KR20240057300A - Use for regulating calcium homeostasis in microglia of astrocytes - Google Patents
Use for regulating calcium homeostasis in microglia of astrocytes Download PDFInfo
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- KR20240057300A KR20240057300A KR1020220175714A KR20220175714A KR20240057300A KR 20240057300 A KR20240057300 A KR 20240057300A KR 1020220175714 A KR1020220175714 A KR 1020220175714A KR 20220175714 A KR20220175714 A KR 20220175714A KR 20240057300 A KR20240057300 A KR 20240057300A
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- astrocytes
- microglial cells
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- calcium concentration
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Abstract
본 발명은 별아교세포의 미세아교세포 내 칼슘 항상성 조절 용도; 인 비트로(in vitro) 상에서 별아교세포를 이용하여 활성화된 미세아교세포 내 칼슘 농도 상승을 억제하는 방법; 및 이를 이용한 미세아교세포 내 칼슘 항상성 기능 이상으로 유발되는 신경계 질환 관련 물질의 스크리닝 방법에 관한 것이다. 본 발명의 조성물은 유효성분으로 별아교세포를 포함함으로써 미세아교세포 내 칼슘 항상성을 효과적으로 조절할 수 있는바, 미세아교세포 내 칼슘 항상성 기능 이상으로 유발되는 다양한 신경계 질환을 연구하기 위한 목적이나, 신경계 질환을 예방 또는 치료하기 위한 목적으로 유용하게 사용될 수 있다.The present invention relates to the use of regulating calcium homeostasis within microglial cells of astrocytes; A method of suppressing the increase in calcium concentration in activated microglia using astrocytes in vitro ; and a method for screening substances related to nervous system diseases caused by dysfunction of calcium homeostasis in microglial cells using the same. The composition of the present invention can effectively control calcium homeostasis within microglial cells by containing astrocytes as an active ingredient, and is intended to study various neurological diseases caused by abnormal calcium homeostasis function within microglial cells. It can be usefully used for prevention or treatment.
Description
본 발명은 별아교세포의 미세아교세포 내 칼슘 항상성 조절 용도; 인 비트로(in vitro) 상에서 별아교세포를 이용하여 활성화된 미세아교세포 내 칼슘 농도 상승을 억제하는 방법; 및 이를 이용한 미세아교세포 내 칼슘 항상성 기능 이상으로 유발되는 신경계 질환 관련 물질의 스크리닝 방법에 관한 것이다.The present invention relates to the use of regulating calcium homeostasis within microglial cells of astrocytes; A method of suppressing the increase in calcium concentration in activated microglia using astrocytes in vitro ; and a method for screening substances related to nervous system diseases caused by dysfunction of calcium homeostasis in microglial cells using the same.
세포 내 칼슘 농도의 항상성은 세포 대사, 세포 증식, 세포 분화 등을 포함하는 세포 기능을 유지하는 데 중요한 역할을 하므로 세포내 칼슘 농도는 매우 정교하게 조절되어야 하며, 이러한 항상성의 파괴는 다양한 질환의 원인이 된다(Brini, M. et al., 2013, Interrelations between essential metal ions and human diseases, 81-137).Since homeostasis of intracellular calcium concentration plays an important role in maintaining cellular functions including cell metabolism, cell proliferation, and cell differentiation, intracellular calcium concentration must be very precisely regulated, and disruption of this homeostasis is the cause of various diseases. (Brini, M. et al., 2013, Interrelations between essential metal ions and human diseases, 81-137).
미세아교세포는 중추신경계에 상주하는 면역세포로, 안정화된(resting) 상태에서는 주변 환경을 감시하고 신경을 보호하는 역할을 한다. 하지만, 염증 반응 등에 의한 자극을 받아 지속적으로 활성화된(activating) 미세아교세포는 세포내 칼슘 농도 상승과 함께 신경독성 물질을 분비하여 신경 퇴행을 유발할 수 있다(Hopp, S. C., 2021, Journal of Neuroscience Research, 99(1), 141-162). 예를 들어, 알츠하이머병 마우스 모델의 미세아교세포는 정상 마우스의 미세아교세포와 비교하여 세포내 자발적 칼슘 농도 상승의 크기가 증가되었다(Brawek, B., et al., 2014, Acta Neuropathologica, 127(4), 495-505). 또한, 파킨슨병 마우스 모델의 미세아교세포에서 세포내 칼슘 유입에 주요 역할을 하는 N형 칼슘 통로 유전자 발현을 억제한 결과 행동학적 증상을 완화시키는 결과를 보였다(Saegusa, H. et al., 2020, FEBS letters, 594(17), 2914-2922). 따라서 미세아교세포 내 칼슘 농도 상승 억제는 미세아교세포의 세포 내 칼슘 농도 상승으로 인한 다양한 신경퇴행성질환을 예방 또는 치료하는데 중요할 것으로 사료된다.Microglia are immune cells that reside in the central nervous system, and in a resting state, they monitor the surrounding environment and protect nerves. However, continuously activating microglial cells stimulated by inflammatory reactions, etc. can increase intracellular calcium concentration and secrete neurotoxic substances, causing neurodegeneration (Hopp, S. C., 2021, Journal of Neuroscience Research , 99(1), 141-162). For example, microglia in an Alzheimer's disease mouse model showed an increased magnitude of intracellular spontaneous calcium concentration elevation compared to microglia in normal mice (Brawek, B., et al., 2014, Acta Neuropathologica, 127( 4), 495-505). In addition, inhibition of the expression of the N-type calcium channel gene, which plays a major role in intracellular calcium influx, in microglial cells of a Parkinson's disease mouse model resulted in alleviating behavioral symptoms (Saegusa, H. et al., 2020, FEBS letters, 594(17), 2914-2922). Therefore, suppressing the increase in calcium concentration within microglial cells is believed to be important in preventing or treating various neurodegenerative diseases caused by increased intracellular calcium concentration in microglial cells.
현재까지 신경 퇴행에 관한 연구는 주로 신경세포에 초점을 맞추었으나, 실제로 신경계를 이루는 신경세포, 별아교세포 및 미세아교세포는 긴밀한 상호 작용을 하므로 함께 고려되어야 한다(Ding, Z. et al., 2021, Frontiers in Cellular Neuroscience, 398). 이 중, 별아교세포는 뇌에서 가장 풍부한 신경교세포로써 시냅스 형성, 시냅스 기능, 신경전달물질 유리, 영양 인자를 생산하여 뇌기능에 중요한 역할을 수행한다(Zhou, B. et al., 2019, CNS neuroscience & therapeutics, 25(6), 665-673). 별아교세포의 기능 상실에 따른 다양한 신경계질환에 대한 연구가 진행된바 있으나, 별아교세포가 미세아교세포 내 칼슘 농도 상승을 억제하는 효과에 대해서는 현재까지 전혀 알려진 바 없다.To date, research on neurodegeneration has mainly focused on neurons, but in fact, neurons, astrocytes, and microglia that make up the nervous system interact closely and should be considered together (Ding, Z. et al., 2021 , Frontiers in Cellular Neuroscience, 398). Among these, astrocytes are the most abundant glial cells in the brain and play an important role in brain function by forming synapses, synaptic function, releasing neurotransmitters, and producing trophic factors (Zhou, B. et al., 2019, CNS neuroscience & therapeutics, 25(6), 665-673). Research has been conducted on various neurological diseases caused by loss of astrocyte function, but to date, nothing is known about the effect of astrocytes in suppressing the increase in calcium concentration in microglial cells.
