WO2018043937A1 - Composition for promoting peripheral nerve cell proliferation comprising protein secreted from mesenchymal stem cells - Google Patents

Composition for promoting peripheral nerve cell proliferation comprising protein secreted from mesenchymal stem cells Download PDF

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WO2018043937A1
WO2018043937A1 PCT/KR2017/008548 KR2017008548W WO2018043937A1 WO 2018043937 A1 WO2018043937 A1 WO 2018043937A1 KR 2017008548 W KR2017008548 W KR 2017008548W WO 2018043937 A1 WO2018043937 A1 WO 2018043937A1
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peripheral nerve
cells
mesenchymal stem
stem cells
cell
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French (fr)
Korean (ko)
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최병옥
장종욱
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사회복지법인 삼성생명공익재단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells

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  • the present invention relates to a composition for promoting peripheral nerve cell proliferation, including mesenchymal stem cell secretion protein.
  • the nervous system is largely divided into the central nervous system and the peripheral nervous system. Peripheral nervous system of the central nervous system, the brain and spinal cord and the peripheral body in contact with the nerve transmission.
  • the peripheral nervous system is classified into the somatic nervous system and the autonomic nervous system, the somatic nervous system is divided into the cranial nerve and spinal nerve.
  • the somatic nervous system is functionally divided into afferent or sensory nerve fibers and centrifugal or motility nerve fibers.
  • Afferent or sensory nerve fibers transmit nerve signals from the sensory receptors to the central nervous system.
  • Centrifugal or motility nerve fibers transmit nerve signals from the brain and spinal cord to muscles or glands.
  • Cerebral nerves which are peripheral nerves from the brain, are known in pairs and consist of sensory, motor, or mixed nerve fibers. These 12 pairs of nerve pairs are called optic nerve, optic nerve, middle nerve, trochlear nerve, trigeminal nerve, abduction nerve, facial nerve, inner ear nerve, Yeti nerve, vagus nerve, para nerve and sublingual nerve. Among these, nerves composed of sensory or mixed nerve fibers are known as the rear nerve, optic nerve, trigeminal nerve, facial nerve, inner ear nerve, Yeti nerve, and vagus nerve.
  • the left and right spinal nerves which are peripheral nerves generated from the spinal cord, are known to have 31 pairs on the left and right, and there are 8 pairs of nerves, 12 pairs of thoracic nerves, 5 pairs of lumbar nerves, 5 pairs of osteoclasts and 1 pair of coccyx nerves.
  • the spinal nerves are all composed of mixed nerve fibers, and include sensory fibers to the skin and motor fibers to the skeletal muscle.
  • Sensory nerve fibers or sensory nerves, are responsible for the precise delivery of stimuli, such as light, sound, temperature, or contact, received by sensory receptors such as visual, auditory, olfactory, taste and skin.
  • sensory receptors such as visual, auditory, olfactory, taste and skin.
  • Neural signals transmitted to the central nervous system are finally transmitted to each sensory field of the cerebral cortex, for example, the visual field and the auditory field, and the senses are normally recognized.
  • peripheral nerves may be damaged by the causes of viral infections, tumors, cancer, ischemia, trauma, compression, drugs or radiation therapy.
  • Symptoms include peripheral pain, numbness, burning sensation, lowering of the intrinsic angle of the joint, lowering of the vibration angle, pain, abnormal feeling, cold or burning.
  • peripheral nerve injury is classified as traumatic peripheral nerve injury, congenital peripheral nerve injury, inflammatory peripheral nerve injury, peripheral nerve injury due to toxicity, and other tumorous or idiopathic peripheral nerve injury.
  • Schwann cells are glia (glia or glial) cells capable of myelinizing nerves in the peripheral nervous system.
  • Schwann cells play an important role in nerve regeneration after nerve injury by forming a Bungner band.
  • Schwann cells secrete various growth factors or cytokines that promote regeneration of damaged peripheral nerve fibers.
  • transplantation of Schwann cells in the spinal cord injury model enhances nerve fiber regeneration and promotes functional recovery. Therefore, although transplantation of Schwann cells for nerve regeneration in the central nervous system has been proposed, this approach is problematic in clinical settings as surgical neural tissue cutting is required to obtain Schwann cells. It is also difficult to increase the number of Schwann cells in order to obtain therapeutic cell numbers.
  • the present inventors have made intensive efforts to develop a method for effectively proliferating Schwann cells, and when co-culturing the mesenchymal stem cells and Schwann cells, it was confirmed that the proteins secreted from the mesenchymal stem cells are useful for the proliferation of Schwann cells.
  • the present invention has been completed.
  • an object of the present invention is to provide a composition for promoting peripheral nerve cell proliferation and a method for promoting the proliferation of peripheral nerve cells using the same.
  • the present invention is to provide a peripheral neuronal cells promoted proliferation by the method and a cell therapy comprising the same.
  • the present invention provides a composition for promoting peripheral nerve cell proliferation, comprising a protein secreted from mesenchymal stem cells.
  • the present invention also provides a media composition for promoting peripheral nerve cell proliferation, comprising a protein secreted from mesenchymal stem cells.
  • the present invention also provides a method for promoting peripheral neuronal cell proliferation, comprising adding a protein secreted from mesenchymal stem cells to peripheral neuronal cell culture medium.
  • the present invention provides a peripheral nerve cell promoted by the above method.
  • the present invention also provides a cell therapy agent comprising the peripheral nerve cells.
  • Protein secreted from mesenchymal stem cells effectively proliferates peripheral nerve cells, particularly Schwann cells, and enables the mass production of peripheral nerve cells that can be used as cell therapy, thereby developing an effective therapeutic agent for neurological diseases. It is expected to be beneficial to.
  • FIG. 1 is a schematic diagram of co-culture of S19 Schwann cell line and Wharton's Jelly derived mesenchymal stem cell.
  • Figure 2 shows the results confirming the proliferation of Schwann cells by mesenchymal stem cells.
  • Figure 3 is a diagram confirming the protein secreted by the mesenchymal stem cells in the medium through a protein array.
  • Figure 4 is a diagram showing the effect on the proliferation of Schwann cells of Pref-1.
  • 5 is a diagram showing the protective effect of Schwann cells of Pref-1.
  • One aspect of the present invention provides a composition for promoting peripheral nerve cell proliferation, comprising a protein secreted from mesenchymal stem cells.
  • stem cell refers to an undifferentiated cell having the ability to differentiate into various body tissues, which are totipotent stem cells, pluripotent stem cells, and multipotent stem cells. can be classified as a multipotent stem cell.
  • the term “mesenchymal stem cell (MSC)” has the ability to differentiate into ectoderm cells, such as various mesodermal cells or neurons, including bone, cartilage, fat, and muscle cells. It is a multipotent stem cell.
  • the mesenchymal stem cells may preferably be derived from those selected from the group consisting of umbilical cord, placenta, umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, chorionic and exfoliating membranes.
  • the mesenchymal stem cells may be derived from mammals other than humans, fetuses or humans. Mammals other than humans may be more preferably canine, feline, ape, animal, cow, sheep, pig, horse, rat, mouse or guinea pig, and the like, without limitation.
  • the protein secreted from the mesenchymal stem cells is a protein with increased secretion in the medium when co-cultured with the mesenchymal stem cells and Schwann cell lines, the type is not limited.
