• KSBB Journal
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• 제16권1호
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• pp.30-35
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• 2001

유가식배양과 세라믹 막을 장착한 생물반응기에서 운전인 TCRC 조업을 통해서 Bacillus thuringiensis의 고농도 세포배양을 실시하였다. 유가식 배양에서 B. thuringiensis의 세포 성장은 선형적으로 증가하였고, 이것은 세포성장 모델리의 결과와 잘 맞았다. 낮은 세포 성장속에도 불구하고 유가식 배양동안에 포자형성은 관찰할 수 없었고, 이것은 연속배양의 결과와는 반대였다. 유가식 조업 후에 회분식 배양으로 바꾸면 300 g/L의 포도당 공급 농도를 사용했을 때 2.7$\times$$10^9$ CFU/mL 의 포자농도를 얻었다. 생물반응기내에 세라믹 막을 장착한 TCRC 조업에서 포도당 공급 농도의 영향을 결정하였다. 50 g/L의 포도당 농도를 사용했을 때 TCRC 조업에서 82.5 g-cell/L에 해당하는 최대 세포농도 1.8$\times$$10^{10}$ CFU/mL 를 얻었다. TCRC에서 세포성장은 선형적으로 증가하였고 포도당 농도는 제한되었는데 이것은 세포성장은 모델링의 결과와 잘 일치하였다. 1 g/L의 포도당을 공급한 경우 이외에는 TCRC 조업 동안에 포자형성을 관찰할 수 없었다. 50 g/L의 포도당을 공급한 경우 TCRC조업 후에 회분식 배양으로 전환시키면 1.2$\times$$10^{10}$ CFU/mL 의 포자농도를 얻었고, 이것은 연구된 여러 배양형태 중에 가장 높은 포자농도이다. 이 때 최적의 포도당 공급속도는 0.55 g glucose/h로 공급하였을 때였다.

• 대한기계학회논문집A
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• 제35권2호
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• pp.131-134
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• 2011

본 논문에서는 한시적 세포포집 구조물을 이용한 다세포 스페로이드의 형성 및 추출칩을 제안하였다. 종래의 웰 플레이트와 플라스크는 작은 스페로이드 형성이 어렵고, 기존 마이크로칩은 고정된 세포포집 구조물을 이용하여 스페로이드 추출이 어려운 단점이 있다. 반면, 제안된 칩은 한시적 세포포집 구조물을 이용하여 스페로이드의 형성과 추출이 모두 용이한 장점이 있다. 50kPa 의 박막압력으로 형성된 세포포집 구조물에 145~155Pa 의 세포입력압력으로 유입되는 세포를 포집한 후, 24 시간 배양하여 스페로이드를 형성하였다. 또한, 박막압력 제거 후 5kPa 의 세포입력압력으로 추출된 스페로이드의 지름과 활성도는 각각 $197{\pm}11.7Bm$, $80.3{\pm}7.7%$로 측정되었다. 제안된 칩은 스페로이드의 균일한 형성과 안정적 추출이 용이하여 스페로이드의 후처리에 적용될 수 있다.

• Journal of Chest Surgery
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• 제36권9호
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• pp.666-673
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• 2003

폐암의 주된 원인으로 알려진 흡연은 그 악성세포 발현기전이 아직 정확히 규명된 바 없다. 이에 저자들은 흡연에 의한 발암성의 지표로 흡연 중에 특이적으로 존재하는 강력한 발암물질인 NNK(4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone)를 이용하여 흡연에 따른 폐암의 발생과 그 Protein kinase C (PKC) isoform과 관련된 기전에 관한 연구를 시도하였다. 대상 및 방법: 인체 상피세포를 NNK에 노출시킨 후 saturation density, soft agar colony formation, cell aggregation 및 foci의 출현 등의 양상을 파악하여 세포 발암성 여부를 관찰하였으며 NNK를 15분간 노출시킨 후 PKC의 변화는 세포 내 PKC isoform의 양을 cytosolic fraction과 membrane fraction으로 분리하여 측정하여 분석하였다. 결과: NNK 투여군에서 saturation density, soft agar colony formation, cell aggregation 및 foci의 출현 시기 등의 세포 발암성을 뚜렷이 나타내었으며 PKC isoform분석의 경우 PKC-$\alpha$의 membrane fraction의 뚜렷한 증가를 보였으며 이러한 활성은 용량-의존적인 형태를 유지하였다. PKC-$\varepsilon$은 NNK 처리 시 용량-의존적으로 cytosol fraction의 감소 및 membrane fraction의 증가를 뚜렷하게 보였고 NNK에 의한 PKC-λ의 변화는 감지되지 않았다. 결론: 본 연구는 화학적 발암물질인 NNK가 인체발암화에 관여함을 재차 확인하면서 초기 과정에 관여하는 PKC isoform의 변화를 분석함으로써 total PKC활성이 아닌 isoform 각각에 대한 변화를 확인하였다는 점에서 앞으로 인체상피세포 기원의 폐암 생성 기전 연구에 기여할 것으로 생각한다.