이러한 배경 하에, 미세아교세포 내 칼슘 농도 상승을 억제하는 방법을 개발하기 위하여 노력하였으며, 그 결과 별아교세포가 미세아교세포에서 염증 반응 및 무스카린성 수용체 차단으로 유도한 세포내 칼슘 농도 상승을 효과적으로 억제하는 것을 실험을 통해 확인함으로써 본 발명을 완성하였다.Under this background, efforts were made to develop a method to suppress the increase in calcium concentration in microglial cells, and as a result, astrocytes effectively suppressed the increase in intracellular calcium concentration induced by inflammatory response and muscarinic receptor blockade in microglial cells. The present invention was completed by confirming through experiment.
따라서 본 발명의 목적은 별아교세포를 이용하여 활성화된 미세아교세포 내 칼슘 농도 상승을 효과적으로 억제할 수 있는 조성물을 제공하는 것이다.Therefore, the purpose of the present invention is to provide a composition that can effectively suppress the increase in calcium concentration in microglia activated using astrocytes.
본 발명의 다른 목적은, 별아교세포를 이용하여 활성화된 미세아교세포 내 칼슘 농도 상승을 효과적으로 억제할 수 있는 방법을 제공하는 것이다.Another object of the present invention is to provide a method that can effectively suppress the increase in calcium concentration in activated microglia using astrocytes.
본 발명의 또 다른 목적은, 별아교세포를 이용하여 활성화된 미세아교세포 내 칼슘 농도 상승을 효과적으로 억제할 수 있는 기전을 이용하여 신경계 질환 관련 물질을 스크리닝할 수 있는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for screening substances related to nervous system diseases using a mechanism that can effectively suppress the increase in calcium concentration in microglia activated using astrocytes.
상기와 같은 본 발명의 목적을 달성하기 위해서,In order to achieve the purpose of the present invention as described above,
본 발명은 별아교세포를 유효성분으로 포함하는 미세아교세포 내 칼슘 항상성을 조절하는 조성물을 제공한다.The present invention provides a composition that regulates calcium homeostasis in microglial cells containing astrocytes as an active ingredient.
본 발명의 일실시예에 있어서, 상기 조성물은 활성화된 미세아교세포 내 칼슘 농도 상승을 억제함으로써 미세아교세포 내 칼슘 항상성을 조절할 수 있다.In one embodiment of the present invention, the composition can control calcium homeostasis in microglial cells by suppressing the increase in calcium concentration in activated microglial cells.
또한, 본 발명은 신경세포, 별아교세포 및 미세아교세포로 이루어진 3종의 세포 혼합물에 무스카린성 수용체 차단제 및 염증 반응 유도제를 처리하는 단계를 포함하며, 상기 3종의 세포 혼합물은 중추신경계에서 각 세포의 비율 대비 별아교세포의 세포수를 2배 이상 증대시키는 것을 특징으로 하는, 인 비트로(in vitro) 상에서 별아교세포를 이용하여 활성화된 미세아교세포 내 칼슘 농도 상승을 억제하는 방법을 제공한다.In addition, the present invention includes the step of treating a mixture of three types of cells consisting of neurons, astrocytes, and microglia with a muscarinic receptor blocker and an inflammatory response inducer, and each of the three types of cell mixture is produced in the central nervous system. A method of suppressing the increase in calcium concentration in activated microglial cells using astrocytes in vitro is provided, which is characterized by increasing the number of astrocytes by more than twofold compared to the cell ratio.
본 발명의 일실시예에 있어서, 상기 무스카린성 수용체 차단제는 스코폴라민(scopolamine)일 수 있다.In one embodiment of the present invention, the muscarinic receptor blocker may be scopolamine.
본 발명의 일실시예에 있어서, 상기 염증 반응 유도제는 리포폴리사카라이드(LPS)일 수 있다.In one embodiment of the present invention, the inflammatory response inducer may be lipopolysaccharide (LPS).
본 발명의 일실시예에 있어서, 상기 중추신경계에서 신경세포, 별아교세포 및 미세아교세포 세포의 비율은 7:2:1일 수 있다.In one embodiment of the present invention, the ratio of neurons, astrocytes, and microglial cells in the central nervous system may be 7:2:1.
본 발명의 일실시예에 있어서, 상기 미세아교세포 내 칼슘 농도 상승의 억제는 Na+/Ca2+ 교환기전을 경유하여 나타날 수 있다.In one embodiment of the present invention, the inhibition of the increase in calcium concentration within the microglial cells may occur through the Na + /Ca 2+ exchange mechanism.
또한, 본 발명은 a) 신경세포, 별아교세포 및 미세아교세포가 7:2:1의 세포 비율로 이루어진 3종의 세포 혼합물에서 별아교세포의 세포수를 2배 이상 증대시키는 단계; b) 상기 세포 혼합물에 무스카린성 수용체 차단제 및 염증 반응 유도제를 처리한 후 피검 물질을 처리하는 단계; 및 c) 상기 피검 물질을 처리하기 전과 처리한 후의 미세아교세포 내 칼슘 농도 수준을 측정하여 비교하는 단계를 포함하는, 신경계 질환 관련 물질의 스크리닝 방법을 제공한다.In addition, the present invention includes the steps of a) increasing the number of astrocytes by more than twofold in a mixture of three types of cells consisting of neurons, astrocytes, and microglia at a cell ratio of 7:2:1; b) treating the cell mixture with a muscarinic receptor blocker and an inflammatory response inducer and then treating the test material; and c) measuring and comparing the calcium concentration level in microglial cells before and after processing the test material.
본 발명의 일실시예에 있어서, 피검 물질을 처리하기 전 대비 처리한 후의 미세아교세포 내 칼슘 농도가 증대되는 경우 신경계 질환을 악화시키는 물질로 판별할 수 있다.In one embodiment of the present invention, if the calcium concentration in microglial cells increases after treatment compared to before treatment of the test substance, it can be determined as a substance that worsens neurological disease.
본 발명의 일실시예에 있어서, 피검 물질을 처리하기 전 대비 처리한 후의 미세아교세포 내 칼슘 농도가 감소되는 경우 신경계 질환을 완화시키는 물질로 판별할 수 있다.In one embodiment of the present invention, if the calcium concentration in microglial cells is reduced after treatment compared to before treatment of the test substance, it can be determined as a substance that alleviates neurological diseases.
본 발명의 일실시예에 있어서, 상기 신경계 질환은 미세아교세포 내 칼슘 항상성 기능 이상으로 유발될 수 있다.In one embodiment of the present invention, the neurological disease may be caused by abnormal calcium homeostasis within microglial cells.