  • Examples include Dtk, BMPR-1B (BMP receptor type IB) / ALK6, beta-Defensin 2, CXCR2 / IL-8 RB (Interleukin-8 receptor B), IFN-alpha / beta R1 (interferon- ⁇ / ⁇ receptor 1), FGF R4 (Fibroblast growth factor receptor 4), NeuroD1 (Neurogenic differentiation 1), CXCR6 (CXC chemokine receptor type 6), IL-12 (Interleukin 12) p40, Epirequlin, IL-1 (Interleukin 1) beta, IL -7R (Interleukin-7 receptor) alpha, BMP-15 (Bone morphogenetic protein 15), Pref-1 (Preadipocyte factor-1), MIP-1d (macrophage inflammatory protein-1D), FGF R3 (Fibroblast growth factor receptor 3) , CCR5 (CC chemokine receptor type 5), S100A10m, WIF-1 (WNT inhibitory factor 1), CD40 Ligand / TNFSF
  • the Pref-1 (Preadipocyte factor-1) protein is an epidermal growth factor (EGF) pseudo-repeat in the extracellular domain, a transmembrane protein with a juxtamembrane region, a single transmembrane domain, and a short cytoplasmic tail. to be.
  • Pref-1 is detected in 3T3-L1 preadipocytes, while it is known that the protein disappears after conversion to adipocytes.
  • Pref-1 may also be referred to as Delta-like protein 1 (Dlk1).
  • peripheral nerve cells the nervous system on the outside of the central nervous system (CNS) is called the peripheral nervous system (peripheral nervous system, PNS).
  • peripheral nervous system peripheral nervous system
  • the cell bodies of most neurons are located in the central nervous system, and the protrusions, ie, axons, from these cell bodies form the peripheral nervous system.
  • axons are surrounded by neuroglial cells and connective tissue sheaths.
  • One axon and the myelin sheath surrounding it are called nerve fibers, and the axons that are not enclosed by the myelin sheath are themselves nerve fibers.
  • the nerve fibers wrapped by the myelin sheath are called myelinated nerve fibers, and the nerve fibers not wrapped by the myelin sheath are called unmyelinated nerve fibers.
  • Myelin sheaths are formed by Schwann cells, one of the glial cells.
  • a number of flowing nerve fibers and anhydrous nerve fibers gather to form a bundle of nerve fibers, and the structure of the bundle of nerve fibers wrapped by the connective tissue membrane is a nerve observed with the naked eye.
  • the type of cells constituting the peripheral nervous system is not limited, but may be Schwann cells.
  • Pref-1 exerts the effect of specifically propagating Schwann cells.
  • another aspect of the present invention includes the step of adding a protein secreted from the mesenchymal stem cells to the peripheral nerve cell culture promoting medium composition and peripheral nerve cell culture medium comprising a protein secreted from the mesenchymal stem cells It also provides a method for promoting peripheral nerve cell proliferation.
  • Peripheral nerves particularly Schwann cells, are known to be very slow in proliferation, but when the protein is secreted from the mesenchymal stem cells according to the present invention, it was confirmed that the proliferation of the cells is promoted very effectively.
  • the culture medium is a basic medium used for culturing nerve cells, and may be a conventional medium known in the art, such as DMEM, MEM, K-SFM medium, and the like.
  • the culture medium may include antibiotics, generally penicillin / streptomycin is used, but is not limited thereto.
  • the culture medium may be supplemented with additives that are known in the art, which can promote the proliferation of cells.
  • the medium may also contain neutral buffers (such as phosphates and / or high concentrations of bicarbonate) and protein nutrients (such as essential and non-essential amino acids such as glutamine) in the isotonic solution.
  • lipids fatty acids, cholesterol, HDL or LDL extracts of serum
  • other components found in most preservative media of this kind (such as insulin or transferrin, nucleosides or nucleotides, pyruvate salts, any ionized form or salt)
  • Sugar sources such as glucose, selenium, glucocorticoids such as hydrocortisone and / or reducing agents such as ⁇ -mercaptoethanol.
  • the medium may also contain an anti-clumping agent or the like for the purpose of preventing cells from adhering to each other, adhering to the container wall, or forming too large a bundle.
  • Another aspect of the present invention provides a peripheral nerve promoted by the method and a cell therapy comprising the same.
  • Cell therapies are “a series of methods for proliferating, screening, or altering the biological properties of cells in vitro to autologous, allogenic, or xenogenic cells to restore the function of cells and tissues.
  • Drugs that are used for the purpose of treatment, diagnosis, or prevention through the act of "(Article 2 of 2008-78), Notification of Product Authorization Examination of Biological Products, etc., notified by KFDA”.
  • the cell therapy agent may be used for the treatment of neurological diseases.
  • Neurological diseases may be diseases due to peripheral nerve damage, in particular, and do not limit the kind thereof.
  • it may be peripheral nerve injury, central nerve injury, neuropathy, neuropathic pain or brain disease, and neuropathy or neuropathic pain is for example acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating Multiple peripheral neuropathy, diabetic peripheral neuropathy, shingles, vasculitis neuropathy, hereditary peripheral neuropathy, spinal cord cerebellar degeneration, muscular dystrophy, painful diabetic peripheral neuropathy, trigeminal neuralgia, cerebral cortex or spinal cord It may be selected from the group consisting of cancerous lesions, post-traumatic neuropathy, shingles neuralgia, ring pain, central pain after stroke and hypothalamus that occur accordingly.
  • the brain disease may be selected from, for example, stroke, brain tumor, dementia, Parkinson's disease, Alzheimer's disease, Huntinton's disease, epilepsy or ischemic brain disease.
  • Peripheral or central nerve injury may also be neuronal insufficiency and traumatic nerve injury.
  • the composition may include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers included in the composition are conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, fine Crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.
  • the pharmaceutical composition may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components.
  • the pharmaceutical composition for preventing or treating the neurological disease may be administered orally or parenterally.
  • parenteral administration it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration and rectal administration.
  • oral administration because proteins or peptides are digested, oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach.
  • the composition may be administered by any device in which the active substance may migrate to the target cell.
  • Suitable dosages of the pharmaceutical composition for the prophylaxis or treatment of neurological diseases are factors such as formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to response of the patient. It can be prescribed in various ways. Preferred dosages of the compositions are in the range of 100-100,000,000 (10 2 -10 8 ) cells / kg on an adult basis.
  • pharmaceutically effective amount means an amount sufficient to prevent or treat cancer or to prevent or treat a disease due to angiogenesis.
  • the composition may be prepared in unit dose form or formulated into a multi-dose container by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods readily available to those skilled in the art.
  • the formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
  • the composition may be administered as a separate therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be administered once or additionally if necessary.
  • MSC Mesenchymal stem cells
  • Schwann cell lines were co-cultured and analyzed for their proliferation rate.
  • Human umbilical cord-derived mesenchymal stem cells were used as mesenchymal stem cells.
  • S19 Schwann cell line in the lower chamber and Wharton's Jelly derived mesenchymal stem cells were placed in the lower chamber in the co-culture chamber and co-cultured.
  • the culture schematic is shown in FIG.
  • the recombinant protein of the candidate protein was treated with Schwann cells and other cells instead of stem cells to confirm cell proliferation. After processing each recombinant protein, intracellular signal transduction kinase was analyzed.
  • human umbilical cord-derived mesenchymal stem cells were confirmed to promote the proliferation of Schwann cells. That is, when umbilical cord-derived mesenchymal stem cells were co-cultured with Schwann cells and analyzed for the proliferation of Schwann cells, the proliferation of Schwann cells was promoted over time.
