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올레아놀린산 및 그 유도체가 흰쥐 해마 신경줄기세포의 분화에 미치는 효과

Title
올레아놀린산 및 그 유도체가 흰쥐 해마 신경줄기세포의 분화에 미치는 효과
Other Titles
Effects of Oleanolic Acid and Its Derivatives on the Differentiation of Rat Hippocampal Neural Progenitor Cells
Author
노현우
Alternative Author(s)
Roh, Hyun Woo
Advisor(s)
이창호
Issue Date
2015-02
Publisher
한양대학교
Degree
Master
Abstract
본 연구에서는 oleanolic acid (OA) 및 그 유도체가 흰쥐 해마 신경줄기세포의 분화에 미치는 영향을 평가하였다. 이를 위해, 수상돌기 가짓수 증가, 길이 신장 및 신경줄기세포 분화 시 수상돌기 가시 형성 등에 미치는 OA 및 그 유도체의 생리활성을 측정하였다. 임신 16.5일 된 흰쥐 배아의 해마조직으로부터 신경줄기세포를 분리, 배양하여 OA와 그 유도체의 생물학적 효과를 규명하였다. MTT 시험을 통하여, OA와 그 유도체는 신경줄기세포에 대한 증식 효과 및 세포독성을 나타내지 않음을 확인하였다. 신경줄기세포의 분화를 특징짓는 표지 단백에 대한 항체를 사용하여 면역세포 화학 염색을 수행한 결과, OA와 그 유도체 처치에 의하여 신경줄기세포가 분화하는 동안 수상돌기 가짓수 증가 및 길이 신장, MAP2와 Tuj1 단백발현이 증가됨이 관찰되었는바, 이는 OA 및 그 유도체에 의한 신경줄기세포 분화 유도 효능을 제시한다. 이들 화합물이 신경줄기세포 분화에 미치는 효과의 추가적인 확인을 위해 축삭돌기의 신경발생 시 관여하는 미소관 관련단백인 MAP2, 시냅스 전 말단과 축삭돌기의 주요 구성요소 관련 단백인 GAP43, 시냅스 소포 구성요소 관련 단백인 Synapsin1, 시냅스 소포단백인 synaptophysin 등의 특이적인 항체를 이용하여 western blot 실험을 수행하였다. 면역세포 화학염색과 western blot 결과 등의 분석을 통해 OA, OAA, OAM을 처리 시 수상돌기가시 형성 관련 단백인 Kif1a, 신경 접합부의 성숙과 가소성 관련단백인 PSD-95, 신경발생 시 관여하는 미소관 단백인 MAP2 등과 같은 시냅스 표지 단백발현이 2배 이상 증가됨을 확인하였다. 위 결과들을 종합하면, OA 및 그 유도체가 신경줄기세포 분화 과정에서 새로운 수상돌기 가시와 신경접합부 형성을 유도하였다고 할 수 있다. 결론적으로 OA 및 그 유도체는 신경줄기세포의 분화와 시냅스 형성을 증강시켰다. 이는 해마부위 신경세포 분화 시 수상돌기 형성과 신경 접합부 연결에 대한 OA 및 그 유도체의 신경 영양적 지원 가능성을 제시한다. |Experiments were carried out to determine the effect of oleanolic acid (OA) and its derivatives on the formation of the dendrite and dendritic spine during the hippocampal neural hippocampal cell (NPC) differentiation. Hippocampal NPCs were prepared from the embryonic day 16.5 rat hippocampus and then treated with OA or its derivatives. MTT assay proved that test compounds were not cytotoxic to hippocampal NPCs. Immunocytochemistry analysis was performed with the antibodies against specific marker proteins representing NPC differentiation marker proteins. The results showed that OA and its derivatives caused significant induction on NPC differentiation as evidenced by the 2- to 4-fold increases in the number of dendritic branches of NPCs treated with OAA, and OAM as compared with control group. Among them, OAA was found to be the most active in inducing dendritic branch formation. Moreover, the length of dendrite was measured and analyzed. OA, and OAA caused about 1.4- to 1.8-fold increase as compared with control group. Also the expression of MAP2 and Tuj1 was measured and analyzed. Treatment with OA, OAA or OAM resulted in 1.5- to 2-fold increase compared to control in neural progenitor cell culture during differentiation. In addition, the effect of test compounds on the differentiation of NPCs were examined with antibodies against specific proteins responsible for the synaptic structure and function, which include Kif1a for axonal transportation of synaptic vesicle, PSD-95 for synaptic maturation and plasticity, MAP2 for microtubule in neurogenesis, GAP43 for a crucial component of the axon and presynaptic terminal, synaptophysin for synaptic vesicle glycoprotein which may participate in synaptic transmission, and synapsin1 for synaptic vesicle component protein. Immunocytochemistry and western blot analysis showed that OA, OAA and OAM caused more