KR102411155B1 - Brain-damaged animal model with intra-septally delivered therapeutics treatment and preparation method thereof - Google Patents
Brain-damaged animal model with intra-septally delivered therapeutics treatment and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
본 발명은 뇌손상 줄기세포 치료제의 효과를 확인할 수 있도록, 비강내로 치료제가 이식된 뇌손상 동물 모델 및 이의 제조방법에 관한 것으로, 본 발명에 따른 동물 모델은 다양한 뇌손상 줄기세포 치료제의 파라크라인 효능을 확인할 수 있을 뿐만 아니라, 실제 임상에서 뇌손상 줄기세포 치료제 적용 시에도 폭넓게 활용될 수 있는 장점이 존재한다.The present invention relates to an animal model for brain injury in which a therapeutic agent is intranasally implanted to confirm the effect of a therapeutic agent for brain injury stem cells, and a method for manufacturing the same. Not only can the efficacy be confirmed, but there is an advantage that can be widely used when applying brain damage stem cell treatment in actual clinical practice.
Description
본 발명은 뇌손상을 치료할 수 있는 줄기세포 치료제를 뇌손상 동물 모델의 비강내로 전달할 수 있는 방법 및 이러한 동물모델에 관한 것이다. 더욱 자세하게는 줄기세포 치료제를 뇌손상 치료에 활용시 줄기세포로부터 안정적이고 지속적인 파라크라인 효과를 확인할 수 있도록, 이를 동물 모델(사람을 제외함)의 비중격과 점막 사이로 이식하는 방법 및 이러한 방법으로 제작된 동물 모델에 관한 것이다. The present invention relates to a method for intranasally delivering a stem cell therapeutic agent capable of treating brain injury in an animal model of brain injury, and to such an animal model. In more detail, a method of transplanting it between the nasal septum and the mucous membrane of an animal model (except humans) to confirm the stable and lasting paracrine effect from stem cells when using stem cell therapeutics for brain injury treatment, and production using this method related to animal models.
[과제고유번호] HI20C0408[Project unique number] HI20C0408
[부처명] 보건복지부[Name of Ministry] Ministry of Health and Welfare
[연구관리전문기관] 한국보건산업진흥원[Research and management institution] Korea Health Industry Promotion Agency
[연구사업명] 첨단의료기술개발[Research project name] Advanced medical technology development
[연구과제명] 줄기 세포 뇌질환 치료 향상을 위한 비강내 전달법 및 줄기 세포 기능 강화 하이드로겔 공통 기반 기술 개발[Research project name] Development of intranasal delivery method and stem cell function-enhancing hydrogel common-based technology to improve the treatment of stem cell brain disease
[주관기관] 한림대학교산학협력단[Organization] Hallym University Industry-Academic Cooperation Foundation
[연구기간] 2020-04-23~2022-12-31[Research Period] 2020-04-23~2022-12-31
세계적으로 인구 고령화가 빠르게 진행되면서 알츠하이머병, 파킨슨병, 뇌졸중 등의 퇴행성 신경질환 환자가 급속히 증가하고 있다. 퇴행성 뇌질환 외에도 해마다 교통사고 등에 의한 외상성 뇌손상(TBI)에 의해 수많은 사망자 및 TBI와 관련된 장기적인 장애에 시달리는 환자가 늘고 있다. 각종 뇌손상과 관련하여 많은 비용이 직간접적으로 지출되는 등 사회적, 경제적인 문제가 되고 있다. As the world population is aging rapidly, the number of patients with degenerative neurological diseases such as Alzheimer's disease, Parkinson's disease, and stroke is rapidly increasing. In addition to degenerative brain diseases, the number of deaths due to traumatic brain injury (TBI) caused by traffic accidents and the like is increasing every year, and the number of patients suffering from long-term disability related to TBI is increasing. It has become a social and economic problem, such as direct and indirect expenses related to various brain injuries.
줄기세포치료는 대뇌의 손상된 기전을 복구할 수 있다는 측면에서 중추신경계에 손상을 입은 신경질환의 새로운 치료법으로서 주목 받고 있으며 줄기세포치료의 효과는 직접 분화보다 파라크라인 효과가 주요 요인이라는 보고가 늘고 있다. 즉, 줄기세포는 신경의 생존/재생을 촉진하는 여러 신경영양성인자를 분비하는 능력이 있고, 면역 조절 기능을 하여 환자 대뇌 내부의 미세 환경과 시너지 효과를 내어, 신경세포를 보호(neuroprotection)하고 내부세포의 증식을 촉진시키고, 신경 세포의 재생 (neuroregeneration)능력을 높일 수 있으며 또한 내부의 host 네트워크와 통합하고, 손실된 뉴런을 대체할 수 있는 가능성이 있다.Stem cell therapy is attracting attention as a new treatment for neurological diseases damaged in the central nervous system in that it can repair the damaged mechanism of the brain. have. In other words, stem cells have the ability to secrete various neurotrophic factors that promote the survival/regeneration of nerves, and have a synergistic effect with the microenvironment inside the patient's brain by acting as an immunomodulatory agent to protect nerve cells and internally It can promote cell proliferation, increase the ability of nerve cells to regenerate (neuroregeneration), and also have the potential to integrate with internal host networks and replace lost neurons.
줄기세포 치료가 상당한 잠재력이 있음에도 불구하고, in vivo 연구에서 만족할만한 효과를 보이지 않고 있는데 그 이유는 줄기세포의 주입 루트에 있다. 현재 뇌손상 줄기세포 전달방법은 뇌손상 부위에 직접 투여하거나 뇌척수액 또는 정맥 투여가 시도 되고 있는데, 직접 투여 방식은 손상 부위 및 접근 주변부 뇌손상을 일으키는 합병증을 유발할 수 있으며 뇌척수액을 통한 투여는 효과가 미미하거나 뇌척수 순환 폐쇄의 합병증을 유발할 수 있다. 정맥에 의한 투여시에는 모세혈관계 내로 줄기세포가 포획되어 줄기 세포의 표적 기관으로의 도달량이 부족해질 수 있는데 그렇다고 많은 양의 치료제를 넣으면 전신독성과 같은 또다른 부작용이 생기며, 뇌신경계 조직이 단단한 두개골과 혈액뇌장벽(BBB, blood brain barrier)으로 둘러싸여 있어서 치료 약물의 전달 효율이 매우 낮게 된다. Although stem cell therapy has great potential, it has not shown satisfactory effects in in vivo studies because of the stem cell injection route. Currently, brain injury stem cell delivery methods are either directly administered to the brain injury site, or cerebrospinal fluid or intravenous administration is being attempted. or complications of cerebrospinal circulation obstruction. When administered intravenously, stem cells are trapped in the capillary system and the amount of stem cells reaching the target organ may be insufficient. and the blood brain barrier (BBB), the delivery efficiency of therapeutic drugs is very low.
따라서 현재 뇌손상 치료에 줄기세포를 적용하기 위하여 줄기세포 치료제의 전달 루트로 비강 전달법이 제시되고 있다. 비강 전달법은 우선 뇌의 불필요한 손상을 낼 필요가 없고, 반복투여가 용이하며, 성장인자와 같은 작은 입자들을 전달해줄 수 있는 통로가 될 수 있다. BBB를 우회하여 후각기관(olfactory)과 3차 신경계 통로(trigeminal neural pathways)를 통해 비침습적 방법으로 뇌에 치료물질을 전달할 수 있으며, 정맥 투여시 생길 수 있는 전신독성을 감소시킬 수 있고, 타겟 영역으로 전달 속도를 높일 수 있는 장점을 가지고 있다. 그러나 현존하는 비강 전달법은 원치 않게 뇌로 줄기세포가 이동할 가능성이 있으며, 후구를 통하지 않은 치료제의 경우 BBB에 의해 효능을 발휘할 수 없고 일부는 전신으로 퍼지게 될 가능성이 있다. 또한 현재 동물실험을 통하여 제시되고 있는 비강전달법은 비강내 점막을 효소로 용해하여 줄기 세포를 주입 한 후 비강을 폐쇄하는 방법으로 생리학적 barrier가 제거된 상태가 되므로 임상에서는 적용할 수 없는 방법이다.Therefore, in order to apply stem cells to the treatment of brain injury, a nasal delivery method has been proposed as a delivery route for stem cell therapeutics. The nasal delivery method does not need to cause unnecessary brain damage, is easy to repeat administration, and can be a channel to deliver small particles such as growth factors. By bypassing the BBB, therapeutic substances can be delivered to the brain in a non-invasive way through the olfactory and trigeminal neural pathways, and can reduce systemic toxicity that may occur during intravenous administration, and target area It has the advantage of increasing the transfer speed. However, the existing nasal delivery method has the potential to cause unwanted migration of stem cells to the brain, and in the case of therapeutic agents that do not go through the olfactory bulb, the BBB may not be effective, and there is a possibility that some will spread throughout the body. In addition, the nasal delivery method, which is currently being presented through animal experiments, is a method that dissolves the nasal mucosa with an enzyme, injects stem cells, and then closes the nasal cavity. .
