KR20080068352A - Neuronal regeneration material screening method by ex vivo model - Google Patents
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Abstract
Description
도 1은 리소레시틴(lysolecithin)에 의한 척수조직절편 탈수초화를 나타낸 그림이고,1 is a diagram showing demyelination of spinal cord tissue sections by lysolecithin,
도 2는 중간엽 줄기세포 이식에 의한 탈수초화된 척수조직절편의 재수초화를 나타낸 그림이고,Figure 2 is a diagram showing the remyelination of demyelination spinal cord tissue section by mesenchymal stem cell transplantation,
도 3은 중간엽 줄기세포 이식에 의한 탈수초화된 척수조직절편의 재수초화 확인을 위한 신경미세섬유(150kDa)의 세포핵에 대한 면역형광 결과를 나타낸 그림이고, Figure 3 is a diagram showing the immunofluorescence results for the cell nucleus of the neurofibrils (150kDa) for remyelination of demyelination spinal cord tissue sections by mesenchymal stem cell transplantation,
도 4는 중간엽 줄기세포 이식에 의한 탈수초화된 척수조직절편의 재수초화 확인을 위한 신경미세섬유(150kDa)의 면역형광 결과를 나타낸 그림이고,Figure 4 is a diagram showing the immunofluorescence results of neurofibrils (150kDa) for remyelination of demyelination spinal cord tissue sections by mesenchymal stem cell transplantation,
도 5는 중간엽 줄기세포 이식에 의한 탈수초화된 척수조직절편의 재수초화에 의한 신경미세섬유(150kDa)의 발현량의 증가를 나타낸 그림이고,Figure 5 is a diagram showing the increase in the amount of expression of neurofibrillary (150kDa) by remyelination of demyelination spinal cord tissue section by mesenchymal stem cell transplantation,
도 6은 중간엽 줄기세포 이식에 의한 탈수초화된 척수조직절편의 재수초화 확인을 위한 PI와 CNPase의 면역형광 결과를 나타낸 그림이다.6 is a diagram showing the results of immunofluorescence of PI and CNPase for remyelination of demyelination spinal cord tissue sections by mesenchymal stem cell transplantation.
본 발명은 생체외(ex vivo) 모델에 의한 신경재생물질 탐색 방법에 관한 방법으로, 보다 상세하게는 척수 조직 절편을 이용하여 신경세포 재생 효과를 지니는 물질을 탐색하는 방법에 관한 것이다. The present invention relates to a method for searching for neuroregeneration material by an ex vivo model, and more particularly, to a method for searching for a material having a neuronal regeneration effect using spinal cord tissue sections.
중간엽 줄기세포(Mesenchymal stem cells)는 최초로 쥐의 골수 세포에서 발견되었으며 섬유아세포(fibroblast)의 형태를 띠고 있는 세포이다(Friedenstein et al., Cell Tissue Kinet, 20, 263-272, 1987). 중간엽 줄기세포는 여러 중배엽 계열로 분류될 수 있으며, 이 세포들은 다양한 종류의 세포로 분화될 수 있다. 다시 말해, 중간엽 줄기세포의 특성은 골아세포(osteoblasts; Friedenstein et al., Cell Tissue Kinet, 20, 263-272, 1987; Abdallah et al., Bone, 39, 181-188, 2006), 지방세포(adipocytes; Fink et al., Stem Cells, 22, 1346-1355, 2004; Abdallah et al., Bone, 39, 181-188, 2006), 내피세포(endothelial cells; Kassem et al., Basic Clin Pharmacol Toxicol 95, 209-14, 2004) 및 신경세포(neuronal cells; Woodbury et al., J Neurosci Res, 61, 364-370, 2000; Woodbury et al., J Neurosci Res, 69, 908-917, 2002; Krabbe et al., APMIS, 113, 831-844, 2005)와 같은 다양한 세포 유형으로 분화될 수 있는 능력에 기인한다. Mesenchymal stem cells are the first cells found in rat bone marrow cells and take the form of fibroblasts (Friedenstein et al., Cell Tissue Kinet, 20, 263-272, 1987). Mesenchymal stem cells can be classified into several mesoderm lineages, which can differentiate into various cell types. In other words, the characteristics of mesenchymal stem cells are osteoblasts; Friedenstein et al., Cell Tissue Kinet, 20, 263-272, 1987; Abdallah et al., Bone, 39, 181-188, 2006). (adipocytes; Fink et al., Stem Cells, 22, 1346-1355, 2004; Abdallah et al., Bone, 39, 181-188, 2006), endothelial cells; Kassem et al., Basic Clin Pharmacol Toxicol 95, 209-14, 2004) and neuronal cells; Woodbury et al., J Neurosci Res, 61, 364-370, 2000; Woodbury et al., J Neurosci Res, 69, 908-917, 2002; Krabbe et al., APMIS, 113, 831-844, 2005), due to their ability to differentiate into various cell types.
