• 대한약리학회지
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• 제30권1호
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• pp.39-48
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• 1994

포만중추 (satiety center)를 자극하여 음식섭취를 억제한다고 알려진 CCK-8을 흰쥐 복강에 투여하여, 흰쥐 뇌의 도파민 변화에 대한 CCK-8의 효과를 관찰하였다. 흰쥐 뇌의 부위별 도파민 함량은 HPLC-ECD방법으로 측정하였으며, 시상하부와 흑질에서의 TH-immunoreactive neuron은 면역조직화학법과 영상분석법을 시행하였다. 굶긴 쥐에서는 정상 쥐에 비하여, 도파민 함량이 전두 피질, 해마, 시상하부 및 편도체에서 각각 감소하였다. CCK-8을 투여한 쥐는 정상 쥐와 굶긴 쥐에 비하여, 도파민 함량이 시상하부에서 의미있게 감소하였다. 또한 굶긴 쥐는 정상 쥐와 비교하여, TH-positive neuron의 분포와 수가 뇌실옆핵, 깔때기핵, 정중융기 및 혹질에서 현저히 감소하였다. CCK-8투여시, 시상하부와 흑질에서의 TH-immunoreactive neuron의 수는 굶긴 쥐에 비하여 증가하였다. 이상의 실험 결과로 보아 음식물 섭취를 억제하는 작용이 있는 CCK-8은 시상하부의 도파민 신경계와 일부분 관련되어 있으며, 또한 시상하부와 흑질에 존재하는 TH-positive neuron은 음식물 섭취 행위에 중요한 역할이 있음을 시사하고 있다.

• 대한수의학회지
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• 제40권1호
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• pp.1-16
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• 2000

Nowadays, mongolian gerbil is notably utilized for the research of brain and water deprivation because of a congenital incomplete willis circle structure in the brain, audiogenic seizure in low noise, and special cholesterol metabolism without water absorption for a long time. In this study, we are intend to identify the morphological changes of the catecholaminergic neuron of brain according to the time lapse in the condition of long term water deprivation. 55 mongolian gerbil were divided 10 groups(control, 1, 2, 3, 4, 5, 10, 15, 20, 42th day water deprivation group), of which each group include 5 mongolian gerbils and 5 normal mongolian gerbils in control group were also used for brain atlas as a control. The brains were observed by the immunohistochemical stain using the TH, DBH and PMNT antibody. The results were as followings; 1. The nerve fibers of the TH-immunoreactive neuron were observed only in the and corpus striatum of the telencephalon. 2. Intensity of the immunostain of the nerve fiber in the cerebral cortex and corpus striatum was decreased gradually day by day after water deprivation. 3. The TH-immunoreactive nerve cells were observed in the paraventricular and periventricular nucleus of the 3rd ventricular in the hypothalamus of mongolian gerbil but the number of nerve cells were decreased from the first day of the water deprivation to the 10th day and increased until the 20th day, after than redecreased from the 20th day by the continuous water deprivation. The number of nerve fibers in this area were increased in the first day, but decreased from the 2nd day of water deprivation. The shape and density of the dopamine secreting cells in the brain of mongolian gerbil by the immunoreactive stain were changed in the continuous water deprivation. In this results, we can conclude that dopamine concerned in the water metabolism of mongolian gerbil, and mongolian gerbil could be used as an animal model for the research of water deprivation.

• 대한수의학회지
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• 제40권2호
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• pp.197-211
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• 2000