본 발명의 일실시예에 있어서, 상기 미세아교세포 내 칼슘 항상성 기능 이상으로 유발되는 신경계 질환은 복합부위통증증후군, 다발성 경화증, 근위축성 측삭경화증, 외상성 뇌손상, 외상성 척수질환, 뇌졸중, 알츠하이머병, 루게릭병, 파킨슨병, 전측두엽 치매 및 인지 장애로부터 선택될 수 있다.In one embodiment of the present invention, the neurological diseases caused by abnormal calcium homeostasis in microglial cells include complex regional pain syndrome, multiple sclerosis, amyotrophic lateral sclerosis, traumatic brain injury, traumatic spinal cord disease, stroke, Alzheimer's disease, It can be selected from Lou Gehrig's disease, Parkinson's disease, frontotemporal dementia and cognitive impairment.
본 발명의 조성물은 유효성분으로 별아교세포를 포함함으로써 미세아교세포 내 칼슘 항상성을 효과적으로 조절할 수 있는바, 미세아교세포 내 칼슘 항상성 기능 이상으로 유발되는 다양한 신경계 질환을 연구하기 위한 목적이나, 신경계 질환을 예방 또는 치료하기 위한 목적으로 유용하게 사용될 수 있다.The composition of the present invention can effectively control calcium homeostasis within microglial cells by containing astrocytes as an active ingredient, and is intended to study various neurological diseases caused by abnormal calcium homeostasis function within microglial cells. It can be usefully used for prevention or treatment.
도 1은 신경세포, 별아교세포 및 미세아교세포 3종을 혼합한 세포에서 별아교세포(U373 세포)를 두 배로 증가시켰을 때, 염증 반응 및 무스카린성 수용체 차단에 의한 세포 내 칼슘 농도 상승이 억제되는 효과를 나타낸 것이다(###P < 0.001 vs. Control; ***P < 0.001 vs. Vehicle)
도 2는 신경세포, 별아교세포 및 미세아교세포 3종을 혼합한 세포에서 별아교세포(U373 세포)를 두 배로 증가시켰을 때, 염증 반응 및 무스카린성 수용체 차단에 의한 세포내 칼슘 농도 상승이 억제되는 효과의 기전을 나타낸 것이다(**P < 0.01 vs. Vehicle; +P < 0.05 vs. LPS+Scopolamine group in doubled U373 cells).
도 3은 별아교세포 및 미세아교세포 2종을 혼합한 세포에서 별아교세포(U373 세포)를 두 배로 증가시켰을 때, 염증 반응 및 무스카린성 수용체 차단에 의한 세포내 칼슘 농도 상승이 억제되는 효과를 나타낸 것이다(###P < 0.001 vs. Control; **P < 0.01 vs. Vehicle).Figure 1 shows that when astrocytes (U373 cells) are doubled in a mixture of three types of neurons, astrocytes, and microglia, the increase in intracellular calcium concentration due to inflammatory response and muscarinic receptor blockade is suppressed. This shows the effect ( ### P < 0.001 vs. Control; *** P < 0.001 vs. Vehicle)
Figure 2 shows that when astrocytes (U373 cells) are doubled in a mixture of three types of neurons, astrocytes, and microglia, the increase in intracellular calcium concentration due to inflammatory response and muscarinic receptor blockade is suppressed. The mechanism of effect is shown ( ** P < 0.01 vs. Vehicle; + P < 0.05 vs. LPS+Scopolamine group in doubled U373 cells).
Figure 3 shows the effect of suppressing the increase in intracellular calcium concentration due to inflammatory response and blocking muscarinic receptors when astrocytes (U373 cells) are doubled in a mixture of two types of astrocytes and microglia. ( ### P < 0.001 vs. Control; ** P < 0.01 vs. Vehicle).
본 발명은 별아교세포를 유효성분으로 포함하는 미세아교세포 내 칼슘 항상성을 조절하는 조성물을 제공한다.The present invention provides a composition that regulates calcium homeostasis in microglial cells containing astrocytes as an active ingredient.
본 발명에서, "별아교세포(astrocyte)"란 중추 신경계의 신경 교세포(neuroglia)의 일종이며, 신경 교세포 중 가장 크기가 크다. 뇌와 척수에서 다량으로 존재하며, 별(star) 모양의 형태를 가지고 있다. 별아교세포는 뻗어 나온 돌기로 뉴런 및 혈관 내피세포와 상호작용을 한다. 혈액 뇌 장벽(blood-brain barrier)을 구성하고 있는 내피세포(endothelial cell)의 생화학적 특성을 조절하며, 신경 조직에 영양분을 공급하고, 손상된 뇌와 척수 조직의 재생에 중요한 역할을 한다.In the present invention, “astrocyte” is a type of neuroglia in the central nervous system, and is the largest among neuroglial cells. It is present in large quantities in the brain and spinal cord and has a star-shaped shape. Astrocytes interact with neurons and vascular endothelial cells through extended projections. It regulates the biochemical properties of endothelial cells that make up the blood-brain barrier, supplies nutrients to nervous tissue, and plays an important role in the regeneration of damaged brain and spinal cord tissues.
본 발명에서, "미세아교세포(microglia)"란 신경아교세포의 한 형태로 뇌와 척추 전역에 분포되어 있으며, 뇌에 있는 모든 세포 중의 10-15%를 차지한다. 중추신경계통에 있는 면역계에서 가장 중요한 역할을 하며, 최근 연구 결과에 의하면 미세아교세포는 건강한 상태에서 정상적인 뇌의 기능에서 핵심적인 역할을 하는 것으로 밝혀졌다.In the present invention, “microglia” is a type of glial cell that is distributed throughout the brain and spine and accounts for 10-15% of all cells in the brain. They play the most important role in the immune system in the central nervous system, and recent research has shown that microglia play a key role in normal brain function in healthy conditions.
본 발명에서는 중추신경계를 모방하기 위한 세포 혼합물로서 신경세포, 별아교세포 및 미세아교세포를 혼합함에 있어, 인체 중추신경계에서 각 세포의 비율인 7:2:1 대비 별아교세포의 세포수를 2배 늘려 7:4:1의 비율로 혼합한 뒤 미세아교세포를 활성화켰을 때, 세포 내 칼슘 농도 상승이 효과적으로 억제되는 것을 실험을 통해 확인하였다.In the present invention, when mixing neurons, astrocytes, and microglia as a cell mixture to mimic the central nervous system, the number of astrocytes is doubled compared to the ratio of each cell in the human central nervous system, which is 7:2:1. It was confirmed through experiments that when microglial cells were activated after mixing at a ratio of 7:4:1, the increase in intracellular calcium concentration was effectively suppressed.
그러므로 본 발명에서는 별아교세포가 미세아교세포 내 칼슘 항상성을 조절하는 역할을 할 수 있음을 최초로 규명하였으며, 본 발명은“별아교세포”의 “미세아교세포 내 칼슘 항상성 조절 용도”를 특징으로 한다.Therefore, in the present invention, it was first identified that astrocytes can play a role in regulating calcium homeostasis within microglial cells, and the present invention is characterized by the “use of astrocytes” for “regulating calcium homeostasis within microglial cells.”