이에 본 발명자들은 상기 뇌손상 줄기세포치료제의 종래 전달루트의 문제점을 해결하고 임상에도 활용할 수 있는 줄기세포 치료제가 비강 내로 전달된 동물 모델(이하 “이건 모델”이라 함) 및 이를 제공하는데 그 목적이 있다. Accordingly, the present inventors solve the problems of the conventional delivery route of the brain injury stem cell treatment agent and provide an animal model (hereinafter referred to as "this model") in which a stem cell treatment agent that can be used clinically is delivered intranasally and the same. have.
이에 따라 본 발명이 구체적으로 이루고자 하는 기술적 과제는, 뇌손상 줄기세포 치료제를 비강 내로 전달하여 그 효능을 확인하는데 활용될 수 있는 동물 모델을 제공하는데 그 목적이 있다.Accordingly, an object of the present invention is to provide an animal model that can be used to confirm the efficacy of a brain injury stem cell therapeutic agent by intranasal delivery.
본 발명이 이루고자 하는 또 다른 기술적 과제는 비강내 전달법과 동물 모델을 이용하여 줄기세포 치료제의 스크리닝 방법을 제공하는 것에 그 목적이 있다.Another technical problem to be achieved by the present invention is to provide a method for screening a stem cell therapeutic agent using an intranasal delivery method and an animal model.
본 발명의 일 실시 형태로 치료물질의 성능을 확인하기 위한 동물(사람을 제외함) 모델을 들 수 있는데, 뇌 손상이 형성된 동물(사람을 제외함) 모델의 비중격(nasal septum)과 점막(mucosa) 사이로 치료물질이 주입되는 것을 특징으로 한다. In an embodiment of the present invention, there is an animal (excluding humans) model for confirming the performance of a therapeutic substance, and the nasal septum and mucosa of an animal (excluding humans) model with brain damage. ), characterized in that the therapeutic material is injected between them.
상기 뇌 손상은, 외상성 뇌손상 혹은 신경퇴행성 뇌질환에 기인하는 뇌손상을 포함하며, 신경퇴행성 뇌질환의 구체적인 예로 파킨슨 병, 헌팅톤 병, 뇌경색, 뇌출혈 등을 들 수 있다.The brain damage includes brain damage caused by traumatic brain injury or neurodegenerative brain disease, and specific examples of neurodegenerative brain disease include Parkinson's disease, Huntington's disease, cerebral infarction, cerebral hemorrhage, and the like.
상기 외상성 뇌손상은, 뇌피질 충격 손상 모델(controlled cortical impact (CCI) model); Marmarou 자유 낙하 모델(폐쇄성 뇌손상 모델) 혹은 Feeney 자유 낙하 모델(개방형 뇌손상 모델)의 자유 낙하 모델(weight drop model); 유체 타진 모델(fluid percussion model); 및 시신경 신전 손상 모델(optic nerve stretch injury model); 중 어느 하나에 따른 외상성 뇌손상인 것이 바람직하다.The traumatic brain injury is, cortical impact injury model (controlled cortical impact (CCI) model); the weight drop model of the Marmarou free fall model (obstructed brain injury model) or the Feeney free fall model (open brain injury model); fluid percussion model; and an optic nerve stretch injury model; It is preferable that it is a traumatic brain injury according to any one of them.
상기 동물 모델은 쥐, 토끼 혹은 돼지이고, 상기 뇌 손상은, 뇌피질 충격 손상 모델 (controlled cortical impact (CCI) model)에 따라 동물 모델에 발생된 외상성 뇌손상인 것이 더욱 바람직하다.The animal model is a rat, rabbit or pig, and the brain injury is more preferably a traumatic brain injury generated in an animal model according to a controlled cortical impact (CCI) model.
본 발명의 다른 실시 형태로, 치료물질의 성능을 확인하기 위한 동물(사람을 제외함) 모델의 제조 방법을 들 수 있으며, 동물 모델에 뇌 손상을 형성하는 제1 단계; 및 뇌 손상이 형성된 동물 모델의 비중격(nasal septum)과 점막(mucosa) 사이에 치료물질을 주입하는 제2단계;를 포함하는 것이 바람직하다.In another embodiment of the present invention, there is a method for manufacturing an animal (excluding humans) model for confirming the performance of a therapeutic substance, the first step of forming brain damage in the animal model; and a second step of injecting a therapeutic material between the nasal septum and the mucosa of the animal model in which brain damage is formed.
상기 제2 단계의 치료물질을 주입하는 단계는, 동물 모델의 비골에 유아골절(greenstick fracture)을 형성시키는 단계; 동물 모델의 비강을 통해 비중격을 열어, 비중격과 점막 사이에 포켓을 형성하는 단계; 및 상기 포켓으로 치료물질을 주입하는 단계;를 포함할 수 있다.The step of injecting the therapeutic material in the second step may include: forming a greenstick fracture in the fibula of an animal model; opening the nasal septum through the nasal cavity of the animal model to form a pocket between the nasal septum and the mucous membrane; and injecting a therapeutic material into the pocket.
상기 비중격과 점막 사이에 형성되는 포켓의 부피는 100~150 ㎕인 것이 바람직하고, 상기 포켓으로 주입되는 줄기세포 치료물질은 주사 방식으로 주입되는 것이 더욱 바람직하다.The volume of the pocket formed between the nasal septum and the mucous membrane is preferably 100 to 150 μl, and the stem cell therapeutic material injected into the pocket is more preferably injected by injection.
상기 제2단계 이후에, 형성된 유아골절의 골편을 복구 하고 봉합하는 단계;를 추가로 더 포함하는 것도 가능하다.After the second step, repairing and suturing the bone fragments of the formed infant fractures; it is also possible to further include.
상기 동물 모델에 뇌손상을 형성시킨 후, 약 10분 후에 치료 물질을 주입하는 것이 바람직하며, 상기 치료물질은, 줄기세포의 단독으로 구성된 치료물질 혹은 줄기세포가 포접된 줄기세포 전달체를 포함하는 것이 더욱 바람직하다.After brain damage is formed in the animal model, it is preferable to inject a therapeutic substance about 10 minutes later, and the therapeutic substance includes a therapeutic substance composed of stem cells alone or a stem cell delivery system in which stem cells are encapsulated. more preferably.
또한, 상기 제2단계 이후에, 주입된 줄기세포 치료물질의 효과를 확인하는 제3단계;를 더 포함할 수 있으며, 상기 제3단계는, 뇌손상 형성 후 3일째의 조직학적 분석; 3, 7, 14, 21일째의 MRI 분석; 및 뇌손상 형성 후 7일 동안의 modified Neurological Severity Score (mNSS); 중에서 적어도 어느 하나 이상이 수행되는 것이 바람직하다.In addition, after the second step, a third step of confirming the effect of the injected stem cell therapeutic material; may further include, the third step, histological analysis of the third day after brain damage formation; MRI analysis on
본 발명은, 최소 침습적으로 비강내, 좀 더 구체적으로는 동물 모델의 비중격과 점막 사이에 줄기세포 치료제를 이식함으로써, 비강내 전달법의 혈액뇌장벽(BBB, blood brain barrier)을 우회할 수 있어 직접적인 뇌손상을 피할 수 있는 장점 및 반복적인 투여의 용이성을 갖는다.The present invention can bypass the blood brain barrier (BBB) of the intranasal delivery method by minimally invasively implanting a stem cell therapeutic agent between the nasal septum and the mucous membrane of an animal model, more specifically, intranasally. It has the advantage of avoiding direct brain damage and the ease of repeated administration.