여러 임상적인 시도를 위하여 중간엽 줄기세포의 이해에 대한 관심이 증가하 였고, 이식을 이용한 유전자 치료의 효율성과 안전성의 연구가 진행 중이다(Zhao et al., J Neurol Sci, 233, 87-91, 2005). 즉, 최근에는 뼈, 심근 및 신경 조직 회복을 포함하는 골수 유래의 중간엽 줄기세포의 다양한 치료 응용이 보고되었고(Jiang et al., Zhonghua Er Bi Yan Hou Ke Za Zhi, 37, 137-139, 2002; Kassem et al., Basic Clin Pharmacol Toxicol, 95, 209-214, 2004; Kemp et al., Leuk Lymphoma, 46, 1531-1544, 2005; Biossy et al., Cancer Res 65, 9943-9952, 2005; Krabbe et al., APMIS, 113, 831-844, 2005) 특히, 손상부위로의 줄기세포 이식은 신경 대체의 가능성뿐만 아니라 기존 세포 유지에 중요하기 때문에 여러 신경 질환에 많은 장점을 지니고 있다고 알려져있다(Maragakis et al., Glia, 50, 145-159, 2005).Interest in understanding mesenchymal stem cells has increased for several clinical trials, and research on the efficacy and safety of gene therapy with transplantation is ongoing (Zhao et al., J Neurol Sci, 233, 87-91, 2005). In other words, various therapeutic applications of bone marrow-derived mesenchymal stem cells have recently been reported, including bone, myocardial and neural tissue repair (Jiang et al., Zhonghua Er Bi Yan Hou Ke Za Zhi, 37, 137-139, 2002). Kassem et al., Basic Clin Pharmacol Toxicol, 95, 209-214, 2004; Kemp et al., Leuk Lymphoma, 46, 1531-1544, 2005; Biossy et al., Cancer Res 65, 9943-9952, 2005; Krabbe et al., APMIS, 113, 831-844, 2005) In particular, stem cell transplantation into the damaged area is known to have many advantages for several neurological diseases because it is important for the maintenance of existing cells as well as the possibility of neuronal replacement. Maragakis et al., Glia, 50, 145-159, 2005).
척수신경의 단기 손상 후 만성적인 병리현상을 연구하는데 척수 손상의 여러 in vivo 모델의 특성이 잘 알려져 있으며 널리 사용되어왔다(Krassioukov et al., J Neurotrauma, 19, 1521-1529, 2002; Lee et al., Orthop Clin North Am, 33, 311-315, 2002; Casha et al., Exp Neurol, 196, 390-400, 2005; Goldman, Nat Biotechnol, 23, 862-871, 2005). 손상 부위에 인접한 척수 조직에서, 직접적인 손상과 조직 파괴의 현상은 손상 부분에서 멀어질수록 완화되고(Buss & Schwab, Glia, 42, 424-432, 2003) 척수 손상은 또한 뉴런과 신경교세포(glia)의 손실과 연관되어 있다고 보고되었다(Goldman, Nat Biotechnol, 23, 862-871, 2005). 척수 손상 후에는 유전자 발현과 단백질에 많은 변화가 있으며, 이는 탈수초화를 초래한다고 보고되었다(Di Giovanni et al., Proc Natl Acad Sci U S A., 102, 8333- 8338, 2005; Kang et al., Proteomics, 6, 2797-2812, 2006). 한편, in vivo 시스템을 이용한 척수 손상 실험의 경우, 시스템의 복잡성으로 인하여, 결과의 해석은 더욱 어려워질 수 있어 in vitro 모델이 더 선호되는데, 이는 세포 밖 환경에서 명확한 조절이 가능하며, 세포에서 쉽게 반복 실험이 가능하며, 더 적은 비용이 들기 때문이다. 그러나 in vitro 모델은 생체 활성을 지니고 있지 않다는 단점이 있다.The characteristics of several in vivo models of spinal cord injury are well known and widely used to study chronic pathology after short-term injury to the spinal nerve (Krassioukov et al., J Neurotrauma, 19, 1521-1529, 2002; Lee et al. , Orthop Clin North Am, 33, 311-315, 2002; Casha et al., Exp Neurol, 196, 390-400, 2005; Goldman, Nat Biotechnol, 23, 862-871, 2005). In spinal cord tissue adjacent to the site of injury, the phenomenon of direct injury and tissue destruction is alleviated farther away from the site of injury (Buss & Schwab, Glia, 42, 424-432, 2003) and spinal cord injury also causes neurons and glia. It has been reported to be associated with the loss of (Goldman, Nat Biotechnol, 23, 862-871, 2005). After spinal cord injury there are many changes in gene expression and protein, which have been reported to result in demyelination (Di Giovanni et al., Proc Natl Acad Sci US A., 102, 8333-8338, 2005; Kang et al., Proteomics, 6, 2797-2812, 2006). On the other hand, for spinal cord injury experiments using in vivo systems, due to the complexity of the system, the interpretation of the results may be more difficult, and in vitro models are preferred, which allows for clear control in the extracellular environment and is readily available in cells. Repeated experiments are possible and at a lower cost. However, the in vitro model has the disadvantage that it is not bioactive.
