KR20110125469A - Method for stimulating the secretion of nerve growth factor by stem cell via pre-treatment of nerve growth factor - Google Patents
Method for stimulating the secretion of nerve growth factor by stem cell via pre-treatment of nerve growth factor Download PDFInfo
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Abstract
Description
본 발명은 신경성장인자 전처리를 통한 줄기세포의 신경성장인자 분비 촉진 방법에 관한 것이다. The present invention relates to a method for promoting nerve growth factor secretion of the stem cells through nerve growth factor pretreatment.
최근 줄기세포 치료가 뇌경색, 외상성 신경손상, 근골격계 질환 등 다양한 질환에서 다양한 전임상, 임상연구가 시도되고 있다. 그러나 현재 기술적인 수준은 단순한 줄기세포의 추출 및 배양/증식과 이를 주입하는 정도이다.
Recently, various preclinical and clinical studies have been attempted for stem cell treatment in various diseases such as cerebral infarction, traumatic nerve injury, and musculoskeletal disorders. However, the current technical level is the extraction and culture / proliferation of simple stem cells and the degree of injecting them.
최근까지의 임상연구결과 이러한 줄기세포 치료는 아직 뚜렷한 효과를 보이고 있지 못하다. 효과를 증진하기 위한 다양한 유전자 조작 줄기세포에 대한 연구가 진행되고 있으나, 유전자를 이용한 세포치료는 윤리적인 문제 때문에 인체에서 적용하기는 불가능하다.
Until recently, clinical studies have not shown a clear effect. Research is being conducted on a variety of genetically engineered stem cells to enhance the effect, but cell therapy using genes is impossible to apply in the human body because of ethical issues.
뇌경색, 외상성 뇌손상과 같은 신경계 질환에서 신경성장인자가 회복과정에서 매우 중요한 역할을 하며, 질환에 따라 주요 신경성장인자가 차이가 난다고 알려져 있다.
In neurological diseases such as cerebral infarction and traumatic brain injury, nerve growth factor plays a very important role in the recovery process, and major nerve growth factors are known to be different according to disease.
반면 줄기세포에서 신경성장 인자가 분비되고, 이는 뇌경색, 외상성 뇌손상 등 질환에 의해 촉발되는 것으로 알려져 있다.
On the other hand, nerve growth factors are secreted from stem cells, which are known to be triggered by diseases such as cerebral infarction and traumatic brain injury.
신경성장인자는 거대분자(macromolecule)로 뇌-혈관 장막(blood brain barrier)을 통과하기 어렵다. 따라서 기존에 뇌 내에서 신경성장인자를 주입하거나 그 농도를 올리기 위해서는 뇌 내로 수술적인 요법으로 직접 주입하거나 유전자 치료나 새로운 벡터를 이용한 뇌-혈관 장막 통과와 같은 방법을 이용한 바 있다. 그러나 이러한 방법이 윤리적인 문제나 부작용으로 인해 환자에게 적용하기 어려운 현실이다.
Nerve growth factors are macromolecules that make it difficult to cross the blood brain barrier. Therefore, in order to inject or increase the concentration of nerve growth factors in the brain, a method such as injecting directly into the brain by surgical treatment or by using a gene therapy or a new vector, such as brain-vessel membrane passage, has been used. However, these methods are difficult to apply to patients because of ethical problems or side effects.
따라서 비유전적 방법으로 줄기세포에서 신경성장인자의 분비를 선택적으로 촉진시키는 방법은 매우 중요하며, 이에 관해서는 보고된 바 없다.
Therefore, the method of selectively promoting the secretion of nerve growth factors in stem cells by a non-genetic method is very important, which has not been reported.
이에, 본 발명자들은 '비유전적 방법'으로 줄기세포에서 신경성장인자의 분비를 선택적으로 촉진시키는 연구를 수행하여, 줄기세포 배양과정에서 신경성장인자의 전처리 과정을 통해 신경성장인자의 분비를 선택적으로 증진시킬 수 있는 방법을 발견하고 본 발명을 완성하였다.
Thus, the present inventors conducted a study for selectively promoting the secretion of neuronal growth factors in stem cells in a 'non-genetic method', and selectively secreting the neuronal growth factors through pretreatment of neural growth factors in the stem cell culture process. We have found a way to improve and have completed the present invention.