본 발명의 조성물은 유효성분으로 별아교세포를 포함함으로써 미세아교세포 내 칼슘 항상성을 효과적으로 조절할 수 있는바, 미세아교세포 내 칼슘 항상성 기능 이상으로 유발되는 다양한 신경계 질환을 연구하기 위한 목적이나, 신경계 질환의 치료를 위한 목적으로 하는 조성물로 사용될 수 있다.The composition of the present invention can effectively regulate calcium homeostasis within microglia by containing astrocytes as an active ingredient, and is intended to study various neurological diseases caused by dysfunction of calcium homeostasis within microglial cells. It can be used as a composition intended for treatment.
예를 들어, 인 비트로(in vitro) 또는 인 비보(in vivo) 상에서 상기 조성물을 처리하거나 투여하는 경우에 미세아교세포의 활성화가 어떠한 기작으로 감소되는지, 미세아교세포 내 칼슘 항상성이 어떠한 기작을 통해 유지되는지 연구하는데 유용하게 사용될 수 있다.For example, when the composition is treated or administered in vitro or in vivo, by what mechanism is the activation of microglial cells reduced and by what mechanism is calcium homeostasis in microglial cells? It can be useful to study whether it is maintained.
또한, 상기 조성물은 신경계 질환의 예방 또는 치료를 위한 약제학적 조성물로 사용될 수 있다.Additionally, the composition can be used as a pharmaceutical composition for preventing or treating neurological diseases.
본 발명에서 용어, "예방"은 상기 조성물의 투여로 신경계 질환의 발병을 억제 또는 지연시키는 모든 행위를 의미하며, "치료"는 상기 조성물의 투여로 신경계 질환의 증세가 호전되거나 이롭게 되는 모든 행위를 의미한다.In the present invention, the term "prevention" refers to any action that suppresses or delays the onset of a neurological disease by administering the composition, and "treatment" refers to any action that improves or benefits the symptoms of a neurological disease by administering the composition. it means.
본 발명에서 “치료”란 질환과 관련된 임상적 상황을 억제하거나 완화하거나 이롭게 변경하는 모든 행위를 의미한다. 또한 치료는 치료를 받지 않은 경우 예상되는 생존율과 비교하여 증가된 생존을 의미할 수 있다. 치료는 치료적 수단 이외에 예방적 수단을 동시에 포함한다.In the present invention, “treatment” means any action that suppresses, alleviates, or beneficially changes the clinical situation related to the disease. Treatment may also mean increased survival compared to expected survival without treatment. Treatment includes preventive measures as well as therapeutic measures.
본 발명의 조성물은 신경계 질환에 대한 예방 또는 치료 효과를 나타낸다. 상기 신경계 질환은 미세아교세포 내 칼슘 항상성 기능 이상으로 유발되는 질환으로, 예를 들어 복합부위통증증후군, 다발성 경화증, 근위축성 측삭경화증, 외상성 뇌손상, 외상성 척수질환, 뇌졸중, 알츠하이머병, 루게릭병, 파킨슨병, 전측두엽 치매 및 인지 장애를 포함하는 다양한 신경계 질환이 포함될 수 있으나, 이에 제한되지 않는다.The composition of the present invention exhibits a preventive or therapeutic effect on neurological diseases. The neurological disease is a disease caused by abnormal calcium homeostasis in microglial cells, for example, complex regional pain syndrome, multiple sclerosis, amyotrophic lateral sclerosis, traumatic brain injury, traumatic spinal cord disease, stroke, Alzheimer's disease, Lou Gehrig's disease, This may include, but is not limited to, various neurological diseases including Parkinson's disease, frontotemporal dementia, and cognitive impairment.
본 발명의 조성물은 세포치료제로써 적용될 수 있으며, 약학적으로 허용가능한 담체를 추가로 포함하여 제제화될 수 있다. 본 발명에서 용어, "약학적으로 허용가능한 담체"란 생물체를 상당히 자극하지 않고 투여 성분의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 말한다. 본 발명에 있어서, 세포치료제로써 적용될 수 있는 본 발명의 조성물에 대해 약학적으로 허용 가능한 담체는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제, 기제, 부형제, 윤활제 등 당업계에 공지된 것이라면 제한없이 사용할 수 있다. 본 발명의 약학적 조성물은 각종 제형의 형태로 통용되는 기법에 따라 제조될 수 있다. 본 발명의 조성물인 세포치료제는 질병 부위로 이동을 유도할 수 있다면 어떠한 경로를 통해서든지 투여 가능하다. 경우에 따라서는 별아교세포를 병변으로 향하게 하는 수단을 구비한 비히클에 로딩하는 방안을 고려할 수도 있다. 따라서 본 발명의 조성물은 국소(협측, 설하, 피부 및 안내 투여를 포함), 비경구 (피하, 피내, 근육내, 점적, 정맥 내, 동맥 내, 관절 내 및 뇌척수액 내를 포함) 또는 경피성 투여를 포함한 여러 경로를 통해 투여할 수 있으며, 바람직하게는 발병부위에 직접 투여한다. 일 양태로서 별아교세포는 적합한 희석제에 약 현탁시켜 개체에 투여할 수 있는데, 이 희석제는 세포를 보호 및 유지하고, 목적하는 조직에 주입 시 사용에 용이하도록 하는 용도로 사용된다. 상기 희석제로는 생리식염수, 인산완충용액, HBSS 등의 완충용액, 뇌척수액 등이 있을 수 있다. 또한, 제약 조성물은 활성 물질이 표적 세포로 이동할 수 있도록 임의의 장치에 의해 투여될 수 있다. 바람직한 투여방식 및 제제는 주사제이다. 주사제는 생리식염액, 링겔액, Hank 용액 또는 멸균된 수용액 등의 수성용제, 올리브 오일 등의 식물유, 에틸올레인산 등의 고급 지방산 에스테르 및 에탄올, 벤질알코올, 프로필렌글리콜, 폴리에틸렌글리콜 또는 글리세린 등의 비수성용제 등을 이용하여 제조할 수 있고, 점막 투과를 위해, 통과할 배리어에 적합한 당업계에 공지된 비침투성제가 사용될 수 있으며, 변질방지를 위한 안정화제로 아스코르빈산, 아황산수소나트륨, BHA, 토코페롤, EDTA 등과, 유화제, pH 조절을 위한 완충제, 질산페닐수은, 치메로살, 염화벤잘코늄, 페놀, 크레솔, 벤질알코올 등의 미생물 발육을 저지하기 위한 보존제 등의 약학적 담체를 추가적으로 포함할 수 있다.The composition of the present invention can be applied as a cell therapy agent and can be formulated by additionally containing a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not significantly irritate living organisms and does not inhibit the biological activity and properties of the administered ingredient. In the present invention, pharmaceutically acceptable carriers for the composition of the present invention that can be applied as a cell therapy agent include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants, etc. known in the art. If it is available, it can be used without restrictions. The pharmaceutical composition of the present invention can be prepared according to commonly used techniques in the form of various dosage forms. The cell therapy composition of the present invention can be administered through any route as long as it can induce movement to the disease site. In some cases, loading a vehicle equipped with a means to direct astrocytes to the lesion may be considered. Accordingly, the compositions of the present invention may be administered topically (including buccal, sublingual, dermal, and intraocular administration), parenterally (including subcutaneous, intradermal, intramuscular, instillation, intravenous, intraarterial, intraarticular, and intracerebrospinal fluid), or transdermal administration. It can be administered through various routes, including, and is preferably administered directly to the affected area. In one embodiment, astrocytes can be administered to an individual by suspending them in a suitable diluent. This diluent is used to protect and maintain the cells and to facilitate their use when injected into the target tissue. The diluent may include physiological saline, phosphate buffer solution, buffer solution such as HBSS, cerebrospinal fluid, etc. Additionally, pharmaceutical compositions can be administered by any device that allows movement of the active agent to target cells. The preferred administration method and formulation is injection. Injections include aqueous solvents such as physiological saline solution, Ringer's solution, Hank's solution, or sterilized aqueous solution, vegetable oils such as olive oil, higher fatty acid esters such as ethyl oleic acid, and non-aqueous solvents such as ethanol, benzyl alcohol, propylene glycol, polyethylene glycol, or glycerin. It can be manufactured using, etc., and for mucosal penetration, an impermeable agent known in the art suitable for the barrier to pass through can be used, and as a stabilizer to prevent deterioration, ascorbic acid, sodium bisulfite, BHA, tocopherol, EDTA It may additionally contain pharmaceutical carriers such as emulsifiers, buffers for pH adjustment, and preservatives to inhibit microbial growth such as phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, and benzyl alcohol.