또한, 후구(olfactory bulb)와 가까운 위치에 있는 후각상피에 줄기세포 치료제를 국부 전달시킴으로써, 줄기세포에서 방출되는 신경영양성장인자가 주로 후각기관 경로만을 통하여 뇌에 전달되도록 함으로써, 뇌손상 영역까지 불필요한 세포의 이동 없이 파라크라인 효과가 전달될 수 있는 효과가 존재한다.In addition, by locally delivering a stem cell therapeutic agent to the olfactory epithelium in a location close to the olfactory bulb, the neurotrophic growth factor released from stem cells is mainly delivered to the brain through the olfactory organ route, which is unnecessary to the brain damage area. There is an effect that the paracrine effect can be transmitted without cell migration.
그리고, 뇌손상을 갖는 랫(rat)에 줄기세포 치료제를 비강내로 이식하는, 랫 모델(rat model)의 제작 방법은 다양한 뇌손상 줄기세포 치료제를 평가할 수 있는 플랫폼으로 활용될 수 있으며, 실제 임상에서 뇌손상 줄기세포 치료제 비강내 이식시 효과를 확인할 수 있는 모델로 활용 가치가 높은 장점이 존재한다.In addition, the method for producing a rat model, in which a stem cell therapeutic agent is intranasally transplanted into a rat with brain injury, can be utilized as a platform for evaluating various brain injury stem cell therapeutic agents, and in actual clinical practice As a model that can confirm the effect of brain injury stem cell treatment intranasal transplantation, there is an advantage of high use value.
도 1는 랫에 외상성 뇌손상을 만들기 위하여 뇌피질 충격 손상 모델 (controlled cortical impact (CCI) model)을 제작하는 과정(a) 및 줄기세포 치료제의 이식 단계(b)를 나타낸 것이다.
도 2는 이건 출원발명에 따른 동물 모델을 통하여 줄기세포 치료제(BDNF고발현 사람 골수유래 중간엽줄기세포가 포접된 실크-젤라틴 혼합 하이드로겔, hydrogel encapsulated BDNF-hMSC)를 이식하는 과정을 나타낸 것이다.
도 3a는 이건 출원발명에 따른 동물 모델에 대하여 줄기세포 치료제가 이식되는 위치(비중격과 점막 사이 포켓)를 보여주는 사진이고, 도 3b는 이식된 후의 모습과 PKH26(붉은 형광)으로 레이블링된 줄기세포 치료제를 지정 위치에 이식 한 후, 이식 성공 여부를 확인하고, 시간의 경과에 따라 이식 위치에 지속적으로 위치하고 있는지를 확인한 결과이다.
도 4는 개두술만 진행한 랫(동물 모델)과 개두술 후 외상성 뇌손상 유도 랫에 줄기세포 치료제를 이식한 후 뇌를 채취하여 영역별 신경영양성장인자의 양을 비교한 결과이다.
도 5a와 5b는 외상성 뇌손상으로 인하여 발생되는 신경세포 사멸이 비강 내 hydrogel로 encapsulation된 BDNF-hMSCs 이식에 의하여 억제됨을 보여주는 조직학적 결과이다.
도 6a와 6b는 외상성 뇌손상으로 인하여 발생되는 수상돌기(dendrite) 감소가 비강 내 hydrogel로 encapsulation된 BDNF-hMSCs의 이식에 의하여 억제됨을 보여주는 조직학적 결과이다.
도 7a와 7b는 외상성 뇌손상으로 인하여 발생되는 산화적 손상(Oxidative stress)이 비강 내 hydrogel로 encapsulation된 BDNF-hMSCs의 이식에 의하여 억제됨을 보여주는 조직학적 결과이다.
도 8a와 8b는 외상성 뇌손상으로 인하여 발생되는 지연성 신경세포 사멸 (delayed neuronal death)이 비강 내 hydrogel로 encapsulation된 BDNF-hMSCs의 이식에 의하여 억제됨을 보여주는 조직학적 결과이다.
도 9은 비강 내 hydrogel로 encapsulation된 BDNF-hMSCs의 이식이 뇌의 해마에 BDNF의 발현을 증가시킴을 보여주는 조직학적 결과이다.
도 10a 내지 10d는 비강 내 hydrogel로 encapsulation된 BDNF-hMSCs의 이식 후 modified Neurological Severity Score (mNSS)와 MRI 검사 결과를 시행하는 시점을 보여주는 그림이다.Figure 1 shows the process (a) of making a cortical impact injury model (controlled cortical impact (CCI) model) to create a traumatic brain injury in rats (a) and the transplantation step (b) of a stem cell therapeutic agent.
2 shows the process of transplanting a stem cell therapeutic agent (silk-gelatin mixed hydrogel, hydrogel encapsulated BDNF-hMSC in which BDNF high-expressing human bone marrow-derived mesenchymal stem cells are encapsulated) through an animal model according to the present invention.
Figure 3a is a photograph showing the location (pocket between the nasal septum and the mucous membrane) where the stem cell therapeutic agent is implanted with respect to the animal model according to the present invention, and Figure 3b is the appearance after transplantation and the stem cell therapeutic agent labeled with PKH26 (red fluorescence) This is the result of checking whether the transplant was successful or not after transplanting it to the designated location, and whether it was continuously positioned at the transplant location over time.
4 is a result of comparing the amount of neurotrophic growth factor by region by collecting brains after transplanting a stem cell therapeutic agent into a rat (animal model) that has only undergone craniotomy and a rat for inducing traumatic brain injury after craniotomy.
5A and 5B are histological results showing that neuronal cell death caused by traumatic brain injury is inhibited by intranasal hydrogel-encapsulated BDNF-hMSCs transplantation.
6A and 6B are histological results showing that the reduction of dendrites caused by traumatic brain injury is inhibited by implantation of BDNF-hMSCs encapsulated with intranasal hydrogel.
7a and 7b are histological results showing that oxidative stress caused by traumatic brain injury is inhibited by implantation of BDNF-hMSCs encapsulated with intranasal hydrogel.
8A and 8B are histological results showing that delayed neuronal death caused by traumatic brain injury is inhibited by intranasal hydrogel-encapsulated BDNF-hMSCs transplantation.
9 is a histological result showing that transplantation of BDNF-hMSCs encapsulated with intranasal hydrogel increases the expression of BDNF in the hippocampus of the brain.
10A to 10D are diagrams showing the time point at which the modified Neurological Severity Score (mNSS) and MRI test results are performed after implantation of BDNF-hMSCs encapsulated with intranasal hydrogel.
이하 본 발명의 바람직한 실시 예를 통해 상세히 설명하기에 앞서, 본 명세서 및 청구범위에 사용된 용어나 단어는 통상적이거나 사전적인 의미로 한정하여 해석되어서는 아니 되고, 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야 함을 밝혀 둔다.Hereinafter, prior to being described in detail through preferred embodiments of the present invention, terms or words used in the present specification and claims should not be construed as being limited to conventional or dictionary meanings, but meanings consistent with the technical spirit of the present invention. and should be interpreted as a concept.
본 발명의 명세서 전반에 걸쳐서, 어떤 부분이 어떤 구성요소를 "포함" 한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.Throughout the specification of the present invention, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated. .
또한, 각 단계들에 있어 식별부호가 사용될 경우에, 이는 설명의 편의를 위하여 사용되는 것으로 식별부호는 각 단계들의 순서를 설명하는 것이 아니며, 각 단계들은 문맥상 명백하게 특정 순서를 기재하지 않는 이상 명기된 순서와 다르게 실시될 수 있다. 즉, 각 단계들은 명기된 순서와 동일하게 실시될 수도 있고, 실질적으로 동시에 실시될 수도 있으며, 반대의 순서대로 실시되는 것도 가능하다.In addition, when an identification code is used in each step, it is used for convenience of description, and the identification code does not describe the order of each step, and each step is specified unless a specific order is clearly stated in context. It may be performed in a different order from the prescribed order. That is, each step may be performed in the same order as specified, may be performed substantially simultaneously, or may be performed in the reverse order.
이하에서는 본 발명에 따른 치료물질이 비강 내로 이식된 동물 모델 및 이의 제조방법에 관하여 보다 상세히 설명하고자 한다. 구체적인 실시예에 있어서, 동물 모델로 랫(쥐) 모델을 중심으로 설명하고 있으나, 이에 한정되는 것은 아니며, 쥐 외에도 토끼 혹은 돼지 등 비강 내로 치료물질이 주입될 수 있는 동물 모델이 모두 포함될 수 있다. Hereinafter, an animal model in which the therapeutic substance according to the present invention is implanted into the nasal cavity and a method for manufacturing the same will be described in more detail. In a specific embodiment, the animal model is mainly described as a rat (rat) model, but is not limited thereto, and may include any animal model in which a therapeutic material can be injected into the nasal cavity, such as a rabbit or a pig, in addition to the rat.