  • mesenchymal stem cells have a paracrine effect because they reduce Schwann cell death and promote proliferation. Therefore, by identifying proteins in the medium, identifying the increased protein in the co-culture group inhibits apoptosis. It is assumed that the effect can be explained. Accordingly, the protein was increased more than twice the secretion from umbilical cord-derived mesenchymal stem cells when co-cultured with umbilical cord stem cells through a protein array as shown in FIG.
  • Pref-1 protein which is expected to exhibit the highest activity
  • the protein was recombined, and treated with Schwann cells at various concentrations and times to measure Schwann cell proliferation and signal transduction.
  • Figure 4 is a diagram showing the effect on the proliferation of Schwann cells of Pref-1.
  • S16 Schwann cells are known to be very late proliferation cells, but the pref-1 protein was observed to promote the proliferation of Schwann cells by concentration.
  • Pref-1 Erk-1,2 kinase was activated while Akt was not changed.
  • Pref-1 affects the proliferation of Schwann cells specifically. As a result, it was confirmed that Pref-1 did not affect the proliferation of SHSY-5Y cells, which are neuroblastoma cells, and C2C12 cells, which are muscle cells, and found that Pref-1 specifically works on Schwann cells. It was.

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Abstract

The present invention relates to a composition for promoting peripheral nerve cell proliferation comprising a protein secreted from mesenchymal stem cells, and a medium composition for promoting peripheral nerve cell proliferation comprising the protein. Also, the present invention relates to a method for promoting peripheral nerve cell proliferation using the protein secreted from the mesenchymal stem cells, a peripheral nerve cell of which proliferation is promoted thereby, and a cell therapeutic agent comprising the peripheral nerve cells. The protein secreted from the mesenchymal stem cells according to the present invention can effectively proliferate the peripheral nerve cells, especially Schwann cells, and enables mass production of peripheral nerve cells that can be used as an agent for cell therapy, and thus, is expected to be effective in the development of a therapeutic agent effective for neurological diseases.

Description

중간엽 줄기세포 분비 단백질을 포함하는 말초신경 세포 증식 촉진용 조성물Peripheral nerve cell proliferation promoting composition comprising mesenchymal stem cell secretion protein
본 발명은 중간엽 줄기세포 분비 단백질을 포함하는, 말초신경 세포 증식 촉진용 조성물에 관한 것이다. The present invention relates to a composition for promoting peripheral nerve cell proliferation, including mesenchymal stem cell secretion protein.
신경계는 크게 중추신경계, 말초신경계로 구분된다. 그 중 말초신경계는 중추신경계인 뇌 및 척수와 신체 말초를 연락하며 신경전달을 담당한다.The nervous system is largely divided into the central nervous system and the peripheral nervous system. Peripheral nervous system of the central nervous system, the brain and spinal cord and the peripheral body in contact with the nerve transmission.
이러한 말초신경계는 체성신경계와 자율신경계로 분류되고, 체성신경계는 뇌신경과 척수신경으로 나누어진다. 또한, 체성신경계는 기능적으로 분류하면, 구심성 또는 감각성의 신경섬유와 원심성 또는 운동성의 신경섬유로 나뉜다. 구심성 또는 감각성 신경섬유는 감각수용기로부터 발생한 신경 신호를 중추신경에 전달하고, 원심성 또는 운동성의 신경섬유는 뇌·척수로부터 근육이나 분비선 등에 신경 신호를 전달하는 한다.The peripheral nervous system is classified into the somatic nervous system and the autonomic nervous system, the somatic nervous system is divided into the cranial nerve and spinal nerve. In addition, the somatic nervous system is functionally divided into afferent or sensory nerve fibers and centrifugal or motility nerve fibers. Afferent or sensory nerve fibers transmit nerve signals from the sensory receptors to the central nervous system. Centrifugal or motility nerve fibers transmit nerve signals from the brain and spinal cord to muscles or glands.
뇌로부터 나오는 말초신경인 뇌신경은 12쌍이 알려져 있고, 감각성, 운동성, 또는 혼합성의 신경섬유로 이루어져 있다. 이러한 12쌍의 신경쌍은 각각 후신경, 시신경, 동안신경, 활차신경, 삼차신경, 외전신경, 안면신경, 내이신경, 설인신경, 미주신경, 부신경, 설하신경이라 불린다. 이들 중, 감각성 또는 혼합성의 신경섬유로 이루어진 신경은 후신경, 시신경, 삼차신경, 안면신경, 내이신경, 설인신경, 미주신경이 알려져 있다.Cerebral nerves, which are peripheral nerves from the brain, are known in pairs and consist of sensory, motor, or mixed nerve fibers. These 12 pairs of nerve pairs are called optic nerve, optic nerve, middle nerve, trochlear nerve, trigeminal nerve, abduction nerve, facial nerve, inner ear nerve, Yeti nerve, vagus nerve, para nerve and sublingual nerve. Among these, nerves composed of sensory or mixed nerve fibers are known as the rear nerve, optic nerve, trigeminal nerve, facial nerve, inner ear nerve, Yeti nerve, and vagus nerve.
척수로부터 발생되는 말초신경인 척수신경은 좌우 31쌍이 알려져 있고, 8쌍의 경신경, 12쌍의 흉신경, 5쌍의 요신경, 5쌍의 선골신경과 1쌍의 미골신경이 알려져 있다. 척수신경은 전부 혼합성의 신경섬유로 이루어지고, 피부 등으로 가는 감각섬유와 골격근으로 가는 운동섬유를 포함하고 있다. The left and right spinal nerves, which are peripheral nerves generated from the spinal cord, are known to have 31 pairs on the left and right, and there are 8 pairs of nerves, 12 pairs of thoracic nerves, 5 pairs of lumbar nerves, 5 pairs of osteoclasts and 1 pair of coccyx nerves. The spinal nerves are all composed of mixed nerve fibers, and include sensory fibers to the skin and motor fibers to the skeletal muscle.
감각성의 신경섬유, 즉 감각신경은 시각기, 청각기, 후각기, 미각기 및 피부 등의 감각 수용기가 수취한 빛, 소리, 온도나 접촉 등의 자극을 중추신경계에 정확히 전달하는 기능을 담당하고 있다. 중추신경계에 전해진 신경 신호는 최종적으로는 대뇌피질의 각 감각야, 예컨대, 시각야, 청각야 등에 전달되며, 정상적으로 감각이 인식된다.Sensory nerve fibers, or sensory nerves, are responsible for the precise delivery of stimuli, such as light, sound, temperature, or contact, received by sensory receptors such as visual, auditory, olfactory, taste and skin. Neural signals transmitted to the central nervous system are finally transmitted to each sensory field of the cerebral cortex, for example, the visual field and the auditory field, and the senses are normally recognized.
그러나, 이러한 말초신경이 바이러스감염, 종양, 암, 허혈, 외상, 압박, 약물이나 방사선 요법 등의 원인에 의해 손상 받을 수 있다. 그 증상은 말초부의 자통, 저림, 작열감, 관절의 고유각 저하, 진동각 저하, 동통, 이상감각, 냉기 또는 화끈거림 등이 있다.However, these peripheral nerves may be damaged by the causes of viral infections, tumors, cancer, ischemia, trauma, compression, drugs or radiation therapy. Symptoms include peripheral pain, numbness, burning sensation, lowering of the intrinsic angle of the joint, lowering of the vibration angle, pain, abnormal feeling, cold or burning.