조직절편배양(organotypic slice culutures)은 국소빈혈(ischemia)과 세포독성 연구와 같은 여러 손상 모델에 사용되어 왔다(Krassioukov et al., J Neurotrauma, 19, 1521-1529, 2002). 상기와 같은 방식으로 실시된 척수 조직 배양체는, 각각 식별할 수 있는 배각(dorsal horn)과 전각(ventral horn)을 띄며 전체적으로 우수한 형태를 유지하며, 뉴런 분포의 확인이 용이하다(Ulich et al., J Neurophysiol., 72, 861-871, 1994; Takuma et al., Neuroscience, 109, 359-370, 2002; Hilton et al., Brain Res Brain Res Rev, 46, 191-203, 2004). Tissue section cultures (organotypic slice culutures) have been used in several damage models, such as ischemia and cytotoxicity studies (Krassioukov et al., J Neurotrauma, 19, 1521-1529, 2002). Spinal cord tissue cultures carried out in the above manner have distinct dorsal and ventral horns and maintain excellent overall shape, and facilitate the identification of neuronal distribution (Ulich et al., J Neurophysiol., 72, 861-871, 1994; Takuma et al., Neuroscience, 109, 359-370, 2002; Hilton et al., Brain Res Brain Res Rev, 46, 191-203, 2004).
구조적으로 손상되지 않은 축색돌기의 탈수초화와 도약신경 전도의 장애는, 척수 손상으로 인한 기능적 결함의 원인으로 여겨진다(Nicot, Brain 126, 398-412, 2003). 리소레시틴은 세정제와 유사한 단백질을 포함하는 지질이며 수초화된 세포에 특이적인 독성을 가지는 막 가용성 작용제와 같은 활성으로 인하여 탈수초화를 유발한다(Franklin et al., J Neurosci Res 58, 207-213, 1999; Arnett et al., Science, 306, 2111-2115, 2004). 리소레시틴은 in vivo에서 척수와 같은 특이적으로 수초화된 섬유에 주입되었을 때 탈수초화를 일으킴으로서 결과적으로 척수손상을 유발하는 것으로 알려졌다. 한편, 희소돌기교세포(oligodendrocyte)의 손실 은 다발성경화증(Multiple Sclerosis)만큼 다양한 성인 탈수초화 질환의 원인이 됨을 통해, 희소돌기교세포의 척수의 수초화와의 관계성을 증명하였다. 즉, 리소레시틴에 의한 손상된 성체 쥐의 뇌에 인간 희소돌기교세포의 전구세포를 이식하였을 때, 이 세포는 희소돌기교세포처럼 빠르게 발달할 수 있었으며, 견고하게 수초화된 것과 비교하여 상대적으로 낮은 효율이지만 노출된 축색돌기를 수초화 시킬 수 있었다(Goldman, Nat Biotechnol, 23, 862-871, 2005). 상기의 결과는 리소레시틴에 의해 유도된 척수 축색돌기의 탈수초화가 중간엽 줄기세포의 이식을 통해서 재수초화될 수 있다는 것을 나타낸다.Demyelination of axons and structural nerve conduction that are not structurally impaired are thought to be the cause of functional defects due to spinal cord injury (Nicot, Brain 126, 398-412, 2003). Lysorecithin is a lipid that contains proteins similar to detergents and causes demyelination due to the same activity as membrane soluble agents that have specific toxicity to myelinated cells (Franklin et al., J Neurosci Res 58, 207-213, 1999). Arnett et al., Science, 306, 2111-2115, 2004). Lysolecithin is known to cause demyelination when injected into specific myelinated fibers such as the spinal cord in vivo , resulting in spinal cord injury. On the other hand, the loss of oligodendrocytes caused various demyelination diseases such as multiple sclerosis, and proved the relationship with myelination of spinal cord of oligodendrocytes. When progenitor cells of human oligodendrocytes were transplanted into the brains of damaged adult rats induced by lysolecithin, they could develop as oligodendrocytes as fast as oligodendrocytes. Axons could be myelinized (Goldman, Nat Biotechnol, 23, 862-871, 2005). The results indicate that demyelination of spinal cord axons induced by lysolecithin can be remyelinated through the transplantation of mesenchymal stem cells.
