• 생명과학회지
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• 제19권7호
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• pp.905-916
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• 2009

본 연구는 저산소증에서 반하가 대뇌신경세포의 유전자 표현에 미치는 영향을 알아보기 위하여 배양한 $E_{18}$의 흰쥐 대뇌 신경세포를 반하로 처리하고, 저산소증을 유도한 후 microarray 기법으로 유전자 표현 변화를 조사하였다. Microarray 결과 tubb5, tgfa, ptpn11, n-ras, pdgfa 등 세포의 성장 분화에 관여하는 유전자들의 표현이 증가하였으며, 세포 자연사를 억제하는 mcl-1 유전자의 표현 또한 증가하였다. 한편 세포 자연사를 유도하는 tieg 유전자는 표현이 감소하였다(Fig. 3). 반하에 의하여 수많은 유전자의 표현이 변화되었고, 세포사를 촉진하는 유전자의 표현이 크게 증가되는 경우(예, alox12, faf1)도 있어 본 연구결과만으로 일반적인 결론을 유도하기는 어려웠다. 그러나 대략적으로 반하는 저산소증에서 주로 세포의 성장과 분화를 유지하고, 세포 자연사를 방지하는 유전자들의 표현을 증가시켜 신경 세포사를 보호하는 것으로 이해된다.

• 한국수정란이식학회지
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• 제17권3호
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• pp.239-249
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• 2002

Embryos derived from pig oocytes matured in mSOF are able to develop to blastocysts after IVF. Experiment 1 evaluated the effects of two maturation media (TCM-199 vs mSOF) on maturation rate, fertilization parameters, including penetration, polyspermy, male pronuclear formation, and the mean number of sperm penetrated per oocyte. Experiment 2 and Experiments 3 examined the effects of two maturation media on zona pellucida solubility and cortical granule distribution by transmissible electron microscopy, respectively. Experiment 4 assessed the effects of two maturation media on the in vitro embryo cleavage rate and development to blastocyst. Lastly, experiment 5 examined the cell number of blastocyst. An effect of media (P<0.05) was detected for mSOF on the mean number of sperm per oocyte. In TCM group, zona digestion time (196.5$\pm$15.5 vs 131.6$\pm$20.1 before IVF, 397.5$\pm$30.3s vs 185.3$\pm$16.4s after IVF, p<0.05) was higher in TCM-199 group. No significant effects of media was observed on cortical granule distribution between two groups by TEM. An effect (P<0.05) was observed on embryo development to blastocyst (16% vs 8%) but not on cleavage rates. No significant effects of media was observed on total cell number of blastocyst. We found that the high mean number of sperm penetrated per oocyte and the weaker zona pellucida on the basis of the digestion time was shown in pig oocytes matured in mSOF, however, porcine oocyte maturation with supplemented synthetic oviduct fluid medium (mSOF) resulted in blastocyst cell numbers comparable to those observed with Tissue Culture Medium 199.

• 한국한의학연구원논문집
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• 제4권1호통권4호
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• pp.99-114
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• 1998

The effect of herbal medicine on glutamate mediated neurotoxicity was studied in mouse neurons in primary culture. Immature cerebral cortex neurons (ED14) were maintained for up to 2 weeks in vitro, and we investigated the expression pattern of neuron differentiation and cytotoxicity of cell death, including LDH activity. Neuronal maturation initiated on day 7 and the susceptibility to glutamate-induced cell death was highly sensitive on Day 11 (Fig. 1). Thus, the exposure of the neurons to glutamate caused a dose$(0.1mM{\sim}1mM)$ and time$(4h{\sim}24h)$-dependent neurotoxicity(Fig. 4). Glutamate-induced neurodegeneration was prevented by Shipchondaebotang(SD), Yollyounggobondan(YG), Yugmijihwangwon(YJ) and the death of neurons exposed to glutamate was blocked by the NMDA receptor antagonist MK-801 (Fig. 5).