본 발명의 목적은 신경성장인자 전처리를 통하여 시험관에서 줄기세포의 신경성장인자 분비 촉진 방법을 제공하는 것이다.It is an object of the present invention to provide a method for promoting nerve growth factor secretion of stem cells in vitro through nerve growth factor pretreatment.
상기 목적을 해결하기 위해서, 본 발명은 In order to solve the above object, the present invention
(i) 줄기세포에 신경성장인자를 처리하는 단계; 및(i) treating nerve growth factors on stem cells; And
(ii) 손상된 신경조직을 제공하여 줄기세포를 자극하는 단계를 포함하는, (ii) providing damaged nerve tissue to stimulate stem cells,
시험관에서 줄기세포의 신경성장인자 분비 촉진 방법을 제공한다.
It provides a method for promoting the secretion of stem cell nerve growth factor in vitro.
상기 단계 (ii)에서 손상된 신경조직은 뇌경색, 파킨슨병, 알츠하이머병 및 루게릭병으로 구성된 군에서 선택된 어느 하나의 질환 환자의 뇌조직일 수 있다.
The neural tissue damaged in step (ii) may be brain tissue of any one diseased patient selected from the group consisting of cerebral infarction, Parkinson's disease, Alzheimer's disease and Lou Gehrig's disease.
또한, 상기 단계 (ii)에서 손상된 신경조직은 뇌외상 또는 척수손상으로부터 수득될 수 있다.
In addition, the neural tissue damaged in step (ii) can be obtained from brain trauma or spinal cord injury.
일 구체예로서, 상기 줄기세포는 중간엽 줄기세포일 수 있다.
In one embodiment, the stem cells may be mesenchymal stem cells.
일 구체예로서, 상기 줄기세포는 골수유래 중간엽 줄기세포, 조혈모세포, 지방 줄기세포 및 제대혈 줄기세포로 구성된 군에서 선택된 어느 하나일 수 있다.
In one embodiment, the stem cells may be any one selected from the group consisting of bone marrow-derived mesenchymal stem cells, hematopoietic stem cells, adipose stem cells and cord blood stem cells.
상기 신경성장인자는 BDNF (brain-derived neurotrophic factor), VEGF (vascular endothelial growth factor), HGF (hepatocyte growth factor), NGF (nerve growth factor), bFGF (basic fibroblast growth factor), GDNF (glial cell line-derived neurotrophic factor) 및 PDGF (platelet-derived growth factor) 으로 구성된 군에서 선택된 어느 하나일 수 있다. 그러나, 본 발명의 신경성장인자가 이것들에 한정되는 것은 아니다.
The neuronal growth factor may include a brain-derived neurotrophic factor (BDNF), a vascular endothelial growth factor (VEGF), a hepatocyte growth factor (HGF), a nerve growth factor (NGF), a basic fibroblast growth factor (BFGF), and a glial cell line- (GDNF). It may be any one selected from the group consisting of derived neurotrophic factor (PDGF) and platelet-derived growth factor (PDGF). However, the nerve growth factor of the present invention is not limited to these.
상기 신경성장인자의 처리농도는 10 ~ 100 ng/ml인 것이 바람직하다. 또한, BDNF는 20 ng/ml, VEGF는 50 ng/ml, HGF는 20 ng/ml, NGF는 100ng/ml, bFGF는 10ng/ml 농도로 처리하는 것이 보다 바람직하다.
The treatment concentration of the nerve growth factor is preferably 10 ~ 100 ng / ml. In addition, it is more preferable to treat BDNF at 20 ng / ml, VEGF at 50 ng / ml, HGF at 20 ng / ml, NGF at 100ng / ml, and bFGF at 10ng / ml.
상기 신경성장인자의 처리시간은 24 시간 내지 120 시간인 것이 바람직하다.
The treatment time of the nerve growth factor is preferably 24 hours to 120 hours.
본 발명의 다른 양태로서, 본 발명은As another aspect of the present invention,
(i) 줄기세포에 신경성장인자를 처리하는 단계; 및(i) treating nerve growth factors on stem cells; And
(ii) 손상된 신경조직을 제공하여 줄기세포를 자극하는 단계를 포함하는, (ii) providing damaged nerve tissue to stimulate stem cells,
시험관에서 줄기세포의 성장 촉진 방법을 제공한다.
