser Val Gln Lys Leu Leu Ala 2480 2485 2490 Ala Glu Gln Thr Val Val Arg Asp Leu Lys Ser Asp Leu Cys Glu 2495 2500 2505 Ser Arg Gln Lys Ser Glu Gln Leu Ser Arg Ser Leu Cys Glu Val 2510 2515 2520 Gln Gln Glu Val Leu Gln Leu Arg Ser Met Leu Ser Ser Lys Glu 2525 2530 2535 Asn Glu Leu Lys Ala Ala Leu Gln Glu Leu Glu Ser Glu Gln Gly 2540 2545 2550 Lys Gly Arg Ala Leu Gln Ser Gln Leu Glu Glu Glu Gln Leu Arg 2555 2560 2565 His Leu Gln Arg Glu Ser Gln Ser Ala Lys Ala Leu Glu Glu Leu 2570 2575 2580 Arg Ala Ser Leu Glu Thr Gln Arg Ala Gln Ser Ser Arg Leu Cys 2585 ​​2590 2595 Val Ala Leu Lys His Glu Gln Thr Ala Lys Asp Asn Leu Gln Lys 2600 2605 2610 Glu Leu Arg Ile Glu His Ser Arg Cys Glu Ala Leu Leu Ala Gln 2615 2620 2625 Glu Arg Ser Gln Leu Ser Glu Leu Gln Lys Asp Leu Ala Ala Glu 2630 2635 2640 Lys Ser Arg Thr Leu Glu Leu Ser Glu Ala Leu Arg His Glu Arg 2645 2650 2655 Leu Leu Thr Glu Gln Leu Ser Gln Arg Thr Gln Glu Ala Cys Val 2660 2665 2670 His Gln Asp Thr Gln Ala His His Ala Leu Leu Gln Lys Leu Lys 2675 2680 2685 Glu Glu Lys Ser Arg Val Val Asp Leu Gln Ala Met Leu Glu Lys 2690 2695 2700 Val Gln Gln Gln Ala Leu His Ser Gln Gln Gln Leu Glu Ala Glu 2705 2710 2715 Ala Gln Lys His Cys Glu Ala Leu Arg Arg Glu Lys Glu Val Ser 2720 2725 2730 Ala Thr Leu Lys Ser Thr Val Glu Ala Leu His Thr Gln Lys Arg 2735 2740 2745 Glu Leu Arg Cys Ser Leu Glu Arg Glu Arg Glu Lys Pro Ala Trp 2750 2755 2760 Leu Gln Ala Glu Leu Glu Gln Ser His Pro Arg Leu Lys Glu Gln 2765 2770 2775 Glu Gly Arg Lys Ala Ala Arg Arg Ser Ala Glu Ala Arg Gln Ser 2780 2785 2790 Pro Ala Ala Ala Glu Gln Trp Arg Lys Trp Gln Arg Asp Lys Glu 2795 2800 2805 Lys Leu Arg Glu Leu Glu Leu Gln Arg Gln Arg Asp Leu His Lys 2810 2815 2820 Ile Lys Gln Leu Gln Gln Thr Val Arg Asp Leu Glu Ser Lys Asp 2825 2830 2835 Glu Val Pro Gly Ser Arg Leu His Leu Gly Ser Ala Arg Arg Ala 2840 2845 2850 Ala Gly Ser Asp Ala Asp His Leu Arg Glu Gln Gln Arg Glu Leu 2855 2860 2865 Glu Ala Met Arg Gln Arg Leu Leu Ser Ala Ala Arg Leu Leu Thr 2870 2875 2880 Ser Phe Thr Ser Gln Ala Val Asp Arg Thr Val Asn Asp Trp Thr 2885 2890 2895 Ser Ser Asn Glu Lys Ala Val Met Ser Leu Leu His Thr Leu Glu 2900 2905 2910 Glu Leu Lys Ser Asp Leu Ser Arg Pro Thr Ser Ser Gln Lys Lys 2915 2920 2925 Met Ala Ala Glu Leu Gln Phe Gln Phe Val Asp Val Leu Leu Lys 2930 2935 2940 Asp Asn Val Ser Leu Thr Lys Ala Leu Ser Thr Val Thr Gln Glu 2945 2950 2955 Lys Leu Glu Leu Ser Arg Ala Val Ser Lys Leu Glu Lys Leu Leu 2960 2965 2970 Lys His His Leu Gln Lys Gly Cys Ser Pro Ser Arg Ser Glu Arg 2975 2980 2985 Ser Ala Trp Lys Pro Asp Glu Thr Ala Pro Gln Ser Ser Leu Arg 2990 2995 3000 Arg Pro Asp Pro Gly Arg Leu Pro Pro Ala Ala Ser Glu Glu Ala 3005 3010 3015 His Thr Ser Asn Val Lys Met Glu Lys Leu Tyr Leu His Tyr Leu 3020 3025 3030 Arg Ala Glu Ser Phe Arg Lys Ala Leu Ile Tyr Gln Lys Lys Tyr 3035 3040 3045 Leu Leu Leu Leu Ile Gly Gly Phe Gln Asp Ser Glu Gln Glu Thr 3050 3055 3060 Leu Ser Met Ile Ala His Leu Gly Val Phe Pro Ser Lys Ala Glu 3065 3070 3075 Arg Lys Ile Thr Ser Arg Pro Phe Thr Arg Phe Arg Thr Ala Val 3080 3085 3090 Arg Val Val Ile Ala Ile Leu Arg Leu Arg Phe Leu Val Lys Lys 3095 3100 3105 Trp Gln Glu Val Asp Arg Lys Gly Ala Leu Ala Gln Gly Lys Ala 3110 3115 3120 Pro Arg Pro Gly Pro Arg Ala Arg Gln Pro Gln Ser Pro Pro Arg 3125 3130 3135 Thr Arg Glu Ser Pro Pro Thr Arg Asp Val Pro Ser Gly His Thr 3140 3145 3150 Arg Asp Pro Ala Arg Gly Arg Arg Leu Ala Ala Ala Ala Ser Pro 3155 3160 3165 His Ser Gly Gly Arg Ala Thr Pro Ser Pro Asn Ser Arg Leu Glu 3170 3175 3180 Arg Ser Leu Thr Ala Ser Gln Asp Pro Glu His Ser Leu Thr Glu 3185 3190 3195 Tyr Ile His His Leu Glu Val Ile Gln Gln Arg Leu Gly Gly Val 3200 3205 3210 Leu Pro Asp Ser Thr Ser Lys Lys Ser Cys His Pro Met Ile Lys 3215 3220 3225 Gln

Claims

1. A pharmaceutical composition for preventing or treating cerebral palsy, comprising as an active ingredient leukocytes in umbilical cord blood that express or secrete a polypeptide having the amino acid sequence of SEQ ID NO:

1.

2. The pharmaceutical composition of claim 1 , wherein the cord blood is autologous cord blood or allogeneic cord blood.

3. 10. The pharmaceutical composition of claim 1, further comprising erythropoietin (EPO).

Images