일반적으로 이러한 말초신경손상은 외상성 말초신경손상, 선천성 말초신경손상, 염증성 말초신경손상, 독성에의한 말초신경손상, 기타 종양성 혹은 특발성 말초신경손상으로 분류된다. Generally, such peripheral nerve injury is classified as traumatic peripheral nerve injury, congenital peripheral nerve injury, inflammatory peripheral nerve injury, peripheral nerve injury due to toxicity, and other tumorous or idiopathic peripheral nerve injury.
한편, 슈반세포(Schwann cells)는 말초신경계에서 신경을 수초화 할 수 있는 신경교(glia) (아교 또는 교) 세포이다. 슈반세포는 뷩너 띠(Bungner band)를 형성함으로써 신경 손상 후 신경 재생에 있어 중요한 역할을 한다. 더 나아가, 슈반세포는 손상된 말초 신경섬유의 재생을 촉진시키는 다양한 성장인자 또는 사이토카인(cytokine)을 분비한다. 게다가, 척수손상 모델에서 슈반세포의 이식은 신경섬유 재생을 강화하고 기능적 회복을 증진시킨다. 따라서, 중추신경계에서 신경 재생을 위한 슈반세포의 이식이 제안되어 왔었지만, 이러한 접근은 슈반세포를 얻기 위해 외과적인 신경 조직 절취가 필요하므로 임상적인 세팅에서는 문제시된다. 또한, 치료상의 세포수를 얻기 위해 슈반세포의 수를 늘리는 것도 어렵다.Schwann cells, on the other hand, are glia (glia or glial) cells capable of myelinizing nerves in the peripheral nervous system. Schwann cells play an important role in nerve regeneration after nerve injury by forming a Bungner band. Furthermore, Schwann cells secrete various growth factors or cytokines that promote regeneration of damaged peripheral nerve fibers. In addition, transplantation of Schwann cells in the spinal cord injury model enhances nerve fiber regeneration and promotes functional recovery. Therefore, although transplantation of Schwann cells for nerve regeneration in the central nervous system has been proposed, this approach is problematic in clinical settings as surgical neural tissue cutting is required to obtain Schwann cells. It is also difficult to increase the number of Schwann cells in order to obtain therapeutic cell numbers.
이에, 본 발명자들은 슈반세포를 효과적으로 증식하는 방법을 개발하기 위하여 예의 노력한 결과, 중간엽 줄기세포와 슈반세포를 공동배양하는 경우, 중간엽 줄기세포로부터 분비되는 단백질이 슈반세포의 증식에 유용함을 확인하고, 본 발명을 완성하기 이르렀다.Thus, the present inventors have made intensive efforts to develop a method for effectively proliferating Schwann cells, and when co-culturing the mesenchymal stem cells and Schwann cells, it was confirmed that the proteins secreted from the mesenchymal stem cells are useful for the proliferation of Schwann cells. The present invention has been completed.
따라서, 본 발명의 일 목적은 말초신경 세포 증식 촉진용 조성물 및 이를 이용한 말초신경 세포의 증식 촉진 방법을 제공하는 것이다. 또한 본 발명은 상기 방법에 의하여 증식이 촉진된 말초신경세포 및 이를 포함하는 세포치료제를 제공하는 것이다. Accordingly, an object of the present invention is to provide a composition for promoting peripheral nerve cell proliferation and a method for promoting the proliferation of peripheral nerve cells using the same. In another aspect, the present invention is to provide a peripheral neuronal cells promoted proliferation by the method and a cell therapy comprising the same.
본 발명은 중간엽 줄기세포에서 분비되는 단백질을 포함하는, 말초신경 세포 증식 촉진용 조성물을 제공한다. The present invention provides a composition for promoting peripheral nerve cell proliferation, comprising a protein secreted from mesenchymal stem cells.
또한 본 발명은 중간엽 줄기세포에서 분비되는 단백질을 포함하는, 말초신경 세포 증식 촉진용 배지 조성물을 제공한다. The present invention also provides a media composition for promoting peripheral nerve cell proliferation, comprising a protein secreted from mesenchymal stem cells.
또한 본 발명은 말초신경세포 배양 배지에 중간엽 줄기세포에서 분비되는 단백질을 첨가하는 단계를 포함하는, 말초신경 세포 증식의 촉진 방법을 제공한다. The present invention also provides a method for promoting peripheral neuronal cell proliferation, comprising adding a protein secreted from mesenchymal stem cells to peripheral neuronal cell culture medium.
또한 본 발명은 상기 방법에 의하여 증식이 촉진된 말초신경 세포를 제공한다. In another aspect, the present invention provides a peripheral nerve cell promoted by the above method.
또한 본 발명은 상기 말초 신경 세포를 포함하는 세포 치료제를 제공한다. The present invention also provides a cell therapy agent comprising the peripheral nerve cells.
본 발명에 따른 중간엽 줄기세포에서 분비되는 단백질은 말초신경 세포, 특히 슈반세포를 효과적으로 증식시키는 바, 세포치료제로 이용될 수 있는 말초신경 세포의 대량 생산을 가능하게 하므로, 신경질환의 효과적인 치료제 개발에 유익할 것으로 기대된다.Protein secreted from mesenchymal stem cells according to the present invention effectively proliferates peripheral nerve cells, particularly Schwann cells, and enables the mass production of peripheral nerve cells that can be used as cell therapy, thereby developing an effective therapeutic agent for neurological diseases. It is expected to be beneficial to.
도 1은 S19 슈반세포주와 Wharton's Jelly derived mesenchymal stem cell의 공동배양 모식도이다. 1 is a schematic diagram of co-culture of S19 Schwann cell line and Wharton's Jelly derived mesenchymal stem cell.
도 2는 중간엽 줄기세포에 의한 슈반세포의 증식 촉진을 확인한 결과이다.Figure 2 shows the results confirming the proliferation of Schwann cells by mesenchymal stem cells.
도 3은 배지내 중간엽 줄기세포가 분비한 단백질을 단백질 어레이를 통하여 확인한 도이다. Figure 3 is a diagram confirming the protein secreted by the mesenchymal stem cells in the medium through a protein array.
도 4는 Pref-1의 슈반세포의 증식에 대한 효과를 나타낸 도이다.Figure 4 is a diagram showing the effect on the proliferation of Schwann cells of Pref-1.
도 5는 Pref-1의 슈반세포 보호 효과를 나타낸 도이다.5 is a diagram showing the protective effect of Schwann cells of Pref-1.
본 발명의 일 양상은 중간엽 줄기세포에서 분비되는 단백질을 포함하는, 말초신경 세포 증식 촉진용 조성물를 제공한다. One aspect of the present invention provides a composition for promoting peripheral nerve cell proliferation, comprising a protein secreted from mesenchymal stem cells.
이하 본 발명에 대하여 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 명세서에서 사용된 용어 “줄기세포”는, 다양한 신체 조직으로 분화할수 있는 능력을 갖는 미분화 세포로서, 이는 만능 줄기 세포(totipotent stem cell), 전분화능 줄기세포 (pluripotent stem cell), 다분화능 줄기세포(multipotent stem cell)로 분류될 수 있다. The term “stem cell” as used herein refers to an undifferentiated cell having the ability to differentiate into various body tissues, which are totipotent stem cells, pluripotent stem cells, and multipotent stem cells. can be classified as a multipotent stem cell.