• 생명과학회지
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• 제14권2호
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• pp.291-295
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• 2004

루이보스(Aspalathus linearis; RB)는 콩과식물로서 남아프리카 Cape Province의 북서부 산악지역에 자생한다. 루이보스차 침제는 가는 가지와 잎의 발효산물로서 플리보노이드류와 페놀산이 있어 강한 항산화활성을 갖는 것으로 알려져 있다. 본 연구에서는 저산소증모델에서 루이보스차 침제가 배양한 흰쥐 대뇌세포의 스트레스를 완화하는지에 대한 연구하였다. 배지로 누출된 LDH의 정량실험에 의하면 루이보스는 정상산소환경 및 저산소증에서 함량 의존적으로(10-100 $\mu\textrm{g}$/ml) 각각 6-18% 및 2-24%의 세포생존율을 증가시켰다(16 DIV 세포, 처리 후 3일째). CFP-Hsc70 단백질을 표현시킨 신경세포의 모양을 관찰하였을때 루이보스(50 $\mu\textrm{g}$/ml)는 저산소처리 후 5일에 세포체에 수포가 있는 세포의 수를 대조군(55.4$\pm$4.59%)에 비하여 유의하게 감소시켰다(40.9$\pm$6.3%). 이러한 결과들은 루이보스차가 저산소증에서 신경세포를 보호함을 의미하며, 신경세포 손상을 예방 또는 치료하는데 응용될 수 있을 것으로 보인다.

• 생명과학회지
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• 제11권4호
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• pp.346-353
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• 2001

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) a prototype of the highly toxid halogenated arylhydrocarbons, bioaccumulates in the food chain and induces a complex spectrum of pathological responses. However, its effect on the nerve system is relatively not well studied. In this study we evaluated TCDDs cytotoxicity on the cortical cell and investigated its effect on the expression 2,3-cyclic nucleotide 3-phosphodiesterase(CNPase), a marker for oilgodendrocytes, The survival rates of 4 DIV cortical cells, that are dissociated from E18 rat cortex and maintained in the presence of TCDD, were 88.8, 83.6, 78.5, and 78.6%(5,10, 20 and 50 nM, respectively) where the reduction in 20 and 50mM TCDD were statistically very significant(p<0.01). Imunocytochemistry of cultured cells revealed that the intensities of immunostaining with an anti-CNP1&2 antibody depended on the concentrations of the toxin. Immunoblot analysis also showed differential expression of CNP1 and CNP2 in the presence of TCDD; the CNP1 expression was dose-dependently decreased. Interestingly, the expression of CNP2 in the presence if TDCC; the CNP1 expression was dose-dependently decreased. Interestingly, the expression of CNP2 fluctuated with the TCDD concentration. These results indicated that CNP1 and 2 are differentially regulated by TCDD, implying the functions of oligodendrocytes are modulated by the toxin.

• 생명과학회지
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• 제17권1호
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• pp.150-161
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• 2007

Acori graminei Rhizomn (AGR) is a perennial herb which has been used clinically as a traditional oriental medicine against stroke, Alzheimer's disease, and vascular dementia. We investigated the effect of AGR on the modulation of gene expression profile in a hypoxic model of cultured rat cortical cells. Rat cerebrocortical cells were grown in Neurobasal medium. On DIV12, cells were treated with AGR $(10ug/m\ell)$, given a hypoxic shock (2% $O_2$, 3 hr) on DIV14, and total RNAs were prepared one day after shock. Microarray analyses indicated that the expression levels of most genes were altered within the global M values +0.5 and -0.5, i.e., 40% increase or decrease. There were 750 genes which were upregulated by < global M +0,2, while 700 genes were downregulated by > global M -0.2. The overall profile of gene expression suggests that AGR suppresses apoptosis (upregulation of anti-apopotic genes such as TEGT, TIEG, Dad, p53, and downregulation of pro-apopotic genes such as DAPK, caspase 2, pdcd8), ROS (upregulation of RARa, AhR), and that AGR has neurotrophic effects (upregulation of Aktl, Akt2). These results provide a platform for investigation of the molecular mechanism of the effect of AGR in neuroprotection.