본 발명에서 용어, "세포치료제"는 대상체로부터 분리, 배양 및 특수한 저작을 통해 제조된 세포 및 조직으로 치료, 진단 및 예방의 목적으로 사용되는 의약품(미국 FDA규정)으로서, 세포 혹은 조직의 기능을 복원시키기 위하여 살아있는 자가, 동종, 또는 이종세포를 체외에서 증식, 선별하거나 다른 방법으로 세포의 생물학적 특성을 변화시키는 등의 일련의 행위를 통하여 치료, 진단 및 예방의 목적으로 사용되는 의약품을 지칭한다.In the present invention, the term "cell therapy product" refers to a medicine (US FDA regulations) used for the purposes of treatment, diagnosis, and prevention with cells and tissues manufactured through isolation, culture, and special manipulation from a subject, and is used to enhance the function of cells or tissues. It refers to medicines used for the purposes of treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro to restore them, or changing the biological characteristics of cells by other methods.
본 발명에서 용어, "투여"는 어떠한 적절한 방법으로 환자에게 본 발명의 조성물을 도입하는 것을 의미하며, 본발명의 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있다. 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 경구 투여, 국소 투여, 비내 투여, 폐내 투여, 직장내 투여 될 수 있으나, 이에 제한되지는 않는다.In the present invention, the term "administration" means introducing the composition of the present invention into a patient by any appropriate method, and the route of administration of the composition of the present invention is through various oral or parenteral routes as long as it can reach the target tissue. may be administered. It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonaryly, or rectally, but is not limited thereto.
본 명세서에서, "유효량"은 목적하는 치료되어야 할 특정 질환의 발병 또는 진행을 지연하거나 전적으로 중지시키는 데 필요한 양을 의미한다. 본 발명에서 조성물은 약학적 유효량으로 투여될 수 있다. 상기 세포의 1일 투여량은 1.0×104 내지 1.0×1010 세포/kg 체중을 1회 또는 수회로 나누어 투여할 수 있다. 그러나, 유효성분의 실제 투여량은 치료하고자 하는 질환, 질환의 중증도, 투여경로, 환자의 체중, 연령 및 성별 등의 여러 관련 인자에 비추어 결정되어야 하는 것으로 이해되어야 하며, 따라서, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.As used herein, “effective amount” means the amount necessary to delay or completely stop the onset or progression of the specific disease to be treated. In the present invention, the composition can be administered in a pharmaceutically effective amount. The daily dose of the cells may be 1.0×10 4 to 1.0×10 10 cells/kg of body weight once or in several divided doses. However, it should be understood that the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age, and gender, and therefore, the dosage should be determined in any way. It does not limit the scope of the present invention in any way.
또한, 본 발명은 상기 조성물을 대상체에 투여하는 단계를 포함하는 미세아교세포 내 칼슘 항상성 기능 이상으로 유발되는 신경계 질환의 치료 방법을 제공한다.Additionally, the present invention provides a method of treating neurological diseases caused by dysfunction of calcium homeostasis in microglial cells, comprising the step of administering the composition to a subject.
본 명세서에 있어서, "대상체"는 치료, 관찰 또는 실험의 대상인 척추동물, 바람직하게는 포유동물, 예를 들어, 개, 고양이, 생쥐, 인간 등일 수 있다.In the present specification, the “subject” may be a vertebrate animal, preferably a mammal, such as a dog, cat, mouse, human, etc., that is the subject of treatment, observation, or experiment.
또한, 본 발명은 신경세포, 별아교세포 및 미세아교세포로 이루어진 3종의 세포 혼합물에 무스카린성 수용체 차단제 및 염증 반응 유도제를 처리하는 단계를 포함하며, 상기 3종의 세포 혼합물은 중추신경계에서 각 세포의 비율 대비 별아교세포의 세포수를 2배 이상 증대시키는 것을 특징으로 하는, 인 비트로(in vitro) 상에서 별아교세포를 이용하여 활성화된 미세아교세포 내 칼슘 농도 상승을 억제하는 방법을 제공한다.In addition, the present invention includes the step of treating a mixture of three types of cells consisting of neurons, astrocytes, and microglia with a muscarinic receptor blocker and an inflammatory response inducer, and each of the three types of cell mixture is produced in the central nervous system. A method of suppressing the increase in calcium concentration in activated microglial cells using astrocytes in vitro is provided, which is characterized by increasing the number of astrocytes by more than twofold compared to the cell ratio.
상기 무스카린성 수용체 차단제는 스코폴라민(scopolamine)일 수 있으며, 상기 염증 반응 유도제는 리포폴리사카라이드(LPS)일 수 있다.The muscarinic receptor blocker may be scopolamine, and the inflammatory response inducer may be lipopolysaccharide (LPS).
일반적으로 인체 중추신경계에서 신경세포, 별아교세포 및 미세아교세포는 7:2:1의 세포 비율을 보이는바, 본 발명의 방법에서는 별아교세포의 세포수를 2배 ~ 5배 증대시켜 상기 3종의 세포를 7:4:1 내지 7:10:1의 세포 비율로 혼합할 수 있다.Generally, in the human central nervous system, neurons, astrocytes, and microglia show a cell ratio of 7:2:1. In the method of the present invention, the number of astrocytes is increased by 2 to 5 times to produce the three types of cells. Cells can be mixed at a cell ratio of 7:4:1 to 7:10:1.