than 2-fold increase in dendritic spine formation and the expression of synaptic marker proteins such as PSD-95, synapsin1 and synaptophysin. The actions of OA and its derivatives might have resulted in the formation of new spines and synapses. Taken together, it was proposed that OA and its derivatives were able to enhance the differentiation and synaptic formation of the NPCs, which implied that these compounds might provide neurotrophic support on the dendrite formation and synaptic connectivity during the neural differentiation in the hippocampus.; Experiments were carried out to determine the effect of oleanolic acid (OA) and its derivatives on the formation of the dendrite and dendritic spine during the hippocampal neural hippocampal cell (NPC) differentiation. Hippocampal NPCs were prepared from the embryonic day 16.5 rat hippocampus and then treated with OA or its derivatives. MTT assay proved that test compounds were not cytotoxic to hippocampal NPCs. Immunocytochemistry analysis was performed with the antibodies against specific marker proteins representing NPC differentiation marker proteins. The results showed that OA and its derivatives caused significant induction on NPC differentiation as evidenced by the 2- to 4-fold increases in the number of dendritic branches of NPCs treated with OAA, and OAM as compared with control group. Among them, OAA was found to be the most active in inducing dendritic branch formation. Moreover, the length of dendrite was measured and analyzed. OA, and OAA caused about 1.4- to 1.8-fold increase as compared with control group. Also the expression of MAP2 and Tuj1 was measured and analyzed. Treatment with OA, OAA or OAM resulted in 1.5- to 2-fold increase compared to control in neural progenitor cell culture during differentiation. In addition, the effect of test compounds on the differentiation of NPCs were examined with antibodies against specific proteins responsible for the synaptic structure and function, which include Kif1a for axonal transportation of synaptic vesicle, PSD-95 for synaptic maturation and plasticity, MAP2 for microtubule in neurogenesis, GAP43 for a crucial component of the axon and presynaptic terminal, synaptophysin for synaptic vesicle glycoprotein which may participate in synaptic transmission, and synapsin1 for synaptic vesicle component protein. Immunocytochemistry and western blot analysis showed that OA, OAA and OAM caused more than 2-fold increase in dendritic spine formation and the expression of synaptic marker proteins such as PSD-95, synapsin1 and synaptophysin. The actions of OA and its derivatives might have resulted in the formation of new spines and synapses. Taken together, it was proposed that OA and its derivatives were able to enhance the differentiation and synaptic formation of the NPCs, which implied that these compounds might provide neurotrophic support on the dendrite formation and synaptic connectivity during the neural differentiation in the hippocampus.
URI
https://repository.hanyang.ac.kr/handle/20.500.11754/128807http://hanyang.dcollection.net/common/orgView/200000426406
Appears in Collections:
GRADUATE SCHOOL OF BIOMEDICAL SCIENCE AND ENGINEERING[S](의생명공학전문대학원) > BIOMEDICAL SCIENCE(의생명과학과) > Theses (Master)
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