이에, 본 발명자들은 독성 화합물에 의해 탈수초화된 척수조직절편에 골수 유래 줄기세포를 이식함으로써, 척수조직절편으로부터 중간엽 줄기세포 쪽으로 재수초화에 의한 신경세포의 재생을 확인하고, 상기의 방향성 있는 신경세포재생의 능력이 새로운 치료법으로 활용할 수 있음을 확인함으로써 본 발명을 완성하였다.Thus, the present inventors transplanted bone marrow-derived stem cells into spinal cord tissue sections demyelinated by toxic compounds, thereby confirming the regeneration of nerve cells by remyelination from spinal cord tissue sections to mesenchymal stem cells, and the directed nerve The present invention was completed by confirming that the ability of cell regeneration can be utilized as a new treatment.
본 발명의 목적은 독성 화합물에 의해 탈수초화된 척수조직절편에 중간엽 줄기세포를 이식함으로써 나타나는 방향성 있는 신경세포의 재생능력을 신경 질환의 치료에 이용하고, 상기의 모델을 신경세포재생 능력을 갖는 신약 탐색에 활용하는 것이다.An object of the present invention is to use the neuronal regeneration of directional neurons, which is obtained by transplanting mesenchymal stem cells into demyelinated spinal cord tissue demyelination by toxic compounds, and to treat the neurological diseases. It is used to search for new drugs.
상기 목적을 달성하기 위하여, 본 발명은 척수 조직 절편을 이용한 손상된 신경 재생 물질의 탐색방법을 제공한다.In order to achieve the above object, the present invention provides a method for searching for damaged nerve regeneration material using spinal cord tissue sections.
또한, 본 발명은 골수 유래 중간엽 줄기세포를 개체의 손상된 척수조직에 투여하는 단계를 포함하는 탈수초화된 척수조직의 재수초화 방법을 제공한다.The present invention also provides a method for remyelination of demyelinated spinal cord tissue comprising administering bone marrow-derived mesenchymal stem cells to an individual's injured spinal cord tissue.
아울러, 본 발명은 골수 유래 중간엽 줄기세포를 포함하는 탈수초화된 척수조직의 재수초화용 세포 조성물을 제공한다.In addition, the present invention provides a cell composition for remyelination of demyelination spinal cord tissue, including bone marrow-derived mesenchymal stem cells.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 The present invention
1) 척수로부터 조직 절편을 수득하여 배양하는 단계;1) obtaining and culturing tissue sections from the spinal cord;
2) 단계 1의 배양중인 조직 절편에 독성 화합물을 처리하여 손상을 가하는 단계;2) damaging the tissue sections in culture of
3) 상기 손상된 조직 절편에 신경재생효과가 기대되는 후보물질을 처리하는 단계; 및3) treating the damaged tissue sections with candidate substances that are expected to have a neuronal regeneration effect; And
4) 상기 후보물질을 처리하지 않은 음성 대조군과 비교하여 유의하게 신경 재생효과를 나타내는 후보물질을 선별하는 단계를 포함하는 손상된 신경 재생 물질의 탐색방법을 제공한다.4) The present invention provides a method for searching for a damaged neuronal regeneration material, comprising selecting a candidate material having a significant neuronal regeneration effect as compared to the negative control which has not been treated with the candidate material.
