• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.231-231
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• 1994

이 연구는 인삼 성분 약물이 도파민 수용체에 미치는 영향을 연구하기 위한 첫 단계로써 그 기본적인 assay system을 정착 시키기 위함이 목적이었다. 우리는 이 연구를 수행하기 위하여 생후 2-4 일의 쥐를 사용하여 흑질, 선조체, 해마구, nucleus accumbens등의 뇌 부위에서 신경세포 배양을 시도하였다. 흑질로 부터 배양한 신경세포들의 경우엔 면역 세포학적방법을 써서 살펴본 결과 대부분의 신경이 도파민성 신경이나 GABA성 신경들 이었다. 또한 이들 세포들의 전기 생리학적 상태를 알아보기 위하여 흑질에서 신호 전달체계가 잘 확립된 GABA 수용체의 작용을 살펴 본 결과 이 신경세포들은 GABA-A 및 GABA-B 수용체의 발현은 물론 이온 채널에 미치는 신호 전달체계를 완전히 갖추고 있었다. 도파민 수용체의 작용을 전기 생리학적으로 연구하기 위하여 배양한 신경세포에 도파민agonist를 가해서 이온 채널에 미치는 효과를 살펴 보았다. 선조체에서 배양한 신경세포들은 Dl 과 D2 agonist에 대해서 상반되는 반응을 나타냈다. 즉 Dl agonist는 선조체 신경세포를 활성화 시켰으나 D2 agonist는 선조체 신경세포들을 억제 하였다. 한편 해마구의 CAI 과 CA3 부위로 부터 배양한 신경세포에 대한 도파민 agonists의 작용은 선조체의 신경세포에 대한도파민 agonists의 작용과는 상반되는 반응 이었다. Dl agonist는 해마구로 부터 배양한 신경세포의 활성을 억제 하였으나 D2 agonist는 이들 신경세포들의 활성을 증가 시켰다. Nucleus accumbens 에서 배양한 신경세포들은 도파민에 의해서 그 활성이 억제 되었다. 이러한 결과로 미루어 봐서 같은 도파민 수용체라도 분포되어 있는 조직에 따라서 신호전달 에 관여 하고 있는 C-단백이나 이차 전령물질이 달라서 신경세포에 대한 작용이 다르든지. 약리학적으로는 구분되지 않으나 뇌의 조직에 따라서 분포가 다른 도파민 수용체의 아그룹이 존재 한다고 생각된다.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.147-147
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• 1993

신생쥐의 DRG 배양에서 접종후 1일째 neurite의 형성을 관찰할수 있었고 배양한 세포가 신경세포임을 확인하기 위하여 신경섬유에 대한 항체를 이용한 조직화학적 실험을 실시한 결과 붉게 염색된 세포를 볼 수 있었으므로 이 세포들이 DRG신경세포임을 확인할 수 있었다. 배양한 신경세포중 capsaicin에 대한 감수성을 갖는 신경세포군을 확인하기 위하여 cobalt stain 방법으로 실험한 결과 capsaicin에 대한 감수성율 갖는 신경세포룰 관찰할 수 있었고 비교를 위하여 함께 실험한 resiniferatoxin, KR 25018, 6-paradol, NE-19550, RDL-201등의 capsaicin 유사체들의 경우도 정도는 달리 하였으나 유사한 반응을 보였으므로 일차배양한 신경세포에 대한 작용이 capsaicin의 작용과 같을 가능성을 보여주었다. 진통효과 검정결과 진통효과가 있는 것으로 확인된 capsaicin 구조 유사체들인 resiniferatoxin, 6-paradol, NE-19550, RDL-201, KR-25018모두 농도 의존적인 반응을 보였다. 또한 칼슘 도입을 위해 요구되는 관능기로는 알킬측쇄와 P-위치의 수산기, 3-methoxy기 및 acyl amide 구조가 중요하였다. 알킬측쇄는 NVA의 경우처럼 탄소 8개 정도가 적당했으나 구조적으로 현저히 다른 치환기를 가진 resiniferatoxin이나 KR-25018의 경우에도 유효하였다. Vanillin환의 P-수산기도 필수적이었고 3-methoxy기의 존재시에는 칼슘 도입효과가 강하였다. Acyl amide 결합도 중요하나 필수적은 아니였다.