The pineal body have been known to be affected by superior cervical ganglia, and most of its nerve fibers containing peptidergic neurotransmitters have been considered to be originated from this ganglia. To confirm this relationships, some peptidergic neurotransmitters were identified in both of pineal body and superior cervical ganglia of the Korean native goat, which were divided into two group; breeding season and non-breeding season. The localizations of two catecholamine-synthesizing enzymes; tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH), were investigated by immunohistochemistry in the superior cervical ganglia and the pineal body of adult Korean native goats. Substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and galanin (GAL) were also identified in these organs by immunohistochemical and double immunofluorescent methods. In superior cervical ganglia, immunoreactivities for TH and DBH were confirmed in the same ganglion cells. The immunoreactivites for SP, VIP(only in male), NPY and GAL were identified in both of ganglion cell bodies and nerve fibers in the ganglia. CGRP immunoreactivity, however, was observed only in nerve fibers. Most NPY- and VIP-immunoreactive(IR) ganglion cells also contained TH. SP and TH were colocalized in the cell bodies, but not in the nerve fibers. TH immunoreactivity was shown in almost all of ganglion cells in the superior cervical ganglia. The immunoreactivity for NPY had some seasonal variation and was stronger in breeding season than in non-breeding season. In pineal body, lots of TH-IR fibers were observed throughout the parenchyma including the pineal stalk and most of them also contained DBH. SP- and NPY-IR fibers were also immunostained with TH or DBH. But a few SP- and NPY-IR fibers were not colocalized with TH or DBH. Exceptionally, a bipolar neuron-like cell was observed to be immunostained with NPY in the pineal body. A few CGRP and GAL-IR fibers were observed, while VIP-IR fibers were not present. It is concluded that most TH- and DBH-IR fibers as well as the peptidergic immunoreactive fibers of the pineal body might be originated from the superior cervical ganglia. Some peptidergic immunoreactive fibers, however, might be come from other regions of brain. We also suggest that NPY in pineal body plays a important role for pineal function. The seasonal variation of NPY immunoreactivity indicates that the synthesis and use of NPY may be different between in breeding and non-breeding seasons.

• 대한수의학회지
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• 제40권3호
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• pp.451-461
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• 2000

The locus coeruleus(LG) is known to be observed a sexual dimorphism in rat CNS. LC is the largest collections of norepinephrine(NE)-containing neurons in the mammalian brain. Especially in rat, all LC neurons contained NE unlike other mammalians, so that specific reactions were found in the tyrosine-hydroxylase(TH) immunoreactive neurons. Sexual dimorphism of rat LC has affected by genes before sex hormone appeared, thereafter affected by sex hormones. In these day, many scientists founded morphological differences between male and female LC morphology, but differences of entire structure was not founded. Thus we investigated sex differences of the LC neuron's morphology in rat by three-dimensional(3-D) reconstruction using Confocal laser scanning microscopy(CLSM). We reported that neuron's shape was relatively-large multipolar neurons and neuron's processes in dorsal LC proceeded to ventral direction in the male and female rat. Male had a longer anterior-posterior length than female had in dorsal LC. In addition to middle-LC, male rat's LC had a more thicker posterior region but had not viewed in a previous study. In reverse, female rat's LC had a thicker anterior region like a previous study. This results using 3-D reconstruction by CLSM showed that the male's LC was more wide-ranging than female's relatively.

• Clinical and Experimental Reproductive Medicine
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• 제31권1호
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• pp.67-74
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• 2004

Objective: This study was to examine in vitro neural cell differentiation pattern of the genetically modified human embryonic stem cells expressing tyrosine hydroxylase (TH). Materials and Methods: Human embryonic stem (hES, MB03) cell was transfected with cDNAs cording for TH. Successful transfection was confirmed by western immunoblotting. Newly transfected cell line (TH#2/MB03) was induced to differentiate by two neurogenic factors retinoic acid (RA) and b-FGF. Exp. I) Upon differentiation using RA, embryoid bodies (EB, for 4 days) derived from TH#2/MB03 cells were exposed to RA ($10^{-6}M$)/AA ($5{\times}10^{-2}mM$) for 4 days, and were allowed to differentiate in N2 medium for 7, 14 or 21 days. Exp. II) When b-FGF was used, neuronal precursor cells were expanded at the presence of b-FGF (10 ng/ml) for 6 days followed by a final differentiation in N2 medium for 7, 14 or 21 days. Neuron differentiation was examined by indirect immunocytochemistry using neuron markers (NF160 & NF200). Results: After 7 days in N2 medium, approximately 80% and 20% of the RA or b-FGF induced Th#2/MB03 cells were immunoreactive to anti-NF160 and anti-NF200 antibodies, respectively. As differentiation continued, NF200 in RA treated cells significantly increased to 73.0% on 14 days compared to that in b-FGF treated cells (53.0%, p<0.05), while the proportion of cells expressing NF160 was similarly decreased between two groups. However, throughout the differentiation, expression of TH was maintained ($\sim$90%). HPLC analyses indicated the increased levels of L-DOPA in RA treated genetically modified hES cells with longer differentiation time. Conclusion: These results suggested that a genetically modified hES cells (TH#2/MB03) could be efficiently differentiated in vitro into mature neurons by RA induction method.