동물을 대상으로 하는 실험적 설계는, 동물 개체를 대상으로 하는 in vivo, 세포 배양액을 대상으로 하는 in vitro 모델, 생조직을 대상으로 하는 ex vivo 모 델 등이 있다. in vivo 모델은 생체 내의 다양한 인자들에 의한 영향의 조절에 대한 확신을 가질 수 없고, 그 결과의 해석 또한 어려워질 수 있다. 그러나 in vitro 모델은 생체 시료의 활성을 보장할 수 없는 단점을 지니고 있다. 본 발명의 척수로부터 수득한 생조직 절편은, 초퍼를 이용한 신속한 수득 단계를 거침으로서 3 주간 배양이 가능하므로, in vivo와 in vitro 시스템의 장점을 포함하는 ex vivo 시스템을 제공할 수 있다. 본 발명의 시스템은 외부적인 자극을 준 후 급성과 준-급성 병리생리학(sub-acute pathophysiology)를 연구하는데 수월하며, 실험목적 이외의 환경을 조절하기가 쉽다. 뿐만 아니라 동물 모델에 비해 재현성이 뛰어나며, 경제적이라는 장점이 있다.Experimental designs for animals include in vivo models for animal subjects, in vitro models for cell cultures, and ex vivo models for living tissues. In vivo models cannot be confident in the regulation of effects by various factors in vivo , and interpretation of the results can be difficult. However, in vitro models have the disadvantage that they cannot guarantee the activity of biological samples. The biotissue sections obtained from the spinal cord of the present invention can be cultured for 3 weeks by undergoing a rapid harvesting step using a chopper, thereby providing an ex vivo system including the advantages of in vivo and in vitro systems. The system of the present invention is easy to study acute and sub-acute pathophysiology after external stimulation, and is easy to control the environment other than the experimental purpose. In addition, it has excellent reproducibility and economicality compared to animal models.
또한, 적당한 배양 조건하에서 상기의 생조직 절편이 대략 3 주 정도 배양이 가능하므로, 본 발명에서 제공하자 하는 척수 조직의 회복에 영향을 줄 수 있는 신규 물질의 탐색방법을 제공할 수 있다. 더욱 상세하게는, 상기의 생조직 절편의 신경을 인공적으로 손상한 후, 상기의 손상을 회복할 수 있을 것으로 기대되는 신규물질을 투여한 뒤, 2 내지 3 주간 배양하면서, 그 회복의 정도 및 특성을 관찰함으로써 신규물질의 활용 가능성을 측정하는 것이다.In addition, since the living tissue sections can be cultured for about 3 weeks under suitable culture conditions, a method for searching for a novel substance that can affect the recovery of spinal cord tissue to be provided by the present invention can be provided. More specifically, after artificially damaging the nerves of the living tissue sections, administering a new substance which is expected to recover the damage, and incubating for 2 to 3 weeks, the extent and characteristics of the recovery By observing this, the possibility of using a new substance is measured.
상기의 척수 조직 절편의 두께는 300 내지 500 ㎛, 바람직하게는 400 ㎛이다. 단계 2의 독성 화합물은 척수 축색돌기의 탈수초화를 유발하는 리소레시틴이 바람직하다. 단계 4의 효능의 증명은 면역형광염색법에 의한 신경교세포 표지 단백질의 발현을 관찰, 조직학적 방법에 의한 뉴런 재생의 관찰, 및 TUNEL 분석법에 의한 자살세포의 확인 등을 포함할 수 있다.The thickness of the spinal cord tissue sections is 300 to 500 µm, preferably 400 µm. The toxic compound of step 2 is preferably lysocithin, which causes demyelination of spinal cord axons. Demonstration of the efficacy of step 4 may include observing expression of glial cell labeled proteins by immunofluorescence staining, observing neuronal regeneration by histological methods, and identifying suicide cells by TUNEL assay.
상기 단계 2의 손상은 척수 내의 모든 신경손상을 포함하며 바람직하게는 탈수초화이고 단계 3에서, 후보물질은 제 1항에 있어서, 후보물질은 화합물, 성장인자, 사이토카인, 중간엽 줄기세포 유래인자, 세포외 기질 단백질(extracellular matrix protein), 축삭돌기 성장에 관여하는 단백질 또는 세포내 신호전달에 관여하는 인자(factors involved in intracellular signal transduction)일 수 있다.The damage of step 2 includes all nerve damage in the spinal cord and is preferably demyelination and in step 3, the candidate is a compound according to
또한, 본 발명은 골수 유래 중간엽 줄기세포를 개체의 손상된 척수조직에 투여하는 단계를 포함하는 탈수초화된 척수조직의 재수초화 방법 및 탈수초화된 척수조직의 재수초화용 세포 조성물을 제공한다.The present invention also provides a method for remyelination of demyelination spinal cord tissue comprising administering bone marrow-derived mesenchymal stem cells to a damaged spinal cord tissue of an individual, and a cell composition for remyelination of demyelination spinal cord tissue.