• 한국수정란이식학회지
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• 제28권2호
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• pp.133-139
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• 2013

The objective of this study was to determine the effect of post-activation treatment with cytoskeletal regulators in combination with or without 6-dimethylaminopurine (DMAP) on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT oocytes were produced by using in vitro-matured pig oocytes and treated for 4 h after electric activation with $0.5{\mu}M$ latrunculin A (LA), $10.4{\mu}M$ cytochalasins B (CB), and $4.9{\mu}M$ cytochalasins D (CD) together with none or 2 mM DMAP. Post-activation treatment of PA oocytes with LA, CB, and CD did not alter embryo cleavage (85.8~88.6%), blastocyst formation (30.7~ 32.4%), and mean cell number of blastocysts (33.5~33.8 cells/blastocyst). When PA oocytes were treated with LA, CB, and CD in combination with DMAP, blastocyst formation was significantly (P<0.05) improved by CB+DMAP (42.5%) compared to LA+DMAP (28.0%) and CD+DMAP (25.1%), but no significant differences were found in embryo cleavage (77.5~78.0%) and mean blastocyst cell number (33.6~35.0 cells) among the three groups. In SCNT, blastocyst formation was significantly (P<0.05) increased by post-activation treatment with LA+DMAP (32.9%) and CD+DMAP (35.0%) compared to CB+DMAP (22.0%) while embryo cleavage (85.5~85.7%) and blastocyst cell number (41.1~43.8 cells) were not influenced. All three treatments (LA, CB, and CD with DMAP) effectively inhibited pseudo-polar body extrusion in SCNT oocytes. The proportions of oocytes showing single pronucleus formation were 89.6%, 83.9%, and 93.3%, respectively with the increased tendency (P<0.1) by LA+DMAP and CD+ DMAP compared to CB+DMAP. Our results demonstrate that post-activation treatment with LA or CD in combination with DMAP improves pre-implantation development of SCNT embryos and the stimulating effect of cytoskeletal modifiers on embryonic development is differentially shown depending on the origin (PA or SCNT) of embryos in pigs.

• Journal of Periodontal and Implant Science
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• 제32권4호
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• pp.733-744
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• 2002

The fibroblasts are the principal cells in the periodontal ligament of peridontium. As the periodontal ligament fibroblasts (PDLF) show similar phenotype with osteoblasts, the PDLF are thought to play an important role in alveolar bone remodeling. Cell-to-cell contacted signaling is crucial for osteoclast formation. Recently it has been reported that PDLJ enhance the bone resorbing activity of osteoclasts differentiated from hematopoietic preosteoclasts. The aims of this study were to $clarify\;^{1)}$ the mechanism of PDLF-induced osteoclastogenesis $and\;^{2)}$ whether we can use preosteoclast cell line instead of primary hematopoietic preosteoclast cells for studying the mechanism of PDLF-induced osteoclastogenesis. Osteoclastic differentiation of mouse macrophage cell line RAW264.7 was compared with that of mouse bone marrow-derived M-CSF dependent cell (MDBM), a well-known hematopoietic preosteoclast model, by examining, 1) osteoclast-specific gene expression such as calcitonin receptor, M-CSF receptor (c-fms), cathepsin K, receptoractivator nuclear factor kappa B (RANK) ,2) generation of TRAP(+) multinucleated cells (MNCs), and 3) generation of resorption pit on the $OAAS^{TM}$ plate. RAW264.7 cultured in the medium containing of soluble osteoclast differentiation Factor (sODF) showed similar phenotype with MDBM-derived osteoclasts, those are mRNA expression pattern of osteoclast-specific genes, TRAP(+) MNCs generation, and bone resorbing abivity. Formation of resorption pits by osteoclastic MNCs differentiated from sODF-treated RAW264.7, was completely blocked by the addition of osteoprotegerin (OPG), a soluble decoy receptor for ODF, to the sODF-containing culture me야um. The effects of PDLF on differentiation of RAW264.7 into　the TRAP(+) multinucleated osteoclast-like cells were examined using coculture system. PDLF were fxed with paraformaldehyde, followed by coculture with RAW264.7, which induced formation of TRAP(+) MNCs in the absence of additional treatment of sODF. When compared with untreated and fixed PDLF (fPDLF), IL-1 ${\beta}$-treated, or lipopolysaccha-ride-treated and then fixed PDLF showed two-folld increase in the supporting activity of osteoclastogenesis from RAW264.7 coculture system. There were no TRAP(+) MNCs formation in coculture system of RAW264.7 with PDLF of no fixation. These findigs suggested that we can replace the primary hematopoietic preosteoclasts for RAW264. 7 cell line for studying the mechanism of PDLF-induced osteoclastogenesis, and we hypothesize that PDLF control osteoclastogenesis through ODF expression which might be enhanced by inflammatory signals.