본 발명의 일구체예에서, 상기 미세아교세포 내 칼슘 농도 상승의 억제는 Na+/Ca2+ 교환기전을 경유하여 나타날 수 있다.In one embodiment of the present invention, the inhibition of the increase in calcium concentration in the microglial cells may occur through the Na + /Ca 2+ exchange mechanism.
또한, 본 발명은 a) 신경세포, 별아교세포 및 미세아교세포가 7:2:1의 세포 비율로 이루어진 3종의 세포 혼합물에서 별아교세포의 세포수를 2배 이상 증대시키는 단계; b) 상기 세포 혼합물에 무스카린성 수용체 차단제 및 염증 반응 유도제를 처리한 후 피검 물질을 처리하는 단계; 및 c) 상기 피검 물질을 처리하기 전과 처리한 후의 미세아교세포 내 칼슘 농도 수준을 측정하여 비교하는 단계를 포함하는, 신경계 질환 관련 물질의 스크리닝 방법을 제공한다.In addition, the present invention includes the steps of a) increasing the number of astrocytes by more than twofold in a mixture of three types of cells consisting of neurons, astrocytes, and microglia at a cell ratio of 7:2:1; b) treating the cell mixture with a muscarinic receptor blocker and an inflammatory response inducer and then treating the test material; and c) measuring and comparing the calcium concentration level in microglial cells before and after processing the test material.
본 명세서에서, "신경계 질환 관련 물질"은 신경계 질환을 악화시키거나, 예방, 완화 또는 치료할 수 있는 물질로서 신경계 질환에 영향을 미치는 물질을 의미한다.In this specification, “substance related to nervous system disease” refers to a substance that affects nervous system disease as a substance that can worsen, prevent, alleviate, or treat nervous system disease.
본 발명의 스크리닝 방법에서 피검 물질을 처리하기 전 대비 처리한 후의 미세아교세포 내 칼슘 농도가 증대되는 경우 신경계 질환을 악화시키는 물질로 판별할 수 있으며, 피검 물질을 처리하기 전 대비 처리한 후의 미세아교세포 내 칼슘 농도가 감소되는 경우 신경계 질환을 완화시키는 물질로 판별할 수 있다.In the screening method of the present invention, if the calcium concentration in microglia increases after treatment compared to before treating the test substance, it can be determined as a substance that worsens neurological diseases, and the microglia after treatment compared to before treatment of the test substance can be determined to be an increase. If intracellular calcium concentration is reduced, it can be identified as a substance that alleviates nervous system diseases.
상기 신경계 질환은 미세아교세포 내 칼슘 항상성 기능 이상으로 유발되는 질환으로, 예를 들어 복합부위통증증후군, 다발성 경화증, 근위축성 측삭경화증, 외상성 뇌손상, 외상성 척수질환, 뇌졸중, 알츠하이머병, 루게릭병, 파킨슨병, 전측두엽 치매 및 인지 장애를 포함하는 다양한 신경계 질환이 포함될 수 있으나, 이에 제한되지 않는다.The neurological disease is a disease caused by abnormal calcium homeostasis in microglial cells, for example, complex regional pain syndrome, multiple sclerosis, amyotrophic lateral sclerosis, traumatic brain injury, traumatic spinal cord disease, stroke, Alzheimer's disease, Lou Gehrig's disease, This may include, but is not limited to, various neurological diseases including Parkinson's disease, frontotemporal dementia, and cognitive impairment.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited to these examples.
<실시예><Example>
1. 실험방법1. Experimental method
세포 준비 및 실험 전 처치Cell preparation and pre-experimental treatment
본 발명에서 사용한 신경세포(SH-SY5Y 세포), 별아교세포(U373 세포) 및 미세아교세포(BV2 세포)는 모두 ATCC(American Type Culture Collection)에서 구입하여 실험하였다. 모든 세포는 DMEM(Dulbecco’s Modified Eagle Medium)에 1% 페니실린/스트렙토마이신과 10% 소태아혈청(FBS)을 첨가한 배지에 배양하였으며, 일정한 온도(37℃), 습도, CO2 농도(5%)를 유지하였다. 신경세포, 별아교세포 및 미세아교세포를 합한 경우(하기에서 간략하게 ‘3종의 세포 혼합물’로 표기함)에는 선행문헌을 바탕으로 하여 실제 인체의 중추신경계에서 각 세포의 비율을 반영한 7:2:1로 혼합하여 사용하였다(Karlsen, A. S., & Pakkenberg, B., 2011, Cerebral cortex, 21(11), 2519-2524). 세포 내 칼슘 농도를 측정하기 전 무스카린성 수용체를 충분히 차단하기 위하여 스코폴라민(scopolamine) 0.1mM을 1시간 동안 처리하였으며, 염증 반응을 유도하기 위하여 지질다당류(lipopolysaccharide, LPS) 1μg/ml을 200초 동안 처치하였다.Neurons (SH-SY5Y cells), astrocytes (U373 cells), and microglia (BV2 cells) used in the present invention were all purchased from ATCC (American Type Culture Collection) and tested. All cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) at constant temperature (37°C), humidity, and CO2 concentration (5%). was maintained. When neurons, astrocytes, and microglia are combined (simply referred to as 'three types of cell mixture' below), the ratio is 7:2, which reflects the ratio of each cell in the actual human central nervous system based on prior literature. :1 was mixed and used (Karlsen, AS, & Pakkenberg, B., 2011, Cerebral cortex, 21(11), 2519-2524). Before measuring intracellular calcium concentration, 0.1mM scopolamine was treated for 1 hour to sufficiently block muscarinic receptors, and lipopolysaccharide (LPS) 1μg/ml was added at 200% to induce an inflammatory response. Treated for seconds.
세포 내 칼슘 농도 측정Measurement of intracellular calcium concentration
세포 내 칼슘 농도를 측정하기 위해 약 100만개의 세포에 Fura-2AM(5 μM; Sigma aldrich, USA)을 상온에서 30분간 부하시킨 후, 형광 분광 광도계를 이용하여 340 nm와 380 nm의 파장으로 세포를 자극시켰을 때 510nm의 파장에서 방출되는 형광의 세기를 측정하였다. 최소 형광 비율(Rmin)은 에틸렌 글리콜 비스(베타-아미노에틸 에테르)-N,N,N',N'-테트라아세틱 에시드(ethylene Glycol Bis(2-aminoethyl Ether)-N,N,N',N'-tetraacetic Acid)를 10mM 첨가하여 측정하였고, 최대 형광 비율(Rmax)은 이오노마이신(ionomycin) 5μM을 첨가하여 측정하였다. 세포 내 칼슘 농도는 선행문헌의 공식을 바탕으로 다음과 같이 계산하였다(You, J. H., et al., 2013, Journal of cardiovascular pharmacology, 61(4), 324-328). To measure intracellular calcium concentration, approximately 1 million cells were loaded with Fura-2AM (5 μM; Sigma aldrich, USA) for 30 minutes at room temperature, and then cells were measured at wavelengths of 340 nm and 380 nm using a fluorescence spectrophotometer. When stimulated, the intensity of fluorescence emitted at a wavelength of 510 nm was measured. The minimum fluorescence ratio (R min ) is ethylene glycol Bis(2-aminoethyl Ether)-N,N,N',N'-tetraacetic acid. ,N'-tetraacetic acid) was added at 10mM, and the maximum fluorescence ratio (R max ) was measured by adding ionomycin (ionomycin) at 5μM. The intracellular calcium concentration was calculated as follows based on the formula in prior literature (You, JH, et al., 2013, Journal of cardiovascular pharmacology, 61(4), 324-328).