• Applied Microscopy
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• 제30권2호
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• pp.193-204
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• 2000

Mongolian gerbil (Meriones unguiculatus)은 선천적으로 불완전한 뇌바닥동백고리와 특이한 콜레스테롤 대사를 가진 동물로서 수분의 섭취없이도 장기간 생존이 가능하기 때문에 최근 뇌신경학 연구방법에 많이 이용되는 동물이다. 따라서 본 연구는 mongolian gerbil을 절수 시켰을 때 시간의 경과에 따라 뇌에서의 catecholaminergic neuron의 형태적인 변화에 관하여 알아보고자 $50\sim80g$의 mongolian gerbil 25마리를 이용하여 5마리는 뇌도보를 작성하였으며 나머지는 정상군, 5일군, 10일군, 20일군으로 각각 5마리씩 나누어 TH, DBH, PNMT항체를 이용하여 면역조직화학염색하여 절수의 시간에 따른 뇌에서의 catecholaminer-gic neuron의 형태적인 변화를 광학 및 전자현미경적으로 관찰하여 다음과 같은 결론을 얻었다. 1. 정상군의 경우 시상하부의 제 3 뇌실주위에 있는 뇌실곁핵과 뇌실주위회백질에 TH면역반응세포들이 나타났고 이들의 신경세포돌기 및 신경섬유들도 많이 분포하였으나 20일군에서 감소되는 양상을 보였다. 2. 중뇌 및 교뇌에서는 catecholamine성 신경세포중 dopamine성 신경세포가 뚜렷히 관찰되었으나 noradrenalin성 신경세포는 약하게 관찰되었다. 3. 흑색질의 치밀부분과 배쪽피개영역에서 TH항체에 대한 면역반응을 보이는 Dopamine성 신경세포 밀집도가 높았으며 그물부분에서도 소수 관찰되었다. 그러나 이 부위에서 dopamine성 신경세포도 절수에 따라 5일군까지 신경세포체와 돌기 및 섬유들이 감소되었으나 10일군에서는 다시 증가하였으며 20일군에서부터 감소하는 양상을 보였다. 4. 절수에 의한 뇌의 TH면역반응세포체의 전자현미경적 소견은 정상군에 비해 5일군에서 핵의 농축 및 변형, 신경연접의 소실, 사립체의 감소, 축삭 수의 감소 등이 나타나 TH의 분비에 영향을 주는 것으로 보인다. 그러나 ER등의 변화는 크게 관찰할 수 없었다. 이러한 연구결과는 정상군의 경우 다른 동물과 유사했으나 다른 동물에서는 청색반점이나 교뇌부위에서 noradrenaline성 신경이 많다고 하였지만 본 연구에서는 그러한 차이를 발견하지 못했으며 이것은 동물간의 유전적, 환경적 차이로 사료된다. 그러므로 mongolian gerbil의 수분대사에 dopamine이 관여할 것으로 보고 탈수 질환모델로 개발하기 위한 기능적인 연구와 더불어 많은 형태학적인 연구가 병행되어야 할 것이다.