Provided are methods for promoting stem cell growth in vitro.
첫째, 본 발명의 방법에 따른 줄기세포 배양과정에서 신경성장인자를 전처리함으로써 줄기세포의 신경성장인자의 분비를 증가시킬 수 있다. 줄기세포는 뇌-혈관 장막을 통과할 수 있으며 선택적으로 뇌경색과 같은 뇌의 손상부위로 이동하기 때문에 신경성장인자 분비능력이 향상된 줄기세포의 임상적용은 보다 큰 치료효과를 기대할 수 있다.First, the secretion of nerve growth factors of stem cells can be increased by pretreatment of nerve growth factors in the stem cell culture process according to the method of the present invention. Stem cells can pass through the brain-vessel membrane and selectively move to damaged areas of the brain such as cerebral infarction, so the clinical application of stem cells with improved ability to secrete nerve growth factors can be expected to have a greater therapeutic effect.
둘째, 본 발명에 따른 전처리 방법으로, 목표하는 신경성장인자를 선택적으로 전처리함으로써 선택적으로 신경성장인자의 분비를 향상시킴으로써, 뇌경색, 파킨슨병, 알츠하이머병, 루케릭병과 같이 질병에서 각 질환과 보다 관련성이 높은 신경성장인자를 선택하여 전처리함으로써 줄기세포의 신경성장인자 분비를 조절하여 줄기세포 치료효과를 증진시킬 수 있을 것이다.
Second, the pretreatment method according to the present invention selectively improves the secretion of nerve growth factors by selectively pretreatment of the target nerve growth factors, and is more related to each disease in diseases such as cerebral infarction, Parkinson's disease, Alzheimer's disease, and Luceric disease. By selecting and pretreating these high nerve growth factors, it is possible to improve stem cell therapeutic effects by controlling the secretion of nerve growth factors in stem cells.
도 1은 본 발명의 일 구체예의 실험방법을 단계적으로 도식화한 것이다.
도 2는 인간 중간엽 줄기세포에서의 신경성장인자의 분비를 나타낸 것이다.
VEGF, 25.03±2.85 pg/ml; BDNF, 7.67±1.04 pg/ml; HGF, 76.39±56.84 pg/ml. *p<0.05 (배양 1일과 비교), +p<0.05 (P2 ischemic과 비교)
도 3은 신경성장인자를 전처리하였을 때 허혈성 자극 후 신경성장인자의 분비 증가를 나타낸 것으로서, 중간엽 줄기세포에서 BDNF, VEGF, HGF의 분비되는 양은 전처리를 하지 않은 군에 비해 유의하게 증가됨이 관찰되었다 (*P<0.001).
도 4는 신경성장인자의 전처리에 따른 중간엽 줄기세포의 표식자의 변화를 나타낸 것이다.
도 5는 신경성장인자의 전처리에 따른 중간엽 줄기세포의 증식능의 변화를 나타낸 것이다.Figure 1 is a schematic diagram of a test method of an embodiment of the present invention.
Figure 2 shows the secretion of nerve growth factors in human mesenchymal stem cells.
VEGF, 25.03 ± 2.85 pg / ml; BDNF, 7.67 ± 1.04 pg / ml; HGF, 76.39 ± 56.84 pg / ml. * p <0.05 (compared to culture day 1), + p <0.05 (compared to P2 ischemic)
Figure 3 shows the increase in the secretion of nerve growth factor after ischemic stimulation when the nerve growth factor pretreatment, it was observed that the amount of BDNF, VEGF, HGF secretion in mesenchymal stem cells increased significantly compared to the group without pretreatment (* P <0.001).
Figure 4 shows the change of the marker of mesenchymal stem cells according to the pre-treatment of nerve growth factor.
Figure 5 shows the change in proliferation capacity of mesenchymal stem cells according to the pre-treatment of nerve growth factor.
이하 본 발명을 보다 구체적으로 이해할 수 있도록 실시예를 들어 설명한다. 그러나 다음의 실시예로써 본 발명의 취지를 제한하는 것은 아니다
Hereinafter, examples will be described so that the present invention can be understood in more detail. However, the following examples are not intended to limit the gist of the present invention.