본 명세서에서 사용된 용어 “중간엽 줄기세포(mesenchymal stem cell, MSC)”는 뼈, 연골, 지방, 근육세포를 포함한 여러 가지 중배엽성 세포 또는 신경세포와 같은 외배엽성 세포로도 분화하는 능력을 가진 다분화능 줄기세포(multipotent stem cell)이다. 상기 중간엽 줄기세포는 바람직하게는 탯줄, 태반, 제대, 제대혈, 골수, 지방, 근육, 신경, 피부, 양막, 융모막 및 탈락막으로 구성된 군에서 선택되는 것으로부터 유래될 수 있다. 또한, 상기 중간엽 줄기세포는 인간, 태아 또는 인간을 제외한 포유동물로부터 유래될 수 있다. 상기 인간을 제외한 포유동물은 보다 바람직하게는 개과 동물, 고양이과 동물, 원숭이과 동물, 소, 양, 돼지, 말, 랫트, 마우스 또는 기니피그 등일 수 있으며, 그 유래를 제한하지 않는다.As used herein, the term “mesenchymal stem cell (MSC)” has the ability to differentiate into ectoderm cells, such as various mesodermal cells or neurons, including bone, cartilage, fat, and muscle cells. It is a multipotent stem cell. The mesenchymal stem cells may preferably be derived from those selected from the group consisting of umbilical cord, placenta, umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, chorionic and exfoliating membranes. In addition, the mesenchymal stem cells may be derived from mammals other than humans, fetuses or humans. Mammals other than humans may be more preferably canine, feline, ape, animal, cow, sheep, pig, horse, rat, mouse or guinea pig, and the like, without limitation.
본 발명에서 상기 중간엽 줄기세포에서 분비되는 단백질은 중간엽 줄기세포와 슈반세포주를 공동배양하였을 때, 배지에서 그 분비가 증가한 단백질로서, 그 종류를 제한하지 않는다.In the present invention, the protein secreted from the mesenchymal stem cells is a protein with increased secretion in the medium when co-cultured with the mesenchymal stem cells and Schwann cell lines, the type is not limited.
그 예로서, Dtk, BMPR-1B (BMP receptor type IB)/ALK6, beta-Defensin 2, CXCR2/IL-8 RB(Interleukin-8 receptor B), IFN-alpha/beta R1 (interferon-α/β receptor 1), FGF R4 (Fibroblast growth factor receptor 4), NeuroD1 (Neurogenic differentiation 1), CXCR6 (C-X-C chemokine receptor type 6), IL-12 (Interleukin 12) p40, Epirequlin, IL-1 (Interleukin 1) beta, IL-7R (Interleukin-7 receptor) alpha, BMP-15 (Bone morphogenetic protein 15), Pref-1 (Preadipocyte factor-1), MIP-1d (macrophage inflammatory protein-1D), FGF R3 (Fibroblast growth factor receptor 3), CCR5 (C-C chemokine receptor type 5), S100A10m, WIF-1 (WNT inhibitory factor 1), CD40 Ligand/TNFSF5/CD154, SDF-1 (stromal cell-derived factor 1)/CXCL12, Dance, 또는 Endocan 일 수 있으며, 바람직하게는 Pref-1일 수 있다.Examples include Dtk, BMPR-1B (BMP receptor type IB) / ALK6, beta-Defensin 2, CXCR2 / IL-8 RB (Interleukin-8 receptor B), IFN-alpha / beta R1 (interferon-α / β receptor 1), FGF R4 (Fibroblast growth factor receptor 4), NeuroD1 (Neurogenic differentiation 1), CXCR6 (CXC chemokine receptor type 6), IL-12 (Interleukin 12) p40, Epirequlin, IL-1 (Interleukin 1) beta, IL -7R (Interleukin-7 receptor) alpha, BMP-15 (Bone morphogenetic protein 15), Pref-1 (Preadipocyte factor-1), MIP-1d (macrophage inflammatory protein-1D), FGF R3 (Fibroblast growth factor receptor 3) , CCR5 (CC chemokine receptor type 5), S100A10m, WIF-1 (WNT inhibitory factor 1), CD40 Ligand / TNFSF5 / CD154, SDF-1 (stromal cell-derived factor 1) / CXCL12, Dance, or Endocan , Preferably Pref-1.
상기 Pref-1 (Preadipocyte factor-1) 단백질은 세포외 도메인에 표피 성장인자 (EGF) 유사-반복절, 막근접 부위 (juxtamembrane region), 단일 막통과 도메인, 및 짧은 세포질막 꼬리를 갖는 막통과 단백질이다. Pref-1는 3T3-L1 지방전구세포 (preadipocyte)에서 검출되는 반면, 지방세포로 전환된 후에는 그 발현이 사라지는 단백질로 알려져 있다. Pref-1 은 Dlk1(Delta-like protein 1) 으로도 지칭될 수 있다. The Pref-1 (Preadipocyte factor-1) protein is an epidermal growth factor (EGF) pseudo-repeat in the extracellular domain, a transmembrane protein with a juxtamembrane region, a single transmembrane domain, and a short cytoplasmic tail. to be. Pref-1 is detected in 3T3-L1 preadipocytes, while it is known that the protein disappears after conversion to adipocytes. Pref-1 may also be referred to as Delta-like protein 1 (Dlk1).
상기 말초신경세포이란, 중추신경계 (CNS)의 바깥쪽에 있는 신경계를 말초신경계 (peripheral nervous system, PNS) 라고 한다. The peripheral nerve cells, the nervous system on the outside of the central nervous system (CNS) is called the peripheral nervous system (peripheral nervous system, PNS).
대부분의 신경원의 세포체는 중추신경계 내에 위치해 있으며, 이 세포체에서 나오는 돌기, 즉 축삭 (axon)이 말초신경계를 형성한다. 말초신경계에서 축삭은 신경아교세포 (neuroglial cell)와 결합조직막 (connective tissue sheath)에 의해 싸여 있다. 하나의 축삭과 이를 둘러싼 수초 (myelin sheath)를 신경섬유 (nerve fiber)라고 하며 수초에 의해 싸여있지 않은 축삭은 그 자체가 하나의 신경섬유이다. 수초에 의해 싸여 있는 신경섬유를 유수신경섬유 (myelinated nerve fiber)라고 하며, 수초에 의해 싸여 있지 않는 신경섬유를 무수신경섬유 (unmyelinated nerve fiber)라고 한다. 수초는 신경아교세포의 하나인 슈반세포 (Schwann cell)에 의해 형성된다. 여러 개의 유수신경섬유와 무수신경섬유가 모여 신경섬유의 다발을 형성하며, 이러한 신경섬유의 다발이 결합조직막에 의해 싸여 있는 구조가 육안으로 관찰되는 신경 (nerve)이다. 상기 말초신경계를 구성하는 세포의 종류를 제한하지 않으나, 바람직하게는 슈반세포일 수 있다. The cell bodies of most neurons are located in the central nervous system, and the protrusions, ie, axons, from these cell bodies form the peripheral nervous system. In the peripheral nervous system, axons are surrounded by neuroglial cells and connective tissue sheaths. One axon and the myelin sheath surrounding it are called nerve fibers, and the axons that are not enclosed by the myelin sheath are themselves nerve fibers. The nerve fibers wrapped by the myelin sheath are called myelinated nerve fibers, and the nerve fibers not wrapped by the myelin sheath are called unmyelinated nerve fibers. Myelin sheaths are formed by Schwann cells, one of the glial cells. A number of flowing nerve fibers and anhydrous nerve fibers gather to form a bundle of nerve fibers, and the structure of the bundle of nerve fibers wrapped by the connective tissue membrane is a nerve observed with the naked eye. The type of cells constituting the peripheral nervous system is not limited, but may be Schwann cells.