• Journal of the Korean Academy of Child and Adolescent Psychiatry
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• 제4권1호
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• pp.79-90
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• 1993

대뇌피질에서 somatostatin 함유 신경세포의 생후 발달과정을 규명하고자 생후 1, 2, 3, 4주 및 2, 3개월의 흰쥐 대뇌를 대상으로 면역조직화학적 염색을 시행하여 신경세포의 형태, 크기 및 분포와 대뇌피질 영역별 세포 수의 변화를 관측하여, 아래와 같은 결과를 얻었다. 1) 형태 및 분포의 변화 생후 1주부터 비교적 염색성이 뚜렷한 다양한 형태의 미성숙 신경세포가 대뇌피질 V, VI층에서 관찰되다가 생후 2주에는 세포질내의 염색성이 균일하게 증가되어 있고 여러개의 돌기를 내는 뭇극성세포가 V, VI층뿐 아니라 제 II층에서도 출현하였다. 생후 3주부터는 신경세포체가 점차 II, III층과 V, VI층에 넓게 분포하였고 미성숙한 신경세포는 II, III층에서 보다 많이 관찰되었으며 생후 4주에는 IV, V, VI층에서의 세포의 감소가 뚜렷하여 주로 II, III, IV층에 분포하는 양상을 보였고 미성숙세포는 거의 관찰되지 않았다. 2) 신경세포체의 크기의 변화 세포체의 크기는 생후 2주를 전후하여 일시적으로 증가하여 최대값을 보이다가 이후 점차 감소하여 생후 8주째에 성숙흰쥐에서의 크기가 되었다. 3) 내뇌피질 영역별 신경세포체 수의 변화 전두, 두정 1, 2, 측두 1, 배모양, 섬피질에서는 생후 1주에서 2주째에 걸쳐 큰 차이없이 세포체의 수가 최대로 증가했다가 이후 성숙흰쥐 수준으로 감소하는 양상을 보였으며 측두 3, 후두, 띠, 코주위 피질에서는 생후 2주째에 최대값을 보였다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.79-79
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• 2003

Embryonic stem cells are capable of differentiating into a variety of cell lineages. However, the ultimate results of differentiation in vitro greatly depend on the duration of treatment and kinds of differentiating inducers added. In order to investigate the efficiencies of various differentiation inducers and the methods of treatment, we examined differentiation patterns of human embryonic stem cell (hESC, MB03) according to several different protocols. Exp. I) Upon differentiation using retinoic acid and ascorbic acid (RA/AA), embryoid bodies (EB, for 4days) derived from hESC was exposed to Rh (10$^{-6}$ M) and AA (50 mM) for 4 days, and were allowed to differentiate in N2 medium for 7, 14, 21, or 28 days. Exp. II) When bFGF was used, neuronal precursor cells were selected for 8 days in N2 medium after EB formation. After selection, cells were expanded at the presence of bFGF (20 ng/ml) for another 6 days followed by a final differentiation in N2 medium for 7, 14, 21 or 28 days. Exp. III) In addition, to examine the effects of neurotrophic factors in the production of mature neurons, groups of cells were exposed to either BDNF (5 ng/ml) or TGF-$\alpha$(10 ng/ml) during the 28 days of final differentiation. Differentiation patterns of RA/AA or bFGF treated groups were very similar; approximately 82% and 83% of the cells, respectively, were positive for anti-NF200 antibody, while it was about 10% and 11%, respectively, for anti-NF160 antibody in 28 days in N2 medium. Alsor, cells expressing TH were as low as 5%, while the cells doubled when matured at the presence of either BDNF or TGF-$\alpha$. Cells immunoreactive to anti-GAD antibody were approximately 20%. These results suggest that a maturation step rather than differentiation induction step, which is formation of EB, effects more decisively to the ultimate differentiation pattern.