앞서 살펴본 바와 같이, 이건 발명의 목적을 달성하기 위한 본 발명의 일 실시 형태는, 뇌손상 줄기세포 치료제의 효과를 확인할 수 있도록, 뇌피질 충격 손상 모델(controlled cortical impact (CCI) model)에 따라 외상성 뇌손상이 발생한 랫에게, 비중격과 점막 사이에 주사 방법으로 뇌손상 줄기세포 치료제를 이식하는 랫 모델을 포함한다As described above, one embodiment of the present invention for achieving the object of the present invention is traumatic according to a controlled cortical impact (CCI) model, so as to confirm the effect of a brain injury stem cell therapeutic agent. Includes a rat model in which brain injury stem cell therapy is implanted between the nasal septum and the mucous membrane in rats with brain injury.
상기 비중격과 점막 사이 주사시 랫의 비골에 유아골절(greenstick fracture)을 형성시킨 후 비강을 통해서 비중격을 열어, 비중격과 점막 사이에 포켓을 만든 다음, 뇌손상 줄기세포 치료제를 이식하는 랫 모델을 제작하는 것이 바람직하다.When injecting between the septum and the mucous membrane, after forming a greenstick fracture in the fibula of the rat, the septum is opened through the nasal cavity, a pocket is created between the septum and the mucous membrane, and then a rat model is implanted with a brain injury stem cell treatment. It is preferable to do
특히, 상기 비중격과 점막 사이의 포켓은 부피를 150㎕로 이내로 만들어 뇌손상 줄기세포 치료제를 이식하는 랫 모델로 제작하는 것이 바람직한데, 상기 뇌손상 줄기세포 치료제를 주사하는 단계에서 21 게이지의 주사바늘이 끼워진 실린지를 이용하여 주사하는 방식으로 뇌손상 줄기세포 치료제가 이식된 동물 모델인 랫 모델을 제작하는 것이 바람직하다.In particular, the pocket between the nasal septum and the mucous membrane is preferably manufactured as a rat model in which the brain injury stem cell treatment agent is implanted by making the volume within 150 μl. It is preferable to produce a rat model that is an animal model transplanted with a brain injury stem cell therapeutic agent by injection using the inserted syringe.
상기 뇌손상 줄기세포 치료제를 주사하는 단계에서 주사 후, 최초 형성시킨 유아골절 골편을 복구 하고 봉합하는, 뇌손상 줄기세포 치료제를 이식하는 랫 모델로 제작하는 것이 바람직하다.After the injection in the step of injecting the brain injury stem cell therapeutic agent, it is preferable to produce a rat model implanting the brain injury stem cell therapeutic agent, which repairs and sutures the initially formed infant fracture fragment.
상기 외상성 뇌손상을 입은 랫은, 뇌피질 충격 손상 모델 (controlled cortical impact (CCI) model)에 따라 외상성 뇌손상이 발생한 랫 뿐만 아니라, Marmarou 자유 낙하 모델 (폐쇄성 뇌손상 모델), Feeney 자유 낙하 모델 (개방형 뇌손상 모델) 등의 자유 낙하 모델 (weight drop model), 유체 타진 모델(fluid percussion model) 혹은 시신경 신전 손상 모델(optic nerve stretch injury model) 등에 따라 외상성 뇌손상이 발생한 랫 중에서 적어도 하나 이상을 포함 할 수 있다. The rats with traumatic brain injury, as well as rats with traumatic brain injury according to the cortical impact injury model (controlled cortical impact (CCI) model), Marmarou free fall model (obstructive brain injury model), Feeney free fall model ( Including at least one or more of rats that have suffered traumatic brain injury according to a weight drop model, such as an open brain injury model), a fluid percussion model, or an optic nerve stretch injury model can do.
상기 이건 모델은 외상성 뇌손상이 발생한 랫 뿐만 아니라 파킨슨 병, 헌팅톤 병, 뇌경색, 뇌출혈 등의 다양한 신경퇴행성 뇌질환이 발생한 랫 중에서 적어도 하나 이상을 포함하는 랫으로부터 제작될 수 있다.The model can be produced from rats including at least one or more of rats that have suffered various neurodegenerative brain diseases, such as Parkinson's disease, Huntington's disease, cerebral infarction, and cerebral hemorrhage, as well as rats with traumatic brain injury.
상기 뇌손상 줄기세포 치료제를 이식하는 랫 모델에 대해서, 뇌피질 충격 손상 모델에 따라 외상성 뇌손상을 발생 시킨지 10분 후 뇌손상 줄기세포 치료제를 주사하는 것이 바람직하다.With respect to the rat model transplanting the brain injury stem cell therapeutic agent, it is preferable to inject the brain injury stem cell
본 발명에 사용되는 치료제는, 줄기세포의 단독 구성 치료제, 줄기세포가 포접된 줄기세포 전달체(하이드로겔 등)로 구성된 치료제 등 다양한 액상 형태의 줄기세포 치료제가 사용될 수 있다. 이건 모델은 줄기세포의 종류, 주입될 세포의 수에 따른 다양한 효능을 확인할 수 있는 모델이므로, 특별히 줄기 세포 치료제의 범위가 한정되는 것은 아니며, 다양한 형태의 뇌손상 줄기세포 치료제가 적용될 수 있다. As the therapeutic agent used in the present invention, various liquid-type stem cell therapeutic agents, such as a therapeutic agent composed of stem cells alone, and a therapeutic agent composed of a stem cell carrier (hydrogel, etc.) in which stem cells are encapsulated, may be used. Since this model is a model that can confirm various efficacy according to the type of stem cell and the number of cells to be injected, the scope of the stem cell treatment is not particularly limited, and various types of brain damage stem cell treatment can be applied.
본 발명의 또 다른 실시 형태로, 이 건 모델에 치료제 혹은 치료물질을 적용하는 단계; 및 상기 치료제 혹은 치료물질이 적용된 랫 모델에 대한 치료제 혹은 치료물질의 효과를 확인하는 단계를 추가로 포함하는, 뇌손상 치료제 혹은 치료물질을 스크리닝하는 방법을 포함 한다.In another embodiment of the present invention, the method comprising: applying a therapeutic agent or a therapeutic substance to the tendon model; And it includes a method of screening for a therapeutic agent or therapeutic agent for brain damage, further comprising the step of confirming the effect of the therapeutic agent or therapeutic substance on the rat model to which the therapeutic agent or therapeutic substance is applied.
한편, 본 발명의 또 다른 실시 형태는, 상기 제조 방법으로 제조된 랫 모델에서 효과를 확인하는 단계가 외상성 뇌손상 발생 후 3일째 조직학적 분석, 3, 7, 14, 21일째 MRI 분석, 발생 7일간 modified Neurological Severity Score (mNSS)를 수행하는 것을 특징으로 하는, 뇌손상 줄기세포 치료제를 스크리닝하는 방법을 더 포함한다.On the other hand, in another embodiment of the present invention, the step of confirming the effect in the rat model prepared by the manufacturing method is histological analysis on the 3rd day after the occurrence of the traumatic brain injury, the MRI analysis on the 3rd, 7th, 14th, and 21st days, the
이하에서는, 본 발명에 따른 뇌손상 줄기세포 치료제의 효과를 확인할 수 있는 뇌손상 동물 모델 및 이의 제조 방법에 관하여 하기의 실시예에 의해 보다 상세히 설명하고자 한다. 다만, 하기에 기재된 실시예는 본 발명의 구체적인 구현예를 예시하는 것에 불과하고, 본 발명의 기술적 사상과 내용이 하기 실시예에 의해 한정되어 해석되는 것은 아님을 밝혀두고자 한다.Hereinafter, a brain injury animal model capable of confirming the effect of the brain injury stem cell therapeutic agent according to the present invention and a method for manufacturing the same will be described in more detail with reference to the following examples. However, the examples described below are merely illustrative of specific embodiments of the present invention, and the technical spirit and content of the present invention are not limited to and interpreted by the following examples.