특히, 상기 중간엽 줄기세포에서 분비되는 단백질 중 Pref-1은 슈반세포를 특이적으로 증식하는 효과를 발휘한다. In particular, among the proteins secreted from the mesenchymal stem cells, Pref-1 exerts the effect of specifically propagating Schwann cells.
또한, 본 발명의 다른 양상은 중간엽 줄기세포에서 분비되는 단백질을 포함하는, 말초신경 세포 증식 촉진용 배지 조성물 및 말초신경세포 배양 배지에 중간엽 줄기세포에서 분비되는 단백질을 첨가하는 단계를 포함하는, 말초신경 세포 증식의 촉진 방법을 제공한다. In addition, another aspect of the present invention includes the step of adding a protein secreted from the mesenchymal stem cells to the peripheral nerve cell culture promoting medium composition and peripheral nerve cell culture medium comprising a protein secreted from the mesenchymal stem cells It also provides a method for promoting peripheral nerve cell proliferation.
말초신경, 특히 슈반세포의 경우, 증식이 매우 더딘 세포로 알려져 있으나, 본 발명에 따른 중간엽 줄기세포에서 분비되는 단백질을 처리하는 경우, 세포의 증식이 매우 효과적으로 촉진됨을 확인하였다.Peripheral nerves, particularly Schwann cells, are known to be very slow in proliferation, but when the protein is secreted from the mesenchymal stem cells according to the present invention, it was confirmed that the proliferation of the cells is promoted very effectively.
상기 배양 배지는 신경세포를 배양하는데 사용되는 기본 배지로서, 당업계에서 신경세포 배양에 적합하다고 알려져 있는 통상적인 배지, 예를 들면 DMEM, MEM, K-SFM 배지 등을 사용할 수 있다. The culture medium is a basic medium used for culturing nerve cells, and may be a conventional medium known in the art, such as DMEM, MEM, K-SFM medium, and the like.
또한, 상기 배양 배지에는 항생제가 포함될 수 있으며, 일반적으로 페니실린/스트렙토마이신을 사용하지만 이에 제한되지 않는다. In addition, the culture medium may include antibiotics, generally penicillin / streptomycin is used, but is not limited thereto.
또한, 상기 배양배지는 당업계에 공지된, 세포의 증식을 촉진할 수 있는 첨가제가 보충될 수 있다. 또한, 배지는 등장액 중의 중성 완충제(예컨대 인산염 및/또는 고농도 중탄산염) 및 단백질 영양분(예를 들면 필수 아미노산 및 비필수 아미노산, 예컨대 글루타민)을 함유할 수 있다. 나아가, 지질(지방산, 콜레스테롤, 혈청의 HDL 또는 LDL 추출물) 및 이 종류의 대부분의 보존액 배지에서 발견되는 기타 성분(예컨대 인슐린 또는 트랜스페린, 뉴클레오시드 또는 뉴클레오티드, 피루빈산염, 임의의 이온화 형태 또는 염인 당원, 예컨대 글루코스, 셀레늄, 글루코코르티코이드, 예컨대 히드로코르티존 및/또는 환원제, 예컨대 β-메르캅토에탄올)을 함유할 수 있다. 또한, 배지는 세포가 서로 유착하거나, 용기벽에 유착하거나, 너무 큰 다발을 형성하는 것을 방지할 목적으로, 항응집제 (anti-clumping agent) 등을 포함할 수도 있다.In addition, the culture medium may be supplemented with additives that are known in the art, which can promote the proliferation of cells. The medium may also contain neutral buffers (such as phosphates and / or high concentrations of bicarbonate) and protein nutrients (such as essential and non-essential amino acids such as glutamine) in the isotonic solution. Furthermore, lipids (fatty acids, cholesterol, HDL or LDL extracts of serum) and other components found in most preservative media of this kind (such as insulin or transferrin, nucleosides or nucleotides, pyruvate salts, any ionized form or salt) Sugar sources such as glucose, selenium, glucocorticoids such as hydrocortisone and / or reducing agents such as β-mercaptoethanol. The medium may also contain an anti-clumping agent or the like for the purpose of preventing cells from adhering to each other, adhering to the container wall, or forming too large a bundle.
본 발명의 다른 양상은 상기 방법에 의하여 증식이 촉진된 말초신경 및 이를 포함하는 세포치료제를 제공한다. Another aspect of the present invention provides a peripheral nerve promoted by the method and a cell therapy comprising the same.
세포치료제란 “세포와 조직의 기능을 복원하기 위하여 살아있는 자가(autologous), 동종(allogenic), 혹은 이종(xenogenic)의 세포를 체외에서 증식, 선별하거나 여러 가지 방법으로 세포의 생물학적 특성을 변화시키는 일련의 행위를 통하여 치료, 진단, 예방의 목적으로 사용하는 의약품 (KFDA가 고시한 생물학적 제재 등의 품목허가 심사고시(2008-78호) 제 2조)"을 의미한다.Cell therapies are “a series of methods for proliferating, screening, or altering the biological properties of cells in vitro to autologous, allogenic, or xenogenic cells to restore the function of cells and tissues. Drugs that are used for the purpose of treatment, diagnosis, or prevention through the act of "(Article 2 of 2008-78), Notification of Product Authorization Examination of Biological Products, etc., notified by KFDA".
상기 세포치료제는 신경질환의 치료에 이용될 수 있다. 신경질환은 특히 말초신경 손상에 기인한 질환일 수 있으며, 그 종류를 제한하지 않는다. 예를 들어, 말초신경손상, 중추신경손상, 신경병증, 신경병증성 통증 또는 뇌질환일 수 있으며, 신경병증 또는 신경병증성 통증은 예를 들면 급성 염증성 탈수초성 다발성말초신경병증, 만성 염증성 탈수초성 다발성말초신경병증, 당뇨병성 말초신경병증, 대상포진, 혈관염신경병증, 유전성 말초신경병증, 척수 소뇌 변성증, 근위축성 측색경화증, 통증성 당뇨병성 말초 신경병증, 삼차 신경통, 대뇌 피질이나 척수시 상로를 따라 발생하는 암성 병변, 외상 후 신경병증, 대상포진후신경통, 환지통, 뇌졸중 후의 중추성 통증 및 시상통으로 이루어진 군에서 선택된 것일 수 있다. 뇌질환은 예를 들면 뇌졸중, 뇌종양, 치매, 파킨슨병, 알츠하이머병, 헌틴톤병, 간질 또는 허혈성 뇌질환에서 선택된 것일 수 있다. 또한, 말초신경손상 또는 중추신경손상은 신경단열증 및 외상성 신경손상일 수 있다. The cell therapy agent may be used for the treatment of neurological diseases. Neurological diseases may be diseases due to peripheral nerve damage, in particular, and do not limit the kind thereof. For example, it may be peripheral nerve injury, central nerve injury, neuropathy, neuropathic pain or brain disease, and neuropathy or neuropathic pain is for example acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating Multiple peripheral neuropathy, diabetic peripheral neuropathy, shingles, vasculitis neuropathy, hereditary peripheral neuropathy, spinal cord cerebellar degeneration, muscular dystrophy, painful diabetic peripheral neuropathy, trigeminal neuralgia, cerebral cortex or spinal cord It may be selected from the group consisting of cancerous lesions, post-traumatic neuropathy, shingles neuralgia, ring pain, central pain after stroke and hypothalamus that occur accordingly. The brain disease may be selected from, for example, stroke, brain tumor, dementia, Parkinson's disease, Alzheimer's disease, Huntinton's disease, epilepsy or ischemic brain disease. Peripheral or central nerve injury may also be neuronal insufficiency and traumatic nerve injury.