실시예Example
실시예Example
1: One:
골수유래Bone marrow
줄기세포 배양과정에서 신경성장인자 전처리 Neural Growth Factor Pretreatment in Stem Cell Culture
1-1. 1-1. 골수유래Bone marrow 줄기세포 채취 및 배양 Stem Cell Collection and Culture
정상인에서 10-20 ml의 골수를 채취하여 phosphate-buffered saline (PBS)에 1:1로 희석하여 원심분리 및 배양을 통해 골수유래 중간엽 줄기세포를 채취하였다. 중간엽 줄기세포는 5% CO2 37℃하에 배양하였으며, 실험에 사용된 세포는 계대배양 2세대 (passage 2, P2) 및 6세대 (P6) 세포를 사용하였다. 실험은 환자의 동의 및 임상연구위원회의 승인하에 진행되었다.
10-20 ml of bone marrow was collected from normal subjects and diluted 1: 1 in phosphate-buffered saline (PBS) to obtain bone marrow-derived mesenchymal stem cells through centrifugation and culture. Mesenchymal stem cells were cultured under 5% CO 2 37 ° C., and the cells used in the experiments were
1-2. 신경성장인자 전처리1-2. Nerve Growth Factor Pretreatment
실험을 위해 1x105개 세포를 60 mm 디쉬에 knockout DMEM (Dulbecco's modified Eagles medium) 및 20% knockout fetal bovine serum (FBS)으로 배양하였다. 배양된 세포에 신경성장인자를 배양과정에서 72 시간 동안 처리하였다. 처리된 신경성장인자는 BDNF (brain-derived neurotrophic factor) 20 ng/ml, VEGF (vascular endothelial growth factor) 50 ng/ml, HGF (hepatocyte growth factor) 20 ng/ml, NGF (Nerve growth factor) 100 ng/ml, bFGF (basic fibroblast growth factor) 10 ng/ml을 처리하였다.
For the
실시예Example
2: 신경성장인자로 전처리한 줄기세포에서 신경성장인자의 분비능력 측정 2: Determination of Nerve Growth Factor Secretion Capacity in Stem Cells Pretreated with Nerve Growth Factor
2-1. 허혈자극2-1. Ischemic stimulation
생쥐 중뇌동맥경색 모델을 통해 허혈-뇌조직 (ischemic brain extract)를 얻어 DMEM에 녹여 실험에 사용하였다. 신경성장인자 또는 대조군으로 PBS를 전처리한 중간엽 줄기세포를 PBS로 3차례 충분히 신경성장인자를 세척한 후 중간엽 줄기세포를 다시 reseeding하였다. 여기에 허혈 뇌조직을 처리한 후 중간엽 줄기세포에서 분비되는 신경성장인자의 농도를 측정하였다. 각 군당 4번 반복 실험되었다. 간략한 실험 방법은 도 1과 같다.
Ischemia-brain tissue (ischemic brain extract) was obtained from the mouse middle cerebral artery infarction model and used in experiments by dissolving in DMEM. Mesenchymal stem cells pretreated with PBS as neural growth factors or controls were washed three times with PBS and then reseeding mesenchymal stem cells. The concentration of nerve growth factor secreted from mesenchymal stem cells after treatment of ischemic brain tissue was measured. Four replicates of each group were performed. A brief experimental method is shown in FIG. 1.
2-2. 줄기세포에서 신경성장인자의 분비능력 측정2-2. Measurement of Nerve Growth Factor Secretion in Stem Cells
줄기세포의 신경성장인자 분비 능력을 줄기세포 배양조건 및 뇌경색 생쥐모델에서 enzyme-linked immunosorbent assay (ELISA) 방법을 통해 확인하였다.
The ability of the stem cells to secrete nerve growth factors was confirmed by enzyme-linked immunosorbent assay (ELISA) in stem cell culture conditions and cerebral infarction mice.
도 2에서 (A) 내지 (C)는 인간 중간엽 줄기세포의 배양조건에서 허혈-뇌조직을 처리했을 때 (in vitro), (D) 내지 (E)는 뇌경색 생쥐모델에 중간엽 줄기세포 꼬리정맥를 주입했을 때 (in vivo)의 신경성장인자의 분비를 확인한 결과이다. 두 경우 모두 대조군에 비해 신경성장인자의 분비가 증가되고 있음을 보여주고 있다. 그 분비량은 P2와 P6 계대배양 세포에서 다소 다른 차이가 있음을 알 수 있으며 이러한 경향은 in vitro 및 in vivo에서 동일한 양상이었다.