[실시예 1][Example 1]
뇌손상의 예시로서 외상성 뇌손상을 일으키기 위하여 뇌피질 충격 손상 모델 (controlled cortical impact (CCI) model)을 기존 방법(R J Hamm 1 , C E Dixon, D M Gbadebo, A K Singha, L W Jenkins, B G Lyeth, R L Hayes, Cognitive Deficits Following Traumatic Brain Injury Produced by Controlled Cortical Impact, J Neurotrauma.. Spring 1992;9(1):11-20. doi: 10.1089/neu.1992.9.11.)으로 제작하였다. As an example of brain injury, a controlled cortical impact (CCI) model was used to induce traumatic brain injury using the existing method (
간략하게 정리하면, 아산화질소(nitrous oxide)와 산소(oxygen)가 70:30으로 혼합된 기체 내로 3% isoflurane을 증발기를 이용하여 흘려보냄으로써, 랫을 흡입 마취 시켰다. 그 후 1-1.5% isoflurane으로 유지하면서 CCI 모델 제작을 진행하였다. Briefly, by flowing 3% isoflurane into a gas in which nitrous oxide and oxygen are mixed at a ratio of 70:30 using an evaporator, the rats were anesthetized by inhalation. After that, the CCI model was manufactured while maintaining 1-1.5% isoflurane.
두부를 동물실험용 뇌 정위 기구에 고정시키고, 우측 반구에서 미드 라인의 우측면으로 2.8 mm, 브레그마의 뒤에서 3 mm 부위에, 휴대용 드릴을 이용하여 5 mm 직경의 개두술 (craniotomy)을 시행하였다. The head was fixed in a stereotaxic apparatus for animal experiments, and a craniotomy with a diameter of 5 mm was performed using a hand-held drill at 2.8 mm from the right hemisphere to the right side of the midline and 3 mm behind the bregma.
이후 뇌피질 충격기구를 사용하여, 3 mm flat tip impactor를 5 m/s 속도로 3 mm 깊이로 하강시켰다. 모든 랫은 수술 동안, 수술 후, 회복 때까지도 온혈동물 blanket control unit을 이용하여 36-37.5℃ 의 온도가 유지되게 하였다(도 1a). 줄기세포 치료제는 손상 후 10분 내로 처리하였으며 손상 후 3, 7, 14, 21일에 손상정도를 측정하였다. Sham-operated group은 개두술만 진행한 것으로 하였다(도 1b).Then, using a cortical impact device, a 3 mm flat tip impactor was lowered to a depth of 3 mm at a speed of 5 m/s. All rats were allowed to maintain a temperature of 36-37.5°C using a warm-blooded blanket control unit during surgery, post-surgery, and until recovery (FIG. 1a). Stem cell therapy was treated within 10 minutes after injury, and the degree of damage was measured at 3, 7, 14, and 21 days after injury. In the Sham-operated group, only craniotomy was performed (Fig. 1b).
[실시예 2][Example 2]
외상성 뇌손상을 일으킨 랫에 줄기세포 치료제 비강내 주입 동물 모델을 제작하였다(도 2 참조). 간략하게, 랫의 콧등 영역을 Povidone-Iodine 10% 스왑스틱으로 소독을 하였다. 코의 배등면을 따라 1% 리도카인 1 ㎖(에피네프린 1:100,000 혼합)을 주사하여 국소적 마취를 시켰다. 오른쪽 비강을 통하여 비중격을 열기 위하여 수술용 가위를 이용해서 유아골절(green stick fracture)을 만든다. An animal model for intranasal injection of stem cell therapeutics into rats with traumatic brain injury was prepared (see FIG. 2 ). Briefly, the nasal region of rats was disinfected with a Povidone-
그 후 비중격과 점막 사이에 엘레베이터를 이용하여 150 ㎕이내의 줄기세포 치료제가 들어갈 수 있는 포켓을 만들어서 줄기세포 치료제가 이식될 수 있게 하였다. 그런 다음 21 G의 뭉툭한 주사바늘이 끼워진 해밀턴 실린지를 이용하여 1 x 106 개의 세포가 함유된 hydrogel 100 ㎕를 포켓 내로 주사하였다. 본 실시예에서 세포 수는 1x106 개로 정하여 진행하였으나 150 ㎕의 부피 내에서 테스트를 원하는 세포 수를 정하여 적용 할 수 있다. Thereafter, using an elevator between the nasal septum and the mucous membrane, a pocket was created in which the stem cell therapeutic agent within 150 μl could be inserted so that the stem cell therapeutic agent could be implanted. Then, using a Hamilton syringe fitted with a 21 G blunt needle, 100 μl of hydrogel containing 1 x 10 6 cells was injected into the pocket. In this example, the number of cells was determined to be 1x10 6 , but the number of cells to be tested can be determined and applied within a volume of 150 μl.
[실시예 3][Example 3]
줄기세포 치료제의 비강내 이식후 안정성을 확인하기 위하여 줄기세포에 PKH26(붉은 형광)을 라벨하여 이식한지 3, 7, 14일째 쥐를 희생하고 이식 부분이 포함된 조직을 절개 후 입체 현미경으로 확인한 후(도 3a), 동결 섹션하여 컨포칼 형광현미경을 통하여 형광 발현도를 확인하였다(도 3b). In order to check the stability of the stem cell therapeutic agent after intranasal transplantation, the stem cells were labeled with PKH26 (red fluorescence) and sacrificed on the 3rd, 7th, and 14th days after transplantation. (FIG. 3a), the degree of fluorescence expression was confirmed through a confocal fluorescence microscope by freezing section (FIG. 3b).
[실시예 4][Example 4]
hydrogel로 encapsulation된 BDNF-hMSCs를 비강내 주사 후 뇌손상 영역에서 BDNF의 발현이 증가되는지의 여부를 확인하였다. 앞선 [실시예 1]에서 개두술만을 진행한 군(sham)과 개두술 후 CCI를 진행한 군에 줄기세포 치료제를 이식한 후 뇌의 여러 부위를 채취하여 western blot으로 BDNF 단백질 발현 정도를 확인하였다(도 4 참조). After intranasal injection of hydrogel-encapsulated BDNF-hMSCs, it was checked whether the expression of BDNF was increased in the brain injury region. In [Example 1], after transplanting the stem cell treatment to the group that had only craniotomy (sham) and the group that had undergone CCI after craniotomy, several parts of the brain were collected and the expression level of BDNF protein was confirmed by western blot (Fig. see 4).
간략하게 설명하면, hydrogel로 encapsulation된 BDNF-hMSCs를 비강내 이식후 3, 7, 14일에 western blot 분석을 위한 뇌 샘플을 채취하여 차가운 식염수에 담갔다. 각 샘플을 액체질소에 얼린 후 1mL의 차가운 RIPA buffer와 섞어서 호모게나이저로 균질화시켰다. 혼합물을 4℃에서 2시간 저장 하고, 상층액을 30분간 15,000 rpm에서 원심분리 하였다. 상청액 내의 단백질은 Bradford assay를 이용하여 ELISA법으로 측정하여 정량하였다. 채취한 조직에서 분리된 단백질 동량을 SDS-polyacrylamide 겔 상에서 전기영동하였고, PVDF 멤브레인에 트랜스퍼 하였다. 비특이적 결합은 10% skim milk로 1시간 가량 실온에서 블로킹하였다. 그 후 멤브레인을 1차 항체 (1:1000 희석비율)를 처리하여 하루 동안 4℃에 저장하였다. Briefly, after intranasal implantation of hydrogel-encapsulated BDNF-hMSCs, brain samples for western blot analysis were collected and immersed in cold saline at 3, 7, and 14 days. Each sample was frozen in liquid nitrogen, mixed with 1 mL of cold RIPA buffer, and homogenized with a homogenizer. The mixture was stored at 4°C for 2 hours, and the supernatant was centrifuged at 15,000 rpm for 30 minutes. Proteins in the supernatant were measured and quantified by ELISA using Bradford assay. The same amount of protein isolated from the collected tissue was electrophoresed on an SDS-polyacrylamide gel and transferred to a PVDF membrane. Non-specific binding was blocked with 10% skim milk at room temperature for about 1 hour. Thereafter, the membrane was treated with a primary antibody (1:1000 dilution ratio) and stored at 4°C for one day.
이후 멤브레인을 상온에서 1시간동안 종에 적합한 2차 항체(goat anti-mouse, goat anti-rabbit IgG)를 처리하여 인큐베이션 시켰다. Enhanced Chemiluminescence (ECL)법으로 각각의 단백질 발현 양을 확인하고 Image J로 밴드의 정량을 시행하였다. Then, the membrane was treated with a species-appropriate secondary antibody (goat anti-mouse, goat anti-rabbit IgG) and incubated for 1 hour at room temperature. The expression level of each protein was confirmed by Enhanced Chemiluminescence (ECL) method, and band quantification was performed using Image J.