세포 내 칼슘 농도 = K d × b × (R - Rmin)/(Rmax - R)Intracellular calcium concentration = K d × b × (R - R min )/(R max - R)
위 공식에서 K d는 Fura-2AM의 해리상수(224 nM)을 나타내며, b는 380 nm의 파장으로 세포를 자극시켰을 때의 형광 강도를 의미한다.In the above formula, K d represents the dissociation constant of Fura-2AM (224 nM), and b represents the fluorescence intensity when cells are stimulated with a wavelength of 380 nm.
세포 내 칼슘 농도 증가 억제 기전 확인Confirmation of mechanism to suppress increase in intracellular calcium concentration
별아교세포가 LPS와 스코폴라민을 전처치한 3종의 세포 혼합물(신경세포+별아교세포+미세아교세포)에서 세포 내 칼슘 농도 증가를 억제하는 효과의 기전을 파악하기 위해 Na+/K+ 펌프 억제제인 우아바인(ouabain) 1μM 또는 Na+/Ca2+ 교환기 억제제인 Ni2+ 100μM을 200초간 전 처치한 후 세포 내 칼슘 농도를 측정하였다.To determine the mechanism of the effect of astrocytes to suppress the increase in intracellular calcium concentration in a mixture of three types of cells (neurons + astrocytes + microglia) pretreated with LPS and scopolamine, the Na + /K + pump was used. After pretreatment with 1 μM of the inhibitor ouabain or 100 μM of the Na + /Ca 2+ exchanger inhibitor Ni 2+ for 200 seconds, the intracellular calcium concentration was measured.
통계 분석statistical analysis
모든 결과는 평균과 표준오차로 나타내었다. 통계 분석은 SPSS 프로그램(ver. 24.0)을 사용하여 수행하였다. 군간 차이 유무는 일원배치 분산분석으로 분석하였으며, 차이가 있을 때 사후분석(Fisher's least significant difference)을 시행하였다. P < 0.05 수준에서 통계적으로 유의미한 것으로 보았다.All results were expressed as mean and standard error. Statistical analysis was performed using the SPSS program (ver. 24.0). The presence or absence of differences between groups was analyzed by one-way analysis of variance, and when there were differences, a post hoc analysis (Fisher's least significant difference) was performed. It was considered statistically significant at the P < 0.05 level.
2. 실험결과2. Experiment results
별아교세포는 미세아교세포에서 염증 반응 및 무스카린성 수용체 차단으로 유도한 세포 내 칼슘 농도 상승을 억제하는 효과Astrocytes have the effect of suppressing the increase in intracellular calcium concentration induced by inflammatory response and muscarinic receptor blockade in microglial cells.
도 1에 개시된 바와 같이, 대조군에 비해 LPS와 스코폴라민을 전처치한 3종의 세포 혼합물(신경세포+별아교세포+미세아교세포)에서 세포 내 칼슘 농도가 유의미하게 증가하였으며(P < 0.001), 별아교세포(U373 세포)를 두 배로 증가시켰을 때 이러한 증가가 억제되어 정상수준으로 회복되는 것을 확인하였다(P < 0.001).As shown in Figure 1, the intracellular calcium concentration was significantly increased in the three cell mixtures (neurons + astrocytes + microglia) pretreated with LPS and scopolamine compared to the control group (P < 0.001). , it was confirmed that when astrocytes (U373 cells) were doubled, this increase was suppressed and restored to normal levels (P < 0.001).
별아교세포가 LPS와 스코폴라민을 전처치한 3종의 세포 혼합물(신경세포+별아교세포+미세아교세포)에서 세포 내 칼슘 농도 증가를 억제하는 효과의 기전을 파악하기 위해 Na+/K+ 펌프 억제제인 우아바인(ouabain) 또는 Na+/Ca2+ 교환기 억제제인 Ni2+을 사용하여 실험을 진행하였다.To determine the mechanism of the effect of astrocytes to suppress the increase in intracellular calcium concentration in a mixture of three types of cells (neurons + astrocytes + microglia) pretreated with LPS and scopolamine, the Na + /K + pump was used. The experiment was conducted using the inhibitor ouabain or the Na + /Ca 2+ exchanger inhibitor Ni 2+ .
그 결과, 도 2에 개시된 바와 같이, LPS와 스코폴라민을 전처치한 3종의 세포 혼합물(신경세포+별아교세포+미세아교세포)에서 별아교세포를 두 배로 증가시켰을 때 세포 내 칼슘 농도가 감소한 효과는 Ni2+에 의해 유의미하게 억제되었다(P = 0.039). 따라서 LPS와 스코폴라민을 전처치한 3종의 세포 혼합물(신경세포+별아교세포+미세아교세포)을 두 배로 증가시켰을 때 세포 내 칼슘 농도가 감소한 효과는 Na+/Ca2+ 교환기를 경유하여 나타난 것임을 확인할 수 있었다.As a result, as shown in Figure 2, when astrocytes were doubled in a mixture of three types of cells (neurons + astrocytes + microglia) pretreated with LPS and scopolamine, the intracellular calcium concentration decreased. The effect was significantly inhibited by Ni 2+ (P = 0.039). Therefore, when the mixture of three types of cells (neurons + astrocytes + microglia) pretreated with LPS and scopolamine is doubled, the effect of reducing intracellular calcium concentration is via the Na + /Ca 2+ exchanger. I was able to confirm that it had appeared.
마지막으로 LPS와 스코폴라민을 전처치한 3종의 세포 혼합물(신경세포+별아교세포+미세아교세포)을 두 배로 증가시켰을 때 세포 내 칼슘 농도가 감소한 효과가 신경세포에 대한 효과인지, 미세아교세포에 대한 효과인지를 명확히 하기 위한 실험을 진행하였다.Lastly, when the mixture of three types of cells (neurons + astrocytes + microglia) pretreated with LPS and scopolamine was doubled, the effect of decreasing intracellular calcium concentration was investigated whether the effect was on neurons or microglia. An experiment was conducted to clarify whether the effect was on cells.
그 결과, 도 3에 개시된 바와 같이, 대조군에 비해 LPS와 스코폴라민을 전처치한 별아교세포 및 미세아교세포의 혼합물에서 세포 내 칼슘 농도가 유의미하게 증가하였으며(P < 0.001), 별아교세포(U373 세포)를 두 배로 증가시켰을 때 이러한 증가가 억제되는 것을 확인하였다(P = 0.008). As a result, as shown in Figure 3, the intracellular calcium concentration was significantly increased in the mixture of astrocytes and microglia pretreated with LPS and scopolamine compared to the control group (P < 0.001), and astrocytes (U373 It was confirmed that this increase was suppressed when the number of cells was doubled (P = 0.008).