In Figure 2 (A) to (C) when ischemic-brain tissue is treated in culture conditions of human mesenchymal stem cells (in vitro), (D) to (E) is a mesenchymal stem cell tail in the cerebral infarction mouse model This is the result of confirming the secretion of nerve growth factor in the intravenous (in vivo). In both cases, the secretion of nerve growth factor was increased compared to the control group. The amount of secretion was slightly different between P2 and P6 subcultured cells, and this tendency was the same in vitro and in vivo.
골수유래 중간엽 줄기세포 배양조건에 허혈-뇌조직을 처리했을 때 신경성장인자의 분비가 증가됨을 확인하였으며, 이는 뇌경색 동물모델에 중간엽 줄기세포를 꼬리정맥으로 주입한 후 뇌조직에서 신경성장인자의 수준 증가와 동일한 양상이었다 (도 2 참조).
When the ischemic-brain tissue was treated under the bone marrow-derived mesenchymal stem cell culture conditions, the secretion of nerve growth factor was increased, which was induced in the cerebral infarct animal model after the injection of the mesenchymal stem cell into the tail vein. It was the same aspect as the level increase (see Fig. 2).
또한, 신경성장인자를 전처리한 경우 줄기세포에서 분비되는 신경성장인자의 양이 전처리하지 않은 군에 비해 유의하게 증가됨을 확인할 수 있었다 (도 3 참조). 이러한 증가는 전처리한 신경성장인자의 종류에 따라 양상의 차이가 있었다. 즉, 전처리한 신경성장인자의 증가가 가장 두드러져 자가분비(autocrine) 양상으로 자극함을 알 수 있었다. 이러한 양상은 뇌경색 모델에서는 최초로 발견된 것으로서, 줄기세포의 독특한 특성을 이용하여 뇌경색에서 줄기세포 적응의 효과를 증진시킬 수 있다는 것을 보여준다.
In addition, the pre-treatment of nerve growth factor was confirmed that the amount of neuronal growth factor secreted from the stem cells significantly increased compared to the group not pretreated (see Figure 3). This increase was different depending on the type of neural growth factor pretreated. In other words, the increase of pre-treated neuronal growth factor was most prominent and stimulated by autocrine. This is the first finding in a model of cerebral infarction and shows that the unique properties of stem cells can be used to enhance the effects of stem cell adaptation on cerebral infarction.
실시예Example
3: 신경성장인자 전처리의 안정성 검사 3: Examination of stability of nerve growth factor pretreatment
신경성장인자를 전처리함으로써 줄기세포의 성향이 변화하는지를 알아보기 위해 줄기세포의 형태학적 변화 및 표지자의 변화를 살펴보았다.
We examined the morphological and marker changes of stem cells to determine whether the propensity of stem cells changes by pretreatment of nerve growth factors.
줄기세포 표지자의 발현을 중간엽줄기세포에 특이성을 보이는 양성 및 음성 표지인자인 CD105, CD34에 대한 표현정도를 유세포분석기(flow cytometry)를 이용하여 분석하였다. 도 4에서 (A)는 전처리하지 않은 줄기세포, (B)는 VEGF를 전처리한 후 줄기세포, (C)는 다양한 신경성장인자를 전처리한 후 줄기세포 표식자의 변화를 나타낸 것이다. 그 결과, 신경성장인자의 전처리에 따른 줄기세포의 형태학적 차이는 없었다 (도 4 참조). Expression of stem cell markers was analyzed using flow cytometry for expression of CD105 and CD34, positive and negative markers specific for mesenchymal stem cells. In Figure 4 (A) is not pre-treated stem cells, (B) is a pre-treatment of VEGF stem cells, (C) shows a change in stem cell markers after pre-treatment of various neuronal growth factors. As a result, there was no morphological difference of stem cells according to pretreatment of nerve growth factors (see FIG. 4).
또한, 신경성장인자를 처리하였을 때 대조군에 비해 계대배양시 줄기세포의 증식속도가 유의하게 증가됨을 확인할 수 있었다 (도 5 참조). 따라서 본 발명의 방법을 이용하여 줄기세포의 성장을 촉진시킬 수 있다.