[실시예 5][Example 5]
이건 모델에 BDNF-hMSC없이 vehicle(hydrogel)만을 처리한 TBI 그룹(TBI-veh), hydrogel로 encapsulation된 BDNF-hMSCs을 처리한 TBI 그룹(TBI-BDNF-hMSCs)을 만들고 퇴화되는 신경세포를 확인하기 위해서 외상성 뇌손상을 일으킨지 3일째 쥐를 희생시켜 Fluoro-Jade B (FJB) 염색을 시행하였다. In this model, we made a TBI group (TBI-veh) treated with vehicle (hydrogel) only without BDNF-hMSC, and a TBI group treated with BDNF-hMSCs encapsulated with hydrogel (TBI-BDNF-hMSCs) and confirmed the degenerating neurons. For this purpose, mice were sacrificed on the 3rd day after traumatic brain injury and Fluoro-Jade B (FJB) staining was performed.
간략하게, 치료제를 이식한 지 3일째 우레탄 1.5 g/kg(in saline of 0.9% NaCl)을 0.01 ㎖/g body weight의 볼륨으로 랫의 복강내로 주사하여 깊게 마취시켰다. 랫의 심장내부로 식염수를 퍼퓨전하였고, 4% 파라 포름알데하이드로 1시간 고정하였으며 cryoprotection을 위해서 30% 수크로스에 침지시켰다. 이후 전체 뇌를 얼려서 30 ㎛ 두께로 화관상 동결섹션을 하였다. Briefly, on the 3rd day after implantation of the therapeutic agent, 1.5 g/kg of urethane (in saline of 0.9% NaCl) was injected intraperitoneally into the rat at a volume of 0.01 ml/g body weight to deeply anesthetize. Saline was perfused into the heart of the rat, fixed with 4% para-formaldehyde for 1 hour, and immersed in 30% sucrose for cryoprotection. After that, the whole brain was frozen and a corolla frozen section was made with a thickness of 30 μm.
각 랫으로부터 꼬리쪽에서 정수리 지점쪽으로 2.92~4.56 ㎜ 영역에서 270 ㎛ 간격의 6개의 화관상 섹션을 얻어 FJB염색을 하였으며 confocal microscope을 이용해서 480 ㎚와 524 ㎚에서 확인하였다. 맹검 관찰자에 의하여 해마 반구에서 해마 CA1, GCL 및 Hilus에서 FJB (+) 뉴런의 총 수를 세었다. 데이터는 영역당 퇴화하고 있는 뉴런의 평균수로 나타냈다.Six corolla-shaped sections with a spacing of 270 μm were obtained from each rat in an area of 2.92 to 4.56 mm from the tail to the parietal point, followed by FJB staining, and confirmed at 480 nm and 524 nm using a confocal microscope. Total number of FJB (+) neurons in hippocampal CA1, GCL and Hilus in hippocampal hemispheres were counted by blinded observers. Data are presented as the average number of degenerating neurons per area.
도 5a는 외상성 뇌손상 후 해마의 CA1, Dentate gyrus (DG)의 Granular cell layer (GCL)와 Hilus 에서 신경세포 사멸 관찰 결과이고, 이에 대한 정량 결과를 도 5b로 제시되어 있다. 비강 내 hydrogel로 encapsulation된 BDNF-hMSCs의 이식으로 상기 해마의 모든 위치에서 이러한 퇴화성 신경세포의 수가 상당히 감소됨을 알 수 있었으며, 이는 비강 내 hydrogel로 encapsulation된 BDNF-hMSCs의 이식이 외상성 뇌손상 후에 일어나는 신경세포 사멸을 억제함을 나타내는 결과이다.FIG. 5a is the result of observation of neuronal cell death in CA1 of the hippocampus, granular cell layer (GCL) of the dentate gyrus (DG), and the hilus after traumatic brain injury, and the quantitative results thereof are shown in FIG. 5b. Transplantation of BDNF-hMSCs encapsulated with intranasal hydrogel significantly reduced the number of these degenerative neurons in all locations in the hippocampus, which indicates that implantation of BDNF-hMSCs encapsulated with intranasal hydrogel occurs after traumatic brain injury. It is a result indicating that it inhibits neuronal cell death.
[실시예 6][Example 6]
이 건 모델에 BDNF-hMSC없이 vehicle(hydrogel)만을 처리한 개두술 그룹(Sham-veh), hydrogel로 encapsulation된 BDNF-hMSCs을 처리한 개두술 그룹(Sham-BDNF-hMSCs), BDNF-hMSC없이 vehicle(hydrogel)만을 처리한 TBI 그룹(TBI-veh), hydrogel로 encapsulation된 BDNF-hMSCs을 처리한 TBI 그룹(TBI-BDNF-hMSCs)을 만들고, 수상돌기 (dendrite) 손상을 확인하기 위해서 외상성 뇌손상을 일으킨지 3일째 쥐를 희생시켜 mirotubule-associated protein 2(MAP-2) 항체로 면역형광염색을 시행하였다. In this model, the craniotomy group treated only with vehicle (hydrogel) without BDNF-hMSC (Sham-veh), the craniotomy group treated with BDNF-hMSCs encapsulated with hydrogel (Sham-BDNF-hMSCs), and without BDNF-hMSC vehicle (hydrogel) ) treated only TBI group (TBI-veh), hydrogel encapsulated BDNF-hMSCs treated TBI group (TBI-BDNF-hMSCs), and whether traumatic brain injury was caused to check dendrite damage On the third day, mice were sacrificed and immunofluorescent staining was performed with mirotubule-associated protein 2 (MAP-2) antibody.
간략하게 설명하면, 앞선 실시예 5의 조직처리 단계를 거친 섹션을, 1.2% H2O2에 20분간 실온처리하여 내재성 퍼록시다아제의 활성을 억제시켰다. 인산완충용액(PBS)로 워싱 후 섹션을 rabbit polyclonal anti-MAP2 항체를 0.3% Triton-X-100함유 PBS기반으로 희석하여 (1:500 희석비율) 4℃에서 하루 동안 저장하였다. PBS 워싱후에 섹션은 fluorescent-conjugated 2차 항체 (1:250 희석비율)를 처리하였다. 섹션은 DAPI(1:1000 희석비율)로 counterstaining 하였다. 형광염색된 섹션은 젤라틴 코팅된 슬라이드위에 마운팅하였으며, DPX로 덮었다. 컨포칼 현미경으로 사진을 촬영하였다. Briefly, the section that had undergone the tissue treatment step of Example 5 was treated with 1.2% H2O2 at room temperature for 20 minutes to inhibit the activity of endogenous peroxidase. After washing with phosphate buffer solution (PBS), the sections were diluted with a rabbit polyclonal anti-MAP2 antibody in PBS containing 0.3% Triton-X-100 (1:500 dilution ratio) and stored at 4°C for one day. After washing in PBS, sections were treated with fluorescent-conjugated secondary antibody (1:250 dilution ratio). Sections were counterstained with DAPI (1:1000 dilution ratio). Fluorescently stained sections were mounted on gelatin-coated slides and covered with DPX. Pictures were taken with a confocal microscope.
도 6a는 외상성 뇌손상 유도 후 즉시 hydrogel로 encapsulation된 BDNF-hMSCs을 비강 내에 이식한 뒤 72시간에 수상돌기 손상 (dendritic damage)을 확인하기 위해 mirotubule-associated protein 2 (MAP-2) 항체로 면역형광염색 후 관찰 결과이고, 도 6b는 이에 대한 정량 결과이다. 외상성 뇌손상 후 해마의 CA1과 DG 모두에서 MAP-2 형광강도 및 발현정도가 감소하였으며 이는 곧 dendritic loss가 일어남을 나타내고 있다. 여기에 비강 내 BDNF-hMSCs의 이식한 실험군에서는 해마의 이러한 모든 위치에서 dendritic loss가 억제됨을 알 수 있으며, 이는 비강 내 hydrogel로 encapsulation된 BDNF-hMSCs의 이식이 외상성 뇌손상 후에 일어나는 수상돌기의 손상을 억제함을 나타내는 결과이다.Figure 6a is immunofluorescence with mirotubule-associated protein 2 (MAP-2) antibody to confirm dendritic damage 72 hours after intranasal implantation of BDNF-hMSCs encapsulated with hydrogel immediately after induction of traumatic brain injury It is an observation result after staining, and FIG. 6b is a quantitative result thereof. After traumatic brain injury, MAP-2 fluorescence intensity and expression were decreased in both CA1 and DG of hippocampus, indicating that dendritic loss occurred soon. Here, in the experimental group in which BDNF-hMSCs were transplanted into the nasal cavity, dendritic loss was suppressed at all these locations in the hippocampus. result indicative of inhibition.