상기 조성물이 신경질환의 치료용 약학적 조성물로 제조되는 경우, 상기 조성물은 약학적으로 허용되는 담체를 포함할 수 있다. 상기 조성물에 포함되는 약학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 상기 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.When the composition is made of a pharmaceutical composition for treating neurological diseases, the composition may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the composition are conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, fine Crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. The pharmaceutical composition may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components.
상기 신경질환의 예방 또는 치료용 약학적 조성물은 경구 또는 비경구로 투여할 수 있다. 비경구 투여인 경우에는 정맥내 주입, 피하 주입, 근육 주입, 복강 주입, 내피 투여, 국소 투여, 비내 투여, 폐내 투여 및 직장내 투여 등으로 투여할 수 있다. 경구 투여시, 단백질 또는 펩타이드는 소화가 되기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화 되어야 한다. 또한, 상기 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.The pharmaceutical composition for preventing or treating the neurological disease may be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration and rectal administration. In oral administration, because proteins or peptides are digested, oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the composition may be administered by any device in which the active substance may migrate to the target cell.
상기 신경질환의 예방 또는 치료용 약학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 상기 조성물의 바람직한 투여량은 성인 기준으로 100-100,000,000 (102-108) cell/kg 범위 내이다. 용어 "약학적 유효량"은 암을 예방 또는 치료하는 데, 또는 혈관신생으로 인한 질환의 예방 또는 치료하는 데 충분한 양을 의미한다.Suitable dosages of the pharmaceutical composition for the prophylaxis or treatment of neurological diseases are factors such as formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to response of the patient. It can be prescribed in various ways. Preferred dosages of the compositions are in the range of 100-100,000,000 (10 2 -10 8 ) cells / kg on an adult basis. The term "pharmaceutically effective amount" means an amount sufficient to prevent or treat cancer or to prevent or treat a disease due to angiogenesis.
상기 조성물은 당해 당업자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액, 시럽제 또는 유화액 형태이거나 엑스제, 산제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다. 또한, 상기 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 또한 단회 또는 필요시 추가 투여될 수 있다.The composition may be prepared in unit dose form or formulated into a multi-dose container by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods readily available to those skilled in the art. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers. In addition, the composition may be administered as a separate therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be administered once or additionally if necessary.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실시예 1. 실험방법Example 1 Experimental Method
중간엽 줄기세포(MSC)와 슈반세포주를 공동배양하고 그 증식률을 분석하였다. 중간엽 줄기세포로는 사람의 탯줄유래 중간엽 줄기세포를 사용하였다.Mesenchymal stem cells (MSC) and Schwann cell lines were co-cultured and analyzed for their proliferation rate. Human umbilical cord-derived mesenchymal stem cells were used as mesenchymal stem cells.
구체적으로 공동배양챔버에서 하부챔버에는 S19 슈반세포주를, 상부챔버에는 Wharton's Jelly derived mesenchymal stem cell을 위치시키고 이를 공동배양 하였다. 그 배양 모식도를 도 1에 나타내었다.Specifically, S19 Schwann cell line in the lower chamber and Wharton's Jelly derived mesenchymal stem cells were placed in the lower chamber in the co-culture chamber and co-cultured. The culture schematic is shown in FIG.
공동배양 후 하부챔버의 슈반세포 증식정도를 세포 계수를 통하여 분석하였다. 이 때의 각 배지를 공동챔버에서 수확하여 농축한 후 protein array로 분석하여 MSC에서 분비가 증가하는 단백질을 동정하였다. After coculture, Schwann cell proliferation in the lower chamber was analyzed by cell count. At this time, each medium was harvested in a cochamber, concentrated, and analyzed by protein array to identify proteins with increased secretion in MSC.
또한, 후보 단백질의 재조합 단백질을 줄기세포 대신 슈반세포 및 타 세포에 처리하여 세포증식유도를 확인하였으며, 각 재조합 단백질을 처리 후 세포 내 신호전달 인산화 효소를 분석하였다. In addition, the recombinant protein of the candidate protein was treated with Schwann cells and other cells instead of stem cells to confirm cell proliferation. After processing each recombinant protein, intracellular signal transduction kinase was analyzed.
실시예 2. 결과 Example 2. Results
실시예 2.1 - 중간엽 줄기세포주의 공동배양에 따른 슈반세포의 증식 촉진 효과 Example 2.1- Mesenchyme Proliferation-promoting Effect of Schwann Cells by Stem Cell Co-culture
하부챔버에는 S19 슈반세포주를, 상부챔버에는 Wharton's Jelly derived mesenchymal stem cell을 위치시키고 이를 공동배양하였으며, 세포를 계수하여 슈반세포의 증식정도를 판단하였다. 그 결과를 도 2에 나타내었다. S19 Schwann cell lines were placed in the lower chamber and Wharton's Jelly derived mesenchymal stem cells were placed and co-cultured in the upper chamber, and the proliferation of Schwann cells was determined by counting the cells. The results are shown in FIG.
도 2에 나타난 바와 같이, 사람의 탯줄유래 중간엽 줄기세포는 슈반 세포의 증식을 촉진시킴을 확인하였다. 즉, 탯줄유래 중간엽 줄기세포를 슈반세포와 공동배양 후 슈반세포의 증식을 분석했을 때, 슈반세포의 증식이 시간이 지남에 따라 촉진되었다. As shown in Figure 2, human umbilical cord-derived mesenchymal stem cells were confirmed to promote the proliferation of Schwann cells. That is, when umbilical cord-derived mesenchymal stem cells were co-cultured with Schwann cells and analyzed for the proliferation of Schwann cells, the proliferation of Schwann cells was promoted over time.
실시예 2.2 - 중간엽 줄기세포주가 분비하는 치료 효능 단백질의 동정Example 2.2 Identification of Therapeutic Efficacy Proteins Secreted by Mesenchymal Stem Cell Lines
중간엽 줄기세포가 슈반세포 사멸을 감소시키고 증식을 촉진시키므로 측분비 (paracrine)효과를 보인다고 설명할 수 있으며, 이에 따라 배지 속의 단백질을 분석하여 공동배양 군에서 증가하는 단백질을 동정하는 것이 세포사멸 억제효과를 설명할 수 있다고 가정하였다. 이에 따라서, 단백질 어레이를 통하여 탯줄 줄기세포와 공동배양 시 탯줄유래 중간엽 줄기세포에서 분비가 2배 이상 늘어난 단백질을 도 3과 같이 분석하였다. It can be explained that mesenchymal stem cells have a paracrine effect because they reduce Schwann cell death and promote proliferation. Therefore, by identifying proteins in the medium, identifying the increased protein in the co-culture group inhibits apoptosis. It is assumed that the effect can be explained. Accordingly, the protein was increased more than twice the secretion from umbilical cord-derived mesenchymal stem cells when co-cultured with umbilical cord stem cells through a protein array as shown in FIG.