마취된 쥐로부터 척수를 추출하여 초퍼(chopper)를 이용하여 일정한 두께로 잘랐다. 척수조직 절편을 적당한 배양액에서 조직절편 배양하였고, 리소레시틴을 처리하여 탈수초화를 유도하였다(Birgbauer et al., J Neurosci Res, 78, 157-166, 2004). 탈수초화 유도 후 리소레시틴이 함유된 배지를 제거하고 새로운 배양액으로 교체한 후, 줄기세포를 척수 후주(dorsal column) 부위에 주입하여 재수초화를 유도하였다. 상기의 줄기세포는 척수 유래 줄기세포를 사용할 수 있고, 바람직하게는 중간엽 줄기세포를 사용한다. 상기의 탈수초군과 재수초군으로부터 면역형광염색법에 의한 신경교 세포 표지 단백질 발현의 관찰, 조직학적 방법에 의한 뉴런 재생의 관찰, 및 PI 분석법에 의한 세포사멸의 확인을 수행하였다.Spinal cords were extracted from anesthetized rats and cut to constant thickness using a chopper. Spinal cord tissue sections were cultured in appropriate cultures and treated with lysocithin to induce demyelination (Birgbauer et al., J Neurosci Res, 78, 157-166, 2004). After induction of demyelination, the medium containing lysocithin was removed and replaced with a new culture medium, and then stem cells were injected into the dorsal column to induce remyelination. The stem cells may be used spinal cord derived stem cells, preferably mesenchymal stem cells. Observation of glial cell labeling protein expression by immunofluorescence staining, neuronal regeneration by histological method, and apoptosis by PI analysis were performed from the demyelination group and remyelination group.
그 결과, 리소레시틴에 의해 척수조직 절편이 탈수초화된 것과(도 1 참조), 중간엽 줄기세포에 의해 재수초화된 것을 조직학적 방법으로 확인할 수 있었다(도 2 참조). 또한, 재수초시 중간엽 줄기세포로부터 척수조직절편으로 방향성을 갖고 신경이 재생되며, 신경교 세포의 발현 증가를 통해 신경세포의 재생을 면역형광법으로 확인할 수 있었다(도 3,4,5 및 6 참조)As a result, it was confirmed by histological method that the spinal cord tissue sections were demyelinated by lysolecithin (see FIG. 1), and remyelinated by mesenchymal stem cells (see FIG. 2). In addition, the regeneration of the nerve from the mesenchymal stem cells from the mesenchymal stem cells to the spinal cord tissue section, and the regeneration of the nerve cells through the expression of glial cells was confirmed by immunofluorescence (see FIGS. 3, 4, 5 and 6). )
본 발명이 적용가능한 개체는 척추동물이고 바람직하게는 포유동물이며, 상기 골수 유래 중간엽 줄기세포를 포함하는 탈수초화된 척수조직의 재수초화 방법 및 세포 조성물이 적용될 수 있는 개체는 척추동물이고 바람직하게는 포유동물이며, 본 발명의 방법 및 조성물은 신경 질환 등의 치료법으로 이용될 수 있다. 적용 가능한 신경 질환으로는 신경세포의 비정상적 소멸에 의해 야기되는 각종 질병은 물론 척수 손상에 의한 운동 장애 등의 치료에도 이용될 수 있다. The subject to which the present invention is applicable is a vertebrate and preferably a mammal, and the subject to which the method and cell composition for remyelination of demyelination spinal cord tissue including the bone marrow-derived mesenchymal stem cells is applicable is a vertebrate. Is a mammal, and the methods and compositions of the present invention can be used for the treatment of neurological diseases and the like. Applicable neurological diseases can be used for the treatment of various diseases caused by abnormal disappearance of nerve cells as well as movement disorders caused by spinal cord injury.
이하, 본 발명을 실시예에 의해 상세히 설명하였다. 단 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용을 한정하지는 않는다.Hereinafter, the present invention has been described in detail by way of examples. However, the following examples are merely to illustrate the present invention, but not to limit the content of the present invention.