In addition, it was confirmed that the growth rate of stem cells significantly increased during subculture when the nerve growth factor was treated (see FIG. 5). Therefore, it is possible to promote the growth of stem cells using the method of the present invention.
지금까지 예시적인 실시 태양을 참조하여 본 발명을 기술하여 왔지만, 본 발명의 속하는 기술 분야의 당업자는 본 발명의 범주를 벗어나지 않고서도 다양한 변화를 실시할 수 있으며 그의 요소들을 등가물로 대체할 수 있음을 알 수 있을 것이다. 또한, 본 발명의 본질적인 범주를 벗어나지 않고서도 많은 변형을 실시하여 특정 상황 및 재료를 본 발명의 교시내용에 채용할 수 있다. 따라서, 본 발명이 본 발명을 실시하는데 계획된 최상의 양식으로서 개시된 특정 실시 태양으로 국한되는 것이 아니며, 본 발명이 첨부된 특허청구의 범위에 속하는 모든 실시 태양을 포함하는 것으로 해석되어야 한다. While the present invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. You will know. In addition, many modifications may be made to adapt a particular situation and material to the teachings of the invention without departing from the essential scope thereof. Accordingly, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention be construed as including all embodiments falling within the scope of the appended claims.
Claims (16)
(ii) 손상된 신경조직을 제공하여 줄기세포를 자극하는 단계를 포함하는,
시험관에서 줄기세포의 신경성장인자 분비 촉진 방법.(i) treating nerve growth factors on stem cells; And
(ii) providing damaged nerve tissue to stimulate stem cells,
Method for promoting the growth of nerve cells in stem cells in vitro.
단계 (ii)에서 손상된 신경조직은 뇌경색, 파킨슨병, 알츠하이머병 및 루게릭병으로 구성된 군에서 선택된 어느 하나의 질환 환자의 뇌조직임을 특징으로 하는, 시험관에서 줄기세포의 신경성장인자 분비 촉진 방법.The method of claim 1,
The neuronal tissue damaged in step (ii) is brain tissue of any one of the disease patients selected from the group consisting of cerebral infarction, Parkinson's disease, Alzheimer's disease and Lou Gehrig's disease.
단계 (ii)에서 손상된 신경조직은 뇌외상 또는 척수손상으로부터 수득된 것을 특징으로 하는, 시험관에서 줄기세포의 신경성장인자 분비 촉진 방법.The method of claim 1,
The nerve tissue injured in step (ii) is characterized in that obtained from brain injury or spinal cord injury, a method for promoting the secretion of growth factors of stem cells in vitro.
상기 줄기세포는 중간엽 줄기세포임을 특징으로 하는, 시험관에서 줄기세포의 신경성장인자 분비 촉진 방법.The method of claim 1,
The stem cell is characterized in that the mesenchymal stem cells, the method of promoting nerve growth factor secretion of stem cells in vitro.
상기 줄기세포는 골수유래 중간엽 줄기세포, 조혈모세포, 지방 줄기세포 및 제대혈 줄기세포로 구성된 군에서 선택된 어느 하나임을 특징으로 하는, 시험관에서 줄기세포의 신경성장인자 분비 촉진 방법.The method of claim 1,
The stem cell is characterized in that any one selected from the group consisting of bone marrow-derived mesenchymal stem cells, hematopoietic stem cells, adipose stem cells and umbilical cord blood stem cells, nerve growth factor secretion promoting method in vitro.
상기 신경성장인자는 BDNF (brain-derived neurotrophic factor), VEGF (vascular endothelial growth factor), HGF (hepatocyte growth factor), NGF (nerve growth factor), bFGF (basic fibroblast growth factor), GDNF (glial cell line-derived neurotrophic factor) 및 PDGF (platelet-derived growth factor) 으로 구성된 군에서 선택된 어느 하나임을 특징으로 하는, 시험관에서 줄기세포의 신경성장인자 분비 촉진 방법.The method of claim 1,
The neuronal growth factor may include a brain-derived neurotrophic factor (BDNF), a vascular endothelial growth factor (VEGF), a hepatocyte growth factor (HGF), a nerve growth factor (NGF), a basic fibroblast growth factor (BFGF), and a glial cell line- (GDNF). A method for promoting nerve growth factor secretion of stem cells in vitro, characterized in that any one selected from the group consisting of derived neurotrophic factor) and platelet-derived growth factor (PDGF).