• Development and Reproduction
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• v.12 no.1
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• pp.87-95
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• 2008

Neural stem/precursor derived from pluripotent human embryonic stem cells (hESCs) has considerable therapeutic potential due to their ability to generate various neural cells which can be used in cell-replacement therapies for neurodegenerative diseases. However, production of neural cells from hESCs remains technically very difficult. Understanding neural-tube like rosette characteristic neural precursor cells from hESCs may provide useful information to increase the efficiency of hESC neural differentiation. Generally, neural rosettes were derived from differentiating hEBs in attached culture system, however this is time-consuming and complicated. Here, we examined if neural rosettes could be formed in suspension culture system by bypassing attachment requirement. First, we tested whether the size of hESC clumps affected the formation of human embryonic bodies (hEBs) and neural differentiation. We confirmed that hEBs derived from $500{\times}500\;{\mu}m$ square sized hESC clumps were effectively differentiated into neural lineage than those of the other sizes. To induce the rosette formation, regular size hEBs were derived by incubation of hESC clumps($500{\times}500\;{\mu}m$) in EB medium for 1 wk in a suspended condition on low attachment culture dish and further incubated for additional $1{\sim}2$ wks in neuroectodermal sphere(NES)-culture medium. We observed the neural tube-like rosette structure from hEBs after $7{\sim}10$ days of differentiation. Their identity as a neural precursor cells was assessed by measuring their expressions of neural precursor markers(Vimentin, Nestin, MSI1, MSI2, Prominin-1, Pax6, Sox1, N-cadherin, Otx2, and Tuj1) by RT-PCR and immunofluorescence staining. We also confirmed that neural rosettes could be terminally differentiated into mature neural cell types by additional incubation for $2{\sim}6$ wks with NES medium without growth factors. Neuronal(Tuj1, MAP2, GABA) and glial($S100{\beta}$ and GFAP) markers were highly expressed after $2{\sim}3$ and 4 wks of incubation, respectively. Expression of oligodendrocyte markers O1 and CNPase was significantly increased after $5{\sim}6$ wks of incubation. Our results demonstrate that rosette forming neural precursor cells could be successfully derived from suspension culture system and that will not only help us understand the neural differentiation process of hESCs but also simplify the derivation process of neural precursors from hESCs.

• The Korean Journal of Zoology
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• v.34 no.2
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• pp.209-216
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• 1991

To examine the effed of Insulin on neuronal differentiation, telencephalic neuroblasts from chick embryonic brains were cultured in a serum-free medium. Indirect immunofluorescence microscopic studies revealed that the spedfic protein, MAP-2, was localized in both cell bodies and neurites of developing neuroblasts. Furthermore, treatinent of increasing concentration of Insulin promoted the MAP-2 synthesis as well as the neurite outgrowth activity. Thus, the enhancement of the morphological and biochemical parameters for neuronal differentiation appears to he closely correlated, and the neurotrophic effect of insulin may play a crucial role in neuronal process formation.

• Journal of Life Science
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• v.12 no.3
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• pp.306-316
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• 2002

The ultrastructural change of sciatic nerve and immunohistochemical changes of NGF, PCNA were studied at the transplanted segment of intravascular cultured neural stem cell in the rat sciatic nerve by 5 months after the sciatic nerve transection. The transplanted intravascular neural stem cells were differentiated into Schwann reals at the 20th day and these cells began to regenerate by the proliferation and hypertrophy. There were many remyelinating Schwann cells in the transplanted nerve in term of stimulation. According to NGF finding, we suggest preexisting Schwann cells may induce the differentiation of neural stem cells into regenerating Schwann cells. Electron microscopic changes were the remyelinating appearance, the increase of intraaxonal microtubules and enlarged mitochondria and contacting tell processes each other.