• 한국발생생물학회지:발생과생식
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• 제13권3호
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• pp.173-181
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• 2009

One of the most extensively studied populations of multipotent adult stem cells are mesenchymal stem cells (MSCs). MSCs derived from the human umbilical cord vein (HUC-MSCs) are morphologically and immunophenotypically similar to MSCs isolated from bone marrow. HUC-MSCs are multipotent stem cells, differ from hematopoietic stem cells and can be differentiated into neural cells. Since neural tissue has limited intrinsic capacity of repair after injury, the identification of alternate sources of neural stem cells has broad clinical potential. We isolated mesenchymal-like stem cells from the human umbilical cord vein, and studied transdifferentiation-promoting conditions in neural cells. Dopaminergic neuronal differentiation of HUC-MSCs was also studied. Neural differentiation was induced by adding bFGF, EGF, dimethyl sulfoxide (DMSO) and butylated hydroxyanisole (BHA) in N2 medium and N2 supplement. The immunoreactive cells for $\beta$-tubulin III, a neuron-specific marker, GFAP, an astrocyte marker, or Gal-C, an oligodendrocyte marker, were found. HUC-MSCs treated with bFGF, SHH and FGF8 were differentiated into dopaminergic neurons that were immunopositive for tyrosine hydroxylase (TH) antibody. HUC-MSCs treated with DMSO and BHA rapidly showed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including NeuroD1, $\beta$-tubulin III, GFAP and nestin was markedly elevated during this acute differentiation. While the stem cell markers such as SCF, C-kit, and Stat-3 were not expressed after neural differentiation, we confirmed the differentiation of dopaminergic neurons by TH/$\beta$-tubulin III positive cells. In conclusion, HUC-MSCs can be differentiated into dopaminergic neurons and these findings suggest that HUC-MSCs are alternative cell source of therapeutic treatment for neurodegenerative diseases.

• 한국발생생물학회지:발생과생식
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• 제12권1호
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• pp.31-39
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• 2008

손상된 뇌신경조직내에서 신경줄기세포로부터 새로운 신경세포로의 분화가 상당히 제한되어 있어 이것이 손상된 뇌신경조직의 복구가 잘 이루어지지 않는 원인이라 여겨지고 있다. 본 연구에서는 세포배양을 통해 지방조직 중간엽 줄기세포를 도파민성 신경세포와 콜린성 신경세포로 분화를 유도하였다. 중간엽 줄기세포를 신경세포로 분화시키기 위해 N2배양액에 bFGF, EGF, dimethyl sulphoxide (DMSO)와 butylated hydroxyanisole (BHA)를 첨가하여 유도하였다. DMSO와 BHA에 처리된 중간엽 줄기세포가 빠르게 신경세포 모양으로 분화하는 것을 관찰하였으며, 이것은 면역조직학적 염색에서 신경세포 특이 표지인 $\beta$-tubulin III, 별아교세포에 대한 특이 표지인 GFAP, 흰돌기아교세포에 대한 특이 표지인 Gal-C에 대해 양성반응을 나타내었다. RT-PCR 분석에서 배양 단계에 따라 신경세포에 특이적인 표지 인자인 neuro D1, $\beta$-tubulin III, GFAP, nestin 등의 발현을 통해, 중간엽 줄기세포가 신경세포로 분화됨을 확인하였다. 그러나 중간엽줄기세포가 신경세포로 분화된 이후에는 줄기세포 표지인 SCF, C-kit와 stat-3 등은 발현되지 않았다. 또한, 중간엽줄기세포에 bFGF, SHH와 FGF8 등을 처리하면 도파민 신경세포로 분화하였다. 중간엽 줄기세포에 bFGF, RA, Shh를 처리하여 콜린성 신경세포로 분화시켰을 때, 신경세포 특이 표지인 $\beta$-tubulin III와 콜린성 신경 특이 표지인 ChAT에 양성반응를 보였다. 결론적으로 사람 지방조직의 중간엽 줄기세포가 도파민성과 콜린성 신경세포로 분화가 가능하고 이러한 잠재성을 가진 지방 유래 중간엽 줄기세포는 퇴행성 신경질환에 대한 세포 치료제로서 가능성을 제시한다.