상기 신경성장인자의 처리농도는 10 ~ 100 ng/ml임을 특징으로 하는, 시험관에서 줄기세포의 신경성장인자 분비 촉진 방법.The method of claim 1,
The treatment concentration of the nerve growth factor is characterized in that 10 ~ 100 ng / ml, method for promoting nerve growth factor secretion of stem cells in vitro.
상기 신경성장인자의 처리시간은 24 시간 내지 120 시간임을 특징으로 하는, 시험관에서 줄기세포의 신경성장인자 분비 촉진 방법.The method of claim 1,
The nerve growth factor treatment time is characterized in that 24 hours to 120 hours, the method for promoting nerve growth factor secretion of stem cells in vitro.
(ii) 손상된 신경조직을 제공하여 줄기세포를 자극하는 단계를 포함하는,
시험관에서 줄기세포의 성장 촉진 방법.(i) treating nerve growth factors on stem cells; And
(ii) providing damaged nerve tissue to stimulate stem cells,
Methods for promoting stem cell growth in vitro.
단계 (ii)에서 손상된 신경조직은 뇌경색, 파킨슨병, 알츠하이머병 및 루게릭병으로 구성된 군에서 선택된 어느 하나의 질환 환자의 뇌조직임을 특징으로 하는, 시험관에서 줄기세포의 성장 촉진 방법.10. The method of claim 9,
The neuronal tissue damaged in step (ii) is brain tissue of any one diseased patient selected from the group consisting of cerebral infarction, Parkinson's disease, Alzheimer's disease and Lou Gehrig's disease.
단계 (ii)에서 손상된 신경조직은 뇌외상 또는 척수손상으로부터 수득된 것을 특징으로 하는, 시험관에서 줄기세포의 성장 촉진 방법.10. The method of claim 9,
The neural tissue damaged in step (ii) is obtained from brain injury or spinal cord injury, the method of promoting growth of stem cells in vitro.
상기 줄기세포는 중간엽 줄기세포임을 특징으로 하는, 시험관에서 줄기세포의 성장 촉진 방법.10. The method of claim 9,
The stem cell is characterized in that the mesenchymal stem cells, the method of promoting growth of stem cells in vitro.
상기 줄기세포는 골수유래 중간엽 줄기세포, 조혈모세포, 지방 줄기세포 및 제대혈 줄기세포로 구성된 군에서 선택된 어느 하나임을 특징으로 하는, 시험관에서 줄기세포의 성장 촉진 방법.10. The method of claim 9,
The stem cell is characterized in that any one selected from the group consisting of bone marrow-derived mesenchymal stem cells, hematopoietic stem cells, adipose stem cells and umbilical cord blood stem cells, method of promoting growth of stem cells in vitro.
상기 신경성장인자는 BDNF (brain-derived neurotrophic factor), VEGF (vascular endothelial growth factor), HGF (hepatocyte growth factor), NGF (nerve growth factor), bFGF (basic fibroblast growth factor), GDNF (glial cell line-derived neurotrophic factor) 및 PDGF (platelet-derived growth factor) 으로 구성된 군에서 선택된 어느 하나임을 특징으로 하는, 시험관에서 줄기세포의 성장 촉진 방법.10. The method of claim 9,
The neuronal growth factor may include a brain-derived neurotrophic factor (BDNF), a vascular endothelial growth factor (VEGF), a hepatocyte growth factor (HGF), a nerve growth factor (NGF), a basic fibroblast growth factor (BFGF), and a glial cell line- (GDNF). A method for promoting stem cell growth in vitro, characterized in that it is any one selected from the group consisting of derived neurotrophic factor (PDGF) and platelet-derived growth factor (PDGF).
상기 신경성장인자의 처리농도는 10 ~ 100 ng/ml임을 특징으로 하는, 시험관에서 줄기세포의 성장 촉진 방법.10. The method of claim 9,
The nerve growth factor treatment concentration is characterized in that 10 ~ 100 ng / ml, method of promoting growth of stem cells in vitro.
상기 신경성장인자의 처리시간은 24 시간 내지 120 시간임을 특징으로 하는, 시험관에서 줄기세포의 성장 촉진 방법.10. The method of claim 9,
The treatment time of the nerve growth factor is characterized in that 24 hours to 120 hours, the method of promoting growth of stem cells in vitro.
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