[실시예 7][Example 7]
이건 모델에 실시예 6과 같이 그룹을 처리한 후, 같은 조직처리 단계를 거쳐 얻은 섹션에 산화적 손상 (Oxidative stress)정도를 확인하기 위하여 mouse monoclonal anti-4-hydroxynonenal (4-HNE)항체와 rabbit polyclonal anti-nitrotyrosine 항체로 면역형광염색을 시행하였다. 항체 희석 배율은 1:500 으로 수행하였다.This model was treated with a group as in Example 6, and then, mouse monoclonal anti-4-hydroxynonenal (4-HNE) antibody and rabbit Immunofluorescence staining was performed with polyclonal anti-nitrotyrosine antibody. The antibody dilution ratio was 1:500.
도 7a와 7b는 각각, 외상성 뇌손상 유도 후 즉시 hydrogel로 encapsulation된 BDNF-hMSCs을 비강 내에 이식한 뒤 72시간에 oxidative stress를 확인하기 위해 4-Hydroxynonenal(4-HNE)와 Nitrotyrosine 항체로 면역형광염색 후 관찰 결과 및 이에 대한 정량 결과이다. 외상성 뇌손상 후 해마의 CA1에서 4-HNE와 Nitrotyrosine 형광강도 및 발현정도가 sham 그룹에 비해 현저하게 증가하였으며 이는 곧 oxidative stress가 과량 발생됨을 의미한다. 하지만 비강 내 BDNF-hMSCs의 이식한 실험군에서는 이러한 oxidative stress가 억제됨을 알 수 있었는데, 이는 비강 내 hydrogel로 encapsulation된 BDNF-hMSCs의 이식이 외상성 뇌손상 후에 일어나는 산화적 손상을 억제함을 의미한다. 7a and 7b respectively show immunofluorescence staining with 4-Hydroxynonenal (4-HNE) and Nitrotyrosine antibody to confirm oxidative stress 72 hours after intranasal implantation of BDNF-hMSCs encapsulated with hydrogel immediately after induction of traumatic brain injury. These are post-observation results and quantitative results. After traumatic brain injury, the fluorescence intensity and expression levels of 4-HNE and Nitrotyrosine in CA1 of the hippocampus were significantly increased compared to that of the sham group, indicating that oxidative stress was excessively generated. However, it was found that this oxidative stress was suppressed in the experimental group in which BDNF-hMSCs were transplanted into the nasal cavity, which means that transplantation of BDNF-hMSCs encapsulated with intranasal hydrogel inhibited oxidative damage that occurs after traumatic brain injury.
[실시예 8][Example 8]
이건 모델에 실시예 6과 같은 그룹을 처리한 후, 동일한 조직처리 단계를 거쳐 얻은 섹션에 지연성 신경세포 사멸 (delayed neuronal death)정도를 확인하기 위하여 mouse monoclonal anti-NeuN 항체로 면역형광염색을 시행하였으며, 항체 희석 배율은 1:500 이다.After treating this model with the same group as in Example 6, immunofluorescence staining was performed with mouse monoclonal anti-NeuN antibody to confirm the degree of delayed neuronal death in sections obtained through the same tissue treatment step. and the antibody dilution ratio is 1:500.
도 8a와 8b는 각각 외상성 뇌손상 유도 후 즉시 hydrogel로 encapsulation된 BDNF-hMSCs을 비강 내에 이식한 뒤 1주일에 delayed neuronal death를 확인하기 위해 Neuronal Nuclei (NeuN) 항체로 면역형광염색 후 관찰 결과와 이에 대한 정량 결과이다. 8a and 8b show the observation results after immunofluorescence staining with Neuronal Nuclei (NeuN) antibody to confirm delayed
외상성 뇌손상 후 해마의 CA1, Dentate gyrus (DG)의 Granular cell layer (GCL)와 Hilus에서 NeuN positive cell의 수가 현저하게 감소하였으며, 이는 곧 외상성 뇌손상 후 delayed neuronal death가 일어남을 의미한다. 여기에 비강 내 BDNF-hMSCs의 이식한 실험군에서는 해마의 이러한 모든 위치에서 신경세포의 감소가 억제되었는데, 이는 비강 내 hydrogel로 encapsulation된 BDNF-hMSCs의 이식이 외상성 뇌손상 후에 일어나는 지연성 신경세포 사멸을 억제함을 나타내었다.After traumatic brain injury, the number of NeuN positive cells in the hippocampal CA1, granular cell layer (GCL) and hilus of the dentate gyrus (DG) was significantly reduced, which means delayed neuronal death after traumatic brain injury. Here, in the experimental group in which BDNF-hMSCs were transplanted into the nasal cavity, the reduction of neurons at all these locations in the hippocampus was inhibited, which indicates that the implantation of BDNF-hMSCs encapsulated with intranasal hydrogel prevented delayed neuronal death that occurs after traumatic brain injury. showed inhibition.
[실시예 9][Example 9]
이건 모델에 대하여 실시예 6과 같은 그룹을 처리한 후, 동일한 조직처리 단계를 거쳐 얻은 섹션에서 BDNF 발현 정도를 확인하기 위하여 guinea pig polyclonal anti-BDNF 항체로 면역형광염색을 시행하였으며, 항체 희석 배율은 1:400 으로 수향되었다.After treating this model with the same group as in Example 6, immunofluorescence staining was performed with guinea pig polyclonal anti-BDNF antibody to check the level of BDNF expression in sections obtained through the same tissue treatment step, and the antibody dilution ratio was It was moved to 1:400.
도 9는 hydrogel로 encapsulation된 BDNF-hMSCs을 비강 내에 이식한 뒤 1주일째 BDNF를 확인하기 위해 BDNF 항체로 면역형광염색 후 관찰 결과이다. Sham 그룹에서 비강 내 BDNF-hMSCs를 이식한 실험군에서 BDNF의 발현이 대조군에 비해 증가됨을 확인하였다. 하지만 외상성 뇌손상 후 해마의 CA1과 DG에서 BDNF의 발현이 현저하게 감소하였으며, 비강 내 BDNF-hMSCs를 이식한 실험군에서는 해마의 이러한 위치에서 BDNF의 발현이 대조군에 비해 증가됨을 알 수 있다. 이는 비강 내 hydrogel로 encapsulation된 BDNF-hMSCs의 이식이 뇌의 해마에 BDNF를 증가시키기 때문으로 해석된다.9 is an observation result after immunofluorescence staining with a BDNF antibody to confirm BDNF at 1 week after intranasal transplantation of BDNF-hMSCs encapsulated with hydrogel. In the Sham group, it was confirmed that the expression of BDNF was increased in the experimental group in which intranasal BDNF-hMSCs were transplanted compared to the control group. However, after traumatic brain injury, the expression of BDNF in CA1 and DG of the hippocampus was significantly reduced, and in the experimental group transplanted with intranasal BDNF-hMSCs, the expression of BDNF at this location in the hippocampus was increased compared to the control group. This is interpreted as the reason that intranasal hydrogel-encapsulated BDNF-hMSCs transplantation increases BDNF in the hippocampus of the brain.