그 결과, 총 23종의 발현증가 단백질을 확인하였으며, 그 중 가장 높은 활성을 나타낼 것으로 예상되는 Pref-1 단백질을 선정하였다.As a result, a total of 23 expression increasing proteins were identified, and Pref-1 protein, which is expected to exhibit the highest activity, was selected.
실시예 2.3 - Pref-1 단백질의 슈반세포 증식촉진 효과 확인Example 2.3-Confirmation of Schwann cell proliferation promoting effect of Pref-1 protein
상기 동정된 단백질 중 Pref-1 단백질의 슈반세포 증식촉진 효과를 확인하였다. It was confirmed that Schwann cell proliferation promoting effect of Pref-1 protein among the identified proteins.
또한, 상기 단백질의 직접적 효과를 확인하기 위하여, 상기 단백질을 재조합하고, 이를 다양한 농도와 시간별로 슈반세포에 처리하여 슈반세포 증식과 신호 전달을 측정하였다. In addition, in order to confirm the direct effect of the protein, the protein was recombined, and treated with Schwann cells at various concentrations and times to measure Schwann cell proliferation and signal transduction.
그 결과를 도 4에 나타내었다. The results are shown in FIG.
도 4는 Pref-1의 슈반세포의 증식에 대한 효과를 나타낸 도이다.Figure 4 is a diagram showing the effect on the proliferation of Schwann cells of Pref-1.
도 4에 나타난 바와 같이, S16 슈반세포는 증식이 매우 늦은 세포로 알려져 있으나 Pref-1 단백질을 농도별로 처리하면 슈반세포의 증식을 촉진함이 관찰되었다. Pref-1의 경우는 Erk-1,2 kinase를 활성화시키는 반면 Akt 에는 변화가 없음을 확인하였다. As shown in Figure 4, S16 Schwann cells are known to be very late proliferation cells, but the pref-1 protein was observed to promote the proliferation of Schwann cells by concentration. In the case of Pref-1, Erk-1,2 kinase was activated while Akt was not changed.
또한, Pref-1이 슈반세포 특이적으로 그 증식에 영향을 미치는지를 확인하였다. 그 결과, Pref-1는 신경아세포종 (neuroblastoma cell)인 SHSY-5Y 세포와 근육세포인 C2C12세포의 증식에 영향을 미치지 못함을 확인하였는 바, Pref-1이 슈반세포에 특이적으로 작용함을 확인하였다. In addition, it was confirmed whether Pref-1 affects the proliferation of Schwann cells specifically. As a result, it was confirmed that Pref-1 did not affect the proliferation of SHSY-5Y cells, which are neuroblastoma cells, and C2C12 cells, which are muscle cells, and found that Pref-1 specifically works on Schwann cells. It was.

Claims (10)

  1. 중간엽 줄기세포에서 분비되는 단백질을 포함하는, 말초신경 세포 증식 촉진용 조성물.A composition for promoting peripheral nerve cell proliferation, comprising a protein secreted from mesenchymal stem cells.
  2. 청구항 1에 있어서,The method according to claim 1,
    상기 중간엽 줄기세포에서 분비되는 단백질은 Dtk, BMPR-1B (BMP receptor type IB)/ALK6, beta-Defensin 2, CXCR2/IL-8 RB(Interleukin-8 receptor B), IFN-alpha/beta R1 (interferon-α/β receptor 1), FGF R4 (Fibroblast growth factor receptor 4), NeuroD1 (Neurogenic differentiation 1), CXCR6 (C-X-C chemokine receptor type 6), IL-12 (Interleukin 12) p40, Epirequlin, IL-1 (Interleukin 1) beta, IL-7R (Interleukin-7 receptor) alpha, BMP-15 (Bone morphogenetic protein 15), Pref-1 (Preadipocyte factor-1), MIP-1d (macrophage inflammatory protein-1D), FGF R3 (Fibroblast growth factor receptor 3), CCR5 (C-C chemokine receptor type 5), S100A10m, WIF-1 (WNT inhibitory factor 1), CD40 Ligand/TNFSF5/CD154, SDF-1 (stromal cell-derived factor 1)/CXCL12, Dance, 및 Endocan으로 이루어진 군으로부터 선택된 1종 이상인, 말초신경 세포 증식 촉진용 조성물.Proteins secreted from the mesenchymal stem cells include Dtk, BMPR-1B (BMP receptor type IB) / ALK6, beta-Defensin 2, CXCR2 / IL-8 RB (Interleukin-8 receptor B), IFN-alpha / beta R1 ( interferon-α / β receptor 1), FGF R4 (Fibroblast growth factor receptor 4), NeuroD1 (Neurogenic differentiation 1), CXCR6 (CXC chemokine receptor type 6), IL-12 (Interleukin 12) p40, Epirequlin, IL-1 ( Interleukin 1) beta, IL-7R (Interleukin-7 receptor) alpha, BMP-15 (Bone morphogenetic protein 15), Pref-1 (Preadipocyte factor-1), MIP-1d (macrophage inflammatory protein-1D), FGF R3 ( Fibroblast growth factor receptor 3), CCR5 (CC chemokine receptor type 5), S100A10m, WIF-1 (WNT inhibitory factor 1), CD40 Ligand / TNFSF5 / CD154, SDF-1 (stromal cell-derived factor 1) / CXCL12, Dance And Endocan is at least one selected from the group consisting of, peripheral nerve cell proliferation promoting composition.
  3. 청구항 1에 있어서, 상기 말초신경 세포는 슈반세포인 것인, 말초신경 세포 증식 촉진용 조성물.The composition of claim 1, wherein the peripheral nerve cells are Schwann cells.
  4. 청구항 3에 있어서, 상기 중간엽 줄기세포에서 분비되는 단백질은 슈반세포 특이적으로 세포 증식을 촉진하는 것을 특징으로 하는, 말초신경 세포 증식 촉진용 조성물.The composition of claim 3, wherein the protein secreted from the mesenchymal stem cells promotes cell proliferation specifically Schwann cells.
  5. 청구항 1에 있어서, 상기 중간엽 줄기세포는 탯줄, 태반, 제대, 제대혈, 골수, 지방, 근육, 신경, 피부, 양막, 융모막, 및 탈락막으로 구성된 군에서 선택되는 것으로부터 유래된 것인, 말초신경 세포 증식 촉진용 조성물.The peripheral cell of claim 1, wherein the mesenchymal stem cells are derived from one selected from the group consisting of umbilical cord, placenta, umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, chorion, and decidual membrane. Neuron cell growth promoting composition.
  6. 중간엽 줄기세포에서 분비되는 단백질을 포함하는, 말초신경 세포 증식 촉진용 배지 조성물.A medium composition for promoting peripheral nerve cell proliferation, comprising a protein secreted from mesenchymal stem cells.
  7. 말초신경세포 배양 배지에 중간엽 줄기세포에서 분비되는 단백질을 첨가하는 단계를 포함하는, 말초신경 세포 증식의 촉진 방법.A method of promoting peripheral nerve cell proliferation, comprising adding a protein secreted from mesenchymal stem cells to peripheral nerve cell culture medium.
  8. 청구항 7의 방법에 의하여 증식이 촉진된 말초신경 세포.Peripheral nerve cell promoted proliferation by the method of claim 7.
  9. 청구항 8의 말초신경 세포를 포함하는 세포치료제. Cell therapy comprising the peripheral nerve cells of claim 8.
  10. 청구항 9에 있어서, 상기 세포치료제는 신경질환 치료용인, 세포치료제.The cell therapy agent according to claim 9, wherein the cell therapy agent is for treating neurological diseases.
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