상기 내용을 종합한 결과, 본 발명의 별아교세포는 미세아교세포에서 염증 반응 및 무스카린성 수용체 차단으로 유도한 세포 내 칼슘 농도 상승을 억제하는 효과가 확인되었는바, 본 발명에 의한 방법은 미세아교세포 내 과도한 칼슘 농도 상승과 관련된 신경계 질환을 예방 또는 치료하는 데 유용할 것임을 알 수 있었다.As a result of summarizing the above, it was confirmed that the astrocytes of the present invention are effective in suppressing the increase in intracellular calcium concentration induced by inflammatory response and muscarinic receptor blockade in microglial cells, and the method of the present invention It was found that it would be useful in preventing or treating neurological diseases related to excessive elevation of intracellular calcium concentration.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.
Claims (12)
상기 조성물은 활성화된 미세아교세포 내 칼슘 농도 상승을 억제함으로써 미세아교세포 내 칼슘 항상성을 조절할 수 있는 것을 특징으로 하는, 조성물. According to paragraph 1,
The composition is characterized in that it can control calcium homeostasis in microglial cells by suppressing the increase in calcium concentration in activated microglial cells.
상기 3종의 세포 혼합물은 중추신경계에서 각 세포의 비율 대비 별아교세포의 세포수를 2배 이상 증대시키는 것을 특징으로 하는, 인 비트로(in vitro) 상에서 별아교세포를 이용하여 활성화된 미세아교세포 내 칼슘 농도 상승을 억제하는 방법.It includes the step of treating a mixture of three types of cells consisting of neurons, astrocytes, and microglia with a muscarinic receptor blocker and an inflammatory response inducer,
The mixture of the three types of cells is characterized in that it increases the number of astrocytes by more than twofold compared to the ratio of each cell in the central nervous system. Calcium in microglia activated using astrocytes in vitro How to suppress concentration rise.
상기 무스카린성 수용체 차단제는 스코폴라민(scopolamine)인 것을 특징으로 하는, 인 비트로(in vitro) 상에서 별아교세포를 이용하여 활성화된 미세아교세포 내 칼슘 농도 상승을 억제하는 방법.According to paragraph 3,
A method of suppressing the increase in calcium concentration in activated microglia using astrocytes in vitro , wherein the muscarinic receptor blocker is scopolamine.
상기 염증 반응 유도제는 리포폴리사카라이드(LPS)인 것을 특징으로 하는, 인 비트로(in vitro) 상에서 별아교세포를 이용하여 활성화된 미세아교세포 내 칼슘 농도 상승을 억제하는 방법.According to paragraph 3,
A method of suppressing the increase in calcium concentration in activated microglia using astrocytes in vitro , wherein the inflammatory response inducer is lipopolysaccharide (LPS).
상기 중추신경계에서 신경세포, 별아교세포 및 미세아교세포 세포의 비율은 7:2:1인 것을 특징으로 하는, 인 비트로(in vitro) 상에서 별아교세포를 이용하여 활성화된 미세아교세포 내 칼슘 농도 상승을 억제하는 방법.According to paragraph 3,
In the central nervous system, the ratio of neurons, astrocytes, and microglial cells is 7:2:1, and the calcium concentration in activated microglial cells is increased using astrocytes in vitro. How to suppress.
상기 미세아교세포 내 칼슘 농도 상승의 억제는 Na+/Ca2+ 교환기전을 경유하여 나타나는 것을 특징으로 하는, 인 비트로(in vitro) 상에서 별아교세포를 이용하여 활성화된 미세아교세포 내 칼슘 농도 상승을 억제하는 방법.According to any one of claims 3 to 6,
Inhibition of the increase in calcium concentration in microglial cells is characterized by an increase in calcium concentration in activated microglial cells using astrocytes in vitro , which is characterized in that it occurs via the Na + /Ca 2+ exchange mechanism. How to suppress.
b) 상기 세포 혼합물에 무스카린성 수용체 차단제 및 염증 반응 유도제를 처리한 후 피검 물질을 처리하는 단계; 및
c) 상기 피검 물질을 처리하기 전과 처리한 후의 미세아교세포 내 칼슘 농도 수준을 측정하여 비교하는 단계를 포함하는, 신경계 질환 관련 물질의 스크리닝 방법.a) increasing the number of astrocytes by more than two-fold in a mixture of three types of cells consisting of neurons, astrocytes, and microglia at a cell ratio of 7:2:1;
b) treating the cell mixture with a muscarinic receptor blocker and an inflammatory response inducer and then treating the test material; and
c) A screening method for substances related to nervous system diseases, comprising the step of measuring and comparing the calcium concentration level in microglial cells before and after processing the test substance.
피검 물질을 처리하기 전 대비 처리한 후의 미세아교세포 내 칼슘 농도가 증대되는 경우 신경계 질환을 악화시키는 물질로 판별하는 것을 특징으로 하는, 신경계 질환 관련 물질의 스크리닝 방법.According to clause 8,
A screening method for a substance related to a nervous system disease, characterized in that it is determined as a substance that worsens a nervous system disease when the calcium concentration in microglial cells increases after processing the test substance compared to before treatment.
피검 물질을 처리하기 전 대비 처리한 후의 미세아교세포 내 칼슘 농도가 감소되는 경우 신경계 질환을 완화시키는 물질로 판별하는 것을 특징으로 하는, 신경계 질환 관련 물질의 스크리닝 방법.According to clause 8,
A screening method for a substance related to a nervous system disease, characterized in that it is determined as a substance that alleviates a nervous system disease when the calcium concentration in microglial cells is reduced before and after the test substance is treated.
상기 신경계 질환은 미세아교세포 내 칼슘 항상성 기능 이상으로 유발되는 것을 특징으로 하는, 신경계 질환 관련 물질의 스크리닝 방법.According to clause 8,
A screening method for substances related to nervous system diseases, characterized in that the neurological disease is caused by dysfunction of calcium homeostasis in microglial cells.
상기 미세아교세포 내 칼슘 항상성 기능 이상으로 유발되는 신경계 질환은 복합부위통증증후군, 다발성 경화증, 근위축성 측삭경화증, 외상성 뇌손상, 외상성 척수질환, 뇌졸중, 알츠하이머병, 루게릭병, 파킨슨병, 전측두엽 치매 및 인지 장애로부터 선택되는 것을 특징으로 하는, 신경계 질환 관련 물질의 스크리닝 방법.According to clause 11,
Neurological diseases caused by abnormal calcium homeostasis within microglial cells include complex regional pain syndrome, multiple sclerosis, amyotrophic lateral sclerosis, traumatic brain injury, traumatic spinal cord disease, stroke, Alzheimer's disease, Lou Gehrig's disease, Parkinson's disease, and frontotemporal dementia. and cognitive disorders.
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