[실시예 10][Example 10]
이건 모델에 hydrogel로 encapsulation된 BDNF-hMSCs의 비강 내 이식시 외상성 뇌손상으로 인한 신경학적 장애에 어떠한 영향을 미치는지 알아보기 위하여, 이건 모델에 실시예 6과 같은 그룹을 처리한 후, 1주일동안 modified Neurological Severity Score (mNSS) 검사를 시행하였고, 손상 유도후 3, 7, 14, 21일에 MRI 검사를 하여 부종과 손상영역 부피의 변화를 확인하였다. 분석 시점에 대한 도표를 도 10a에 나타내었다.In order to examine the effect of intranasal implantation of BDNF-hMSCs encapsulated with hydrogel in this model on neurological disorders caused by traumatic brain injury, the model was treated with the same group as in Example 6, and then modified for 1 week. Neurological Severity Score (mNSS) was performed, and MRI was performed on
도 10b 내지 10c는 이 건 모델에 실시예 6과 같은 그룹을 처리한 후, 1주일 동안 NSS 검사를 실시한 결과 비강 내 BDNF-hMSCs를 이식한 실험군에서 외상성 뇌손상으로 인해 발생되는 신경학적 장애가 억제됨을 확인한 결과이다. 도 10b에서 18점은 모든 임무를 달성하지 못함을 의미하며, 0점은 모든 임무를 성공적으로 달성함을 의미한다. 도 10c는 다양한 시간 간격에서 mNSS의 변화정도를 나타낸 그래프이다.10b to 10c show that the neurological disorder caused by traumatic brain injury was suppressed in the experimental group in which intranasal BDNF-hMSCs were implanted as a result of NSS test for 1 week after treating the same group as in Example 6 in this case model. This is the confirmed result. In FIG. 10B , a score of 18 means that all tasks were not accomplished, and a score of 0 means that all tasks were successfully accomplished. 10c is a graph showing the degree of change in mNSS at various time intervals.
도 10d 내지 10e는 손상 유도 후 3일, 7일, 14일, 21일에 MRI 검사결과 비강 내 BDNF-hMSCs를 이식한 실험군에서 외상성 뇌손상으로 인한 부종(edema) 및 lesion volumes이 현저히 감소됨을 확인한 결과이다. 도 10d는 T2-weighted image 이며, 10e는 21일째 TBI-veh와 TBI-BDNF-hMSCs 그룹에서 대표적인 T2 map을 나타낸 것으로 뇌의 부종은 T2값이 높게 (붉게) 나타났다. 10d to 10e show that 3, 7, 14, and 21 days after injury induction, edema and lesion volumes due to traumatic brain injury were significantly reduced in the experimental group implanted with intranasal BDNF-hMSCs as a result of MRI examination. It is the result. 10d is a T2-weighted image, and 10e shows a representative T2 map in the TBI-veh and TBI-BDNF-hMSCs groups on the 21st day. Brain edema showed a high T2 value (red).
이들은 비강 내 hydrogel로 encapsulation된 BDNF-hMSCs의 이식이 외상성 뇌손상 후에 일어나는 신경학적 장애를 개선시킴을 나타내는 결과들이다.These results indicate that implantation of BDNF-hMSCs encapsulated with intranasal hydrogel improves neurological disorders after traumatic brain injury.
Claims (12)
뇌 손상이 형성된 동물 모델의 비중격(nasal septum)과 점막(mucosa) 사이로 치료물질이 주입되는 것을 특징으로 하는, 치료물질이 비강 내로 이식된 동물 모델.In animal models other than humans, which can confirm the performance of the therapeutic material,
An animal model in which a therapeutic substance is implanted into the nasal cavity, characterized in that the therapeutic substance is injected between the nasal septum and the mucosa of the animal model in which brain damage is formed.
상기 뇌 손상은, 외상성 뇌손상 혹은 신경퇴행성 뇌질환에 기인하는 뇌손상을 포함하는 것을 특징으로 하는, 치료물질이 비강 내로 이식된 동물 모델.According to claim 1,
The brain damage, characterized in that it includes brain damage caused by traumatic brain injury or neurodegenerative brain disease, an animal model in which a therapeutic substance is implanted into the nasal cavity.
상기 외상성 뇌손상은, 뇌피질 충격 손상 모델(controlled cortical impact (CCI) model); Marmarou 자유 낙하 모델과 Feeney 자유 낙하 모델을 포함하는 자유 낙하 모델(weight drop model); 유체 타진 모델(fluid percussion model); 및 시신경 신전 손상 모델(optic nerve stretch injury model); 중 어느 하나에 따른 외상성 뇌손상인 것을 특징으로 하는, 치료물질이 비강 내로 이식된 동물 모델.3. The method of claim 2,
The traumatic brain injury, cortical impact injury model (controlled cortical impact (CCI) model); a weight drop model including the Marmarou free fall model and the Feeney free fall model; fluid percussion model; and an optic nerve stretch injury model; An animal model in which a therapeutic substance is implanted into the nasal cavity, characterized in that the traumatic brain injury according to any one of the above.
상기 동물 모델은 쥐, 토끼 혹은 돼지인 것을 특징으로 하는, 치료물질이 비강 내로 이식된 동물 모델.According to claim 1,
The animal model is a mouse, rabbit or pig, characterized in that the therapeutic material is implanted into the nasal cavity animal model.
상기 뇌 손상은, 뇌피질 충격 손상 모델 (controlled cortical impact (CCI) model)에 따라 동물 모델에 발생된 외상성 뇌손상인 것을 특징으로 하는, 치료물질이 비강 내로 이식된 동물 모델.According to claim 1,
The brain injury, characterized in that the traumatic brain injury occurred in the animal model according to the cortical impact injury model (controlled cortical impact (CCI) model), a therapeutic material is implanted into the nasal cavity animal model.
뇌 손상이 형성된 동물 모델의 비중격(nasal septum)과 점막(mucosa) 사이에 치료물질을 주입하는 제2단계;를 포함하는, 동물 모델의 제조 방법.A method of manufacturing an animal model other than a human for confirming the performance of a therapeutic material, the method comprising: a first step of forming brain damage in the animal model; and
A second step of injecting a therapeutic material between the nasal septum and the mucosa of the animal model in which brain damage is formed;
상기 제2 단계의 치료물질을 주입하는 단계는,
동물 모델의 비골에 유아골절(greenstick fracture)을 형성시키는 단계;
동물 모델의 비강을 통해 비중격을 열어, 비중격과 점막 사이에 포켓을 형성하는 단계; 및
상기 포켓으로 치료물질을 주입하는 단계;를 포함하는, 동물 모델의 제조 방법.7. The method of claim 6,
The step of injecting the therapeutic material of the second step,
forming a greenstick fracture in the fibula of the animal model;
opening the nasal septum through the nasal cavity of the animal model to form a pocket between the nasal septum and the mucous membrane; and
Injecting a therapeutic substance into the pocket; comprising, a method of manufacturing an animal model.
상기 비중격과 점막 사이에 형성되는 포켓의 부피는 100~150 ㎕이고,
상기 포켓으로 주입되는 줄기세포 치료물질은 주사 방식으로 주입되는 것을 특징으로 하는, 동물 모델의 제조 방법.8. The method of claim 7,
The volume of the pocket formed between the septum and the mucous membrane is 100 to 150 μl,
The method for producing an animal model, characterized in that the stem cell treatment material injected into the pocket is injected by injection.
형성된 유아골절의 골편을 복구 하고 봉합하는 단계;를 추가로 더 포함하는 것을 특징으로 하는, 동물 모델의 제조 방법.According to claim 6, After the second step,
Restoring and suturing the bone fragments of the formed infant fractures; Method for producing an animal model, characterized in that it further comprises.
상기 동물 모델에 뇌손상을 형성시킨 후, 10분 후에 치료 물질을 주입하는 것을 특징으로 하는, 동물 모델의 제조 방법.7. The method of claim 6,
After brain damage is formed in the animal model, a method for producing an animal model, characterized in that the therapeutic substance is injected 10 minutes later.
상기 치료물질은, 줄기세포의 단독으로 구성된 치료물질 혹은 줄기세포가 포접된 줄기세포 전달체를 포함하는 것을 특징으로 하는, 동물 모델의 제조 방법.7. The method of claim 6,
The therapeutic material, a method for producing an animal model, characterized in that it comprises a therapeutic material composed of stem cells alone or a stem cell delivery system in which the stem cells are encapsulated.
상기 제2단계 이후에, 주입된 줄기세포 치료물질의 효과를 확인하는 제3단계;를 더 포함하되,
상기 제3단계는, 뇌손상 형성 후 3일째의 조직학적 분석; 3, 7, 14, 21일째의 MRI 분석; 및 뇌손상 형성 후 7일 동안의 modified Neurological Severity Score (mNSS); 중에서 적어도 어느 하나 이상으로 수행되는 것을 특징으로 하는, 동물 모델의 제조 방법.
7. The method of claim 6,
After the second step, a third step of confirming the effect of the injected stem cell therapeutic material; further comprising,
The third step is histological analysis on the 3rd day after brain injury formation; MRI analysis on days 3, 7, 14, and 21; and modified Neurological Severity Score (mNSS) for 7 days after brain injury formation; A method for producing an animal model, characterized in that it is performed by at least any one or more of
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