• Preventive Nutrition and Food Science
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• 제6권3호
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• pp.187-191
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• 2001

Effects of kochujang (Korean red pepper soybean paste) extracts on tumor formation, natural killer (NK) cell activity in spleen and glutathione S-transferase (GST) activity in liver were investigated in the sarcoma-180 cell transplanted mice. Inhibitory effects of these samples on lung metastasis of colon 26-M3.1 cells were also evaluated in the Balb/c mice. The injection of methanol extracts from traditional kochujang I (TK I, 0-day fermented), II (TKII, 6-month fermented), commercial kochujang (CK, 1-month fermented) and red pepper powder (RPP) significantly reduced tumor formation in Balb/c mice (p<0.05), TKII decreased tumor growth by 46% compared with control, resulting in the smallest tumor weight. The transplantation of sarcoma-180 cells increased the spleen/body weight ratio of Balb/c mice, while TKI and TKll significantly decreased this index (p<0.05). The effect of TKll and CK, fermented kochujang, on the NK cell activity of splenocytes was higher than that of sarcoma-180 cells transplanted control group. TK II recovered the activity of hepatic GST that was decreased by the transplantation of sarcoma- 180 cells in to the mice. All kochujang-treated mice had significantly fewer lung metastatic colonies than control mice. TKII was the most effective in inhibiting lung metastasis of colon 26-M3.1 cells. These results indicated that optimally ripened (6-month) TK had more suppressive effects on tumor formation and lung metastasis than RPP and kochujang without fermentation and commercially prepared kochujang in mice.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.24-25
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• 2003

The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. I will talk about positional cloning of two developmental mutants in zebrafish. The first mutant is headless: The vertebrate organizer can induce a complete body axis when transplanted to the ventral side of a host embryo by virtue of its distinct head and trunk inducing properties. Wingless/Wntantagonists secreted by the organizer have been identified as head inducers. Their ectopic expression can promote head formation, whereas ectopic activation of Wnt signalling during early gastrulation blocks head formation. These observations suggest that the ability of head inducers to inhibit Wntsignalling during formation of anterior structures is what distinguishes them from trunk inducers that permit the operation of posteriorizing Wnt signals. I describe the zebrafish headless (hdl) mutant and show that its severe head defects are due to a mutation in T-cell factor-3 (Tcf3), a member of the Tcf/Lef family. Loss of Tcf3 function in the hdl mutant reveals that hdl represses Wnt target genes. I provide genetic evidence that a component of the Wntsignalling pathway is essential in vertebrate head formation and patterning. Second mutant is mind bomb: Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneuralgene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. (중략)

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.100-100
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• 2003

Pluripotent embryonic stem (ES) cells differentiate spontaneously into beating cardiomyocytes via embryo-like aggregates. We describe the use of mouse embryonic stem (mES03) cells as a reproducible differentiation system for cardiomyocyte. To induce cardiomyocytic differentiation, mES03 cells were dissociated and allowed to aggregate (EB formation) at the presence of 0 75% dimethyl sulfoxide (DMSO) for 4 days and then another 4 days without DMSO (4+/4-). Thus treated EBs were plated onto gelatin-coated dish for differentiation. Spontaneously contracting colonies which appeared in approximately 4-5 days upon differentiation. Expression of cardiac-specific genes were determined by RT-PCR. Rebust expression of myosin light chain (MLC-2V), cardiac myosin heavy chain $\alpha$, cardiac muscle heavy polypeptide 7 $\beta(\beta$-MHC), cardiac transcription factor GATA4 and skeletal muscle-specific ${\alpha}_1$-subunit of the L-type calcium channel (${\alpha}_1 CaCh_{sm}$) were detected as early as 8 days after EB formation, but message of cardiac muscle-specific $\alpha$$_1$-subunit of the L-type calcium channel (${\alpha}_1$CaCh) were revealed at a low level. Strikingly, the expression of atrial natriuretic factor (ANF) was not detected. When spontaneous contracting cell masses were examined their electrophysiological features by patch-clamp technique, it showed ventricle-like action potential 17 days after the EB formation. This study indicates that mES03 cell-derived cardiomyocytes displayed biochemical and electrophysiological properties of cardiomyocytes and DMSO enhanced development of cardiomyocytes in 4+/4- method.

• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.705-709
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• 1998

In the course of screening for the substances suppressing bleb formation of K562 cell induced by phorbol 12, 13-dibutyrate (PDBu), an inhibitor, chaetoglobosin A (CgA) was isolated from a cultured broth of unidentified fungus. CgA