• 생명과학회지
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• 제17권5호
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• pp.706-713
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• 2007

Scutellaria baicalensis GEORGI(SB) is used in oriental medicine for the treatment of incipient strokes. Although it has been reported that SB is neuroprotective in a hypoxia model, its mechanism is poorly understood. Here, we investigated the effect of SB on the modulation of heme oxygenase-1(HO-1), which has important biological roles in regulating mitochondrial heme protein turnover and in protecting against conditions such as hypoxia, neurodegenerative diseases, or sepsis. Rat cerebrocortical day In vitro(DIV)12 cells were grown in neurobasal medium. On DIV12 cells were treated with SB($20{\mu}g/ml$) and given a hypoxic shock ($2%\;O_2/5%\;CO_2,\;3\;hr$) on DIV14. In situ hybridization results revealed that SB upregulated HO-1 mRNA in neuronal dendrites in both normoxia and hypoxia(38.5% and 59.2%, respectively). At the protein level, SB upregulated HO-1 in the neuronal soma in both normoxia and hypoxia(22.4% and 15.7%, respectively). Interestingly, most significant increase was associated with astrocytes, which increased HO-1 protein by 77.5% compared to SB-untreated culture. These results indicate that SB upregulates both neuronal and glial HO-1 expression, which contributes to the neuroprotection efficacy in hypoxia).

• Clinical and Experimental Reproductive Medicine
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• 제27권1호
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• pp.23-29
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• 2000

Objective: Zona pellucida (ZP) has been thought to be the barrier of egg to sperm penetration before and after fertilization. The phenomenon of ZP hardening has been considered as a post-fertilization event until now, and it is generally accepted that it is caused by the secretory products of cortical granules released during the cortical reaction. Hardening of ZP could occur "spontaneously" in mammalian oocytes in standard culture conditions, and that it is probably not a consequence of cortical reaction. The purpose of our study was to investigate the effect of human amniotic fluid (HAF) on nuclear maturation (NM) and fertilization ability of mouse immature oocytes. Methods: HAF was obtained from patients undergoing amniocentesis at $16{\sim}20$ weeks of gestation. HAF from five to ten patients was centrifuged and the supernatants was pooled. Cumulusenclosed mouse immature oocytes were incubated in the medium containing HAF, and examined to confirm NM and fertilization. Female ICR mice (about 3 weeks old) were stimulated with 7.5 IU PMSG. Immature oocytes were isolated at $48{\sim}52$ hrs post PMSG injection and cultured in TCM-199 supplemented with 20% HAF for 18 hrs. FBS was used as a control for the examination. Matured oocytes (MII) were fertilized with sperms collected from the epididymis of male mice (over 10 weeks old). Fertilization was in conducted T6 medium containing 15 mg/ml BSA, and confirmed at 6 hrs post-insemination. Fertilization rate was assessed in zona-intact or zona-free oocytes (denuded by trypsin). Evaluation of NM and fertilization was carried out by rapid staining method. ZP hardening was evaluated by incubating cumulus cell-free mature oocytes in 0.001% chymotrypsin at $37^{\circ}C$ for 10 min. Results: There was no significant difference between the effects of HAF (86.6%) and FBS (87.7%) supplements on NM of immature oocytes. When maturation medium was supplemented with HAF, total fertilization rates (7%) were significantly lower (p<0.01) than that of FBS (85.1%). In HAF group, fertilization rate was increased (p<0.01) in zona-free oocytes (7% versus 100%). The resistance of mouse oocyte ZP to digestion by chymotrypsin after maturation in vitro was significantly higher (p<0.01) in HAF group (86.7%) than in FBS (6.7%). To culture oocytes in FBS were very effective in preventing ZP hardening. However cultured oocytes in HAF showed high rate of ZP hardening (p<0.01). Conclusions: These results suggest that HAF can be used as a supplement for the NM of mouse immature oocytes in vitro. However, HAF induces spontaneous hardening of ZP of mouse immaure oocytes during maturation in vitro.