<실시에 1> 중간엽 줄기세포 이식Example 1 Mesenchymal Stem Cell Transplantation
<1-1> 인간 중간엽 줄기세포의 배양 <1-1> Culture of Human Mesenchymal Stem Cells
골수에서 유래한 인간 중간엽 줄기세포는 캠브렉스사(USA)에서 구입하여, 중간엽 줄기세포 성장 배지(MSCGM)에서 2 계대까지 배양하였고, 그 이후로는 10% FBS(Invitrogen, USA), 100 ng/㎖ 페니실린, 및 100 U/㎖ 스트렙토마이신 (Invitrogen, USA)이 함유된 DMEM(Invitrogen, USA)에서 배양하였다. 5% CO2 및 37℃의 조건에서 유지되었다.Bone marrow-derived human mesenchymal stem cells were purchased from Cambrex (USA) and cultured up to 2 passages in mesenchymal stem cell growth medium (MSCGM), after which 10% FBS (Invitrogen, USA), 100 Incubations were made in DMEM (Invitrogen, USA) containing ng / ml penicillin, and 100 U / ml streptomycin (Invitrogen, USA). It was maintained at the conditions of 5% CO 2 and 37 ° C.
<1-2> 척수 조직 절편 제작<1-2> Spinal Cord Tissue Section Fabrication
16일령 성체 SD 쥐를 마취한 후, 척추를 추출한 후 얼음에 식혀놓은 HBSS(Hanks balanced salt solution)에 담아두었다. 초퍼(McIlwain tissue chopper, The Mickle Laboratory Engineering Co. LTD, England)를 사용하여 흉부와 허리 척수를 400 ㎛ 두께로 잘라서, 여러 개의 척수조직 절편을 6 웰 플레이트에 놓인 0.4 ㎛ 필터(Millicell-CM filters, Millipore, USA) 위에 놓았다. 척수 절편은 염(Earl's salts)이 함유된 50% MEM(Invitrogen, USA), 25% HBSS(Invitrogen, USA), 20 mM HEPES(Sigma, USA), 및 6 ㎎/㎖의 D-glucose(Duchefa, Netherlands)가 함유된 25% Horse serum(Invitrogen, USA) 1 ㎖에 배양하였다. 절편은 5% CO2, 37℃의 조건에서 유지되었고 배지는 일주일에 두 번 교체하였다.After 16-day-old adult SD rats were anesthetized, the spine was extracted and placed in iced HBSS (Hanks balanced salt solution). A 0.4 μm filter (Millicell-CM filters, placed on a 6-well plate) using a chopper (McIlwain tissue chopper, The Mickle Laboratory Engineering Co. LTD, England) was used to cut the thoracic and lumbar spinal cords to 400 μm thickness. Millipore, USA). Spinal cord slices were obtained with 50% MEM (Invitrogen, USA) containing Ear's salts, 25% HBSS (Invitrogen, USA), 20 mM HEPES (Sigma, USA), and 6 mg / ml D-glucose (Duchefa, Incubated in 1 ml of 25% Horse serum (Invitrogen, USA). Sections were maintained at 5% CO 2 , 37 ° C. and medium was changed twice a week.
<1-3> 리소레시틴에 의한 탈수초화 유도<1-3> Induction of demyelination by lysocithin
척수 절편을 7일 동안 in vitro(DIV, days in vitro)에서 배양하였다. 0.5 ㎎/㎖의 리소레시틴을 처리한 후, 37℃에서 17시간 동안 배양하여 탈수초화를 유도하였다(Birgbauer et al., J Neurosci Res, 78, 157-166, 2004). 탈수초화 유도 후 리소레시틴이 함유된 배지를 제거하고 새로운 배양 배지 1 ㎖로 교체하였다.Spinal cord sections were incubated for 7 days in vitro (DIV, days in vitro). After treatment with 0.5 mg / ml of lysolecithin, incubation at 37 ° C. for 17 hours induced demyelination (Birgbauer et al., J Neurosci Res, 78, 157-166, 2004). After induction of demyelination, the medium containing lysocithin was removed and replaced with 1 ml of fresh culture medium.
<1-4> 세포 이식<1-4> Cell Transplantation
Eppendorf Cell Tram Injector (Eppendorf, Germany)를 이용하여 실시예 <1-1>의 중간엽 줄기세포를 실시예 <1-2>의 절편의 척수 후주(dorsal column) 부위에 주입함으로써 이식을 수행하였다.Transplantation was performed by injecting the mesenchymal stem cells of Example <1-1> into the dorsal column region of the section of Example <1-2> using an Eppendorf Cell Tram Injector (Eppendorf, Germany).