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.163-163
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• 2005

• Journal of Life Science
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• v.21 no.2
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• pp.176-182
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• 2011

Deep seawater (DSW) generally refers to seawater at depths equal to or greater than 200 meters. DSW is rich in inorganic materials which have attracted attention for its various applications. In this study we investigated the effects of the DSW upwelled from the East Sea, offshore Yang Yang (KangWon-do, Korea), on the expression of ${\mu}$-opioid receptor (MOR) of cultured rat hippocampal neurons. Neurons were grown in a minimal essential medium containing 10% (v/v) fetal bovine serum and either 25% (v/v) distilled water, or hardness (H) 800, or H 1000 DSW. Cultures grown in the presence of DSW with H 800 and H 1000 exhibited robust MOR immunoreactive signals in both neurons and astrocytes. Interestingly, the increase in MOR immunoreactive signals was more dramatic in astrocytes than in neurons. Statistical analysis revealed that the relative intensities for MOR clusters increased approximately 4-fold in astrocytes cultured in H 800 and H 1000 media. These increases were statistically very significant (p<0.001). In contrast, the increase in intensities for MOR immunoreactive signals was relatively less dramatic in neurons, where only the increase in the H 1000 culture was statistically very significant (p<0.001). These results indicated that DSW promotes expression of MOR in both neurons and astrocytes, and more significantly in the latter.

• Applied Microscopy
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• v.29 no.4
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• pp.459-470
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• 1999

To investigate the changes during the differentiation of the cerebral neurons of the embryogenic day (ED) 9 and 10, investigated the ultrastructural changes in the cerebral neurons by Electromicroscope, also cerebral protein, the activity of dehydronases (LDH, MDH and SDH) and changes of adenosine triphosphate concentration were analyzed, the result obtained are as follows. In the ultrastructural changes in the cerebral neurons, chromatin in 9 day-old chick embryos are comparatively distributed to even in neucleoplasm and could investigate very prominently that nuclear membrane is double-layer. Esperially, Rough endoplasmic reticulum (RER) and Golgi complex are developed well, also polysome is investigated and synaptic vesicles were scattered. In 10 day-old chick embryos, chromatin evenly spread and nuclear membrane could be differentiated prominently. Rough endoplasmic reticulum (RER) contain cytoplasm, mitochondria and Golgi complex are comparatively developed well. In 9 day-old cultural group of chick embryo cerebrum were separated 37 polypeptide bands and In 10 day-old cultural group of chick embryo cerebrum were separated 38 poly -peptide bands. The more culture time increase, the more the activity of dehydronases (LDH, MDH and SDH) increase. LDH activity was 11.07 (9th day) and 12.12 (10th day), MDH activity was 11.89 (9th day) and 13.44 (10th day) and SDH activity was 8.45 (9th day) and 10.52 (10th day) respectively. The ATP concentration degreesed 10 day-old cultural group than 9 day-old cultural group.

• Herbal Formula Science
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• v.7 no.1
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• pp.143-151
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• 1999

To evaluate the neurotoxic effect of oxygen radicals in cultured mouse spinal dorsal root ganglion(DRG) neurons, cytotoxicity was determined by MTT assay after cultured DRG neurons were grown in the media containing various concentrations of glucose oxidase(GO). In addition, neuroprotective effect of herb extract, Epimedium Koreanum Nakai was examined by MTT assay in cultured DRG neurons. Cell viability of cultured DRG neurons was remarkably decreased by GO in dose- and time-dependent manner, and Epimedium Koreanum Nakai protected remarkably GO-induced neurotoxicity in these cultures. From the above results, it is suggested that oxygen radicals is toxic in cultured mouse DRG neurons, and herb extracts such as Epimedium Koreanum Nakai are effective in prevention of the neurotoxicity induced by oxygen radicals in cultured mouse DRG neurons.

• Journal of Sasang Constitutional Medicine
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• v.11 no.2
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• pp.251-266
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• 1999

1. Purpose : The purpose of this study was to determine the effects of Chungsimyeunjatang on the cerebral neurons injured by hydrogen peroxide($H_2O_2$). 2. Methods : I observed cell viability in mouse cerebral neurons exposed to hydrogen peroxide by NR assay and MTT assay and determined lipid peroxidation and amounts of LDH release in mouse cerebral neurons exposed to hydrogen peroxide. After administration of Chungsimyeunjatang water extracts, I observed significant changes of cell viability, lipid peroxidation and amounts of LDH release in mouse cerebral neurons exposed to hydrogen peroxide. 3. Results : Hydrogen peroxide showed neurotoxicity. Cell viability in mouse cerebral neurons exposed to hydrogen peroxide decreased in NR assay and MTT assay. Lipid peroxidation and amounts of LDH release in mouse cerebral neurons exposed to hydrogen peroxide increased. Chungsimyeunjatang was very effective in blocking hydrogen peroxide-induced neurotoxicity.