• Clinical and Experimental Pediatrics
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• 제46권11호
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• pp.1101-1106
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• 2003

목 적 : 미노사이클린이 in vivo 연구에서 뇌보호 효과가 있는것으로 알려져 있어 본 연구에서는 미노사이클린이 저산소 상태로 유발된 백서 뇌세포 배양에서 고사를 억제하는 뇌보호 효과가 있는지를 알아보고자 하였다. 방 법: 임신 18일된 백서의 대뇌피질 신경세포를 배양하여 정상산소 상태군과 저산소 상태군으로 두 군으로 나누고, 정상산소 상태는 5% $CO_2$ 배양기에, 저산소 상태는 1% $CO_2$ 배양기에서 세포 수를 세면서 수일간 처리하여 현미경하에서 충분한 손상이 있다고 판단되면 두 군을 각각 대조군, 미노사이클린 $1{\mu}g/mL$, $10{\mu}g/mL$로 처리한 군으로 나누어서 실험하였다. TUNEL 및 DAPI 염색으로 세포 고사 상태를 통계학적으로 처리하였다. 결 과: 정상산소 상태군에서 대조군과 미노사이클린 $1{\mu}g/mL$ 투여군, 미노사이클린 $10{\mu}g/mL$ 투여군 3군 모두에서 통계학적으로 유의한 차이가 있었다(P<0.01). 저산소 상태군에서 대조군과 미노사이클린 투여군에서 통계학적으로 유의한 차이가 있었으나(P<0.01), 미노사이클린 투여군 간에는 통계학적으로 유의한 차이가 없었다(P>0.05). 정상산소 상태군과 저산소 상태군간의 대조군과 미노사이클린 $1{\mu}g/mL$ 투여군의 비교에서는 정상산소 상태군이 저산소 상태군보다 평균이 낮아 통계학적으로 유의한 차이가 있었다(P<0.01). 정상산소 상태군과 저산소 상태군간의 미노사이클린 $10{\mu}g/mL$ 투여군의 비교에서는 정상산소 상태군이 저산소 상태군보다 다소 평균이 낮았으나 통계학적으로 유의성은 없었다(P>0.05). 결 론 : 결론적으로 미노사이클린은 고사를 억제하는 기전으로 저 산소 상태나 정상 산소 상태에서 뇌보호 효과가 있었다.

• Biomolecules & Therapeutics
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• 제24권1호
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• pp.99-107
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• 2016

Triclosan is an antimicrobial or sanitizing agent used in personal care and household products such as toothpaste, soaps, mouthwashes and kitchen utensils. There are increasing evidence of the potentially harmful effects of triclosan in many systemic and cellular processes of the body. In this study, we investigated the effects of triclosan in the survivability of cultured rat neural stem cells (NSCs). Cortical cells from embryonic day 14 rat embryos were isolated and cultured in vitro. After stabilizing the culture, triclosan was introduced to the cells with concentrations ranging from $1{\mu}M$ to $50{\mu}M$ and in varied time periods. Thereafter, cell viability parameters were measured using MTT assay and PI staining. TCS decreased the cell viability of treated NSC in a concentration-dependent manner along with increased expressions of apoptotic markers, cleaved caspase-3 and Bax, while reduced expression of Bcl2. To explore the mechanisms underlying the effects of TCS in NSC, we measured the activation of MAPKs and intracellular ROS. TCS at $50{\mu}M$ induced the activations of both p38 and JNK, which may adversely affect cell survival. In contrast, the activities of ERK, Akt and PI3K, which are positively correlated with cell survival, were inhibited. Moreover, TCS at this concentration augmented the ROS generation in treated NSC and depleted the glutathione activity. Taken together, these results suggest that TCS can induce neurodegenerative effects in developing rat brains through mechanisms involving ROS activation and apoptosis initiation.