<실시예 2> 신경세포 재생 확인Example 2 Neuronal Regeneration Confirmation
<2-1> 면역형광염색법<2-1> Immunofluorescence Staining
척수 절편은 4% 포름알데히드로 밤새 4℃에서 고정되었고 0.5% Triton X-100로 10분 동안 투과되었다. 절편은 0.1% Triton X100이 함유된 3% BSA로 한 시간 동안 차단된 후 1차 항체로서 래빗-MBP 항체(myelin basic protein; Chemicon, USA), 래빗-NF-M 항체(Neurofilament M; Chemicon, USA), 마우스-Hu 항체 (Human Nuclei; Chemicon, USA), PI (propidium iodide, Sigma, USA) 또는 마우스-CNPase 항체 (cyclin nucleotide phosphodiesterase; Covance, USA)로 4℃에서 밤새 배양되었고, 염소-항-마우스 Alexa488 (녹색, molecular probe, USA), 또는 항-래빗 Cy3(빨간색, Jackson Lab, USA)와 같은 2차 항체를 이용하여 배양되었다.Spinal cord sections were fixed at 4 ° C. overnight at 4% formaldehyde and permeated for 10 minutes with 0.5% Triton X-100. Sections were blocked for 1 hour with 3% BSA containing 0.1% Triton X100 and then rabbit-MBP antibody (myelin basic protein; Chemicon, USA), rabbit-NF-M antibody (Neurofilament M; Chemicon, USA) as the primary antibody. ), Mouse-Hu antibody (Human Nuclei; Chemicon, USA), PI (propidium iodide, Sigma, USA) or mouse-CNPase antibody (cyclin nucleotide phosphodiesterase; Covance, USA) and incubated overnight at 4 ° C. The mice were incubated with secondary antibodies such as Alexa488 (green, molecular probe, USA), or anti-rabbit Cy3 (red, Jackson Lab, USA).
그 결과, 리소레시틴에 의해 척수 절편에서 대조군에 비해 MBP의 발현량이 감소한 것을 확인하였다(도 1). 또한, NF-M의 발현을 면역형광법으로 관찰한 결 과, 중간엽 줄기세포로부터 척수절편 쪽으로 방향성을 갖는 재수초화를 관찰할 수 있었다(도 3, 4). 더불어, 신경교세포 표지(CNPase)의 발현량의 증가를 확인할 수 있었고(도 4, 6), PI의 사멸세포의 표지가 중간엽 줄기세포 처리군에서 감소하는 것을 관찰함으로써, 중간엽 줄기세포에 의한 신경재생의 효과를 확인할 수 있었다(도 6).As a result, it was confirmed that the amount of MBP expression was reduced in the spinal cord slices by lysocithin compared to the control (Fig. 1). In addition, as a result of observing the expression of NF-M by immunofluorescence, remyelination with a direction from the mesenchymal stem cells toward the spinal cord fragment was observed (Fig. 3, 4). In addition, an increase in the expression level of glial cell markers (CNPase) was confirmed (FIGS. 4 and 6), and the mesenchymal stem cell induced by mesenchymal stem cell treatment was observed by observing a decrease in the label of PI dead cells. The effect of neuronal regeneration could be confirmed (FIG. 6).
<2-2> 조직학적 분석<2-2> Histological Analysis
척수조직 절편을 10% 포르말린으로 1시간 동안 고정하였다. 그리고 수초의 검출을 위해 조직 절편을 0.1% Luxol fast blue로 염색을 하였고 뉴런을 구별하기 위하여 H&E대비염색제로 착색하였다.Spinal cord sections were fixed with 10% formalin for 1 hour. Tissue sections were stained with 0.1% Luxol fast blue for detection of myelin sheath and stained with H & E counterstainers to distinguish neurons.
그 결과, 리소레시틴에 의해 척수절편에서 대조군에 비해, 심한 탈수초 현상이 나타난 것을 확인할 수 있었고(도 1), 중간엽 줄기세포의 이식에 의해 재수초화 된 것을 확인할 수 있었다(도 2).As a result, it was confirmed that severe demyelination was observed in the spinal cord slices by lysocithin compared to the control group (FIG. 1), and remyelination by transplantation of mesenchymal stem cells (FIG. 2).
본 발명의 척수 유래 줄기세포 이식에 의한 탈수초화된 신경세포의 재생 방법 및 척수 생조직 절편의 생체외(ex vivo) 모델 적용에 의한 신경재생물질의 탐색방법은 신경 질환 등의 치료법으로 유용하게 이용될 수 있다.The method of regeneration of demyelized neurons by spinal cord-derived stem cell transplantation of the present invention and the search method of neuronal regeneration material by applying ex vivo model of spinal cord biotissue sections are useful for the treatment of neurological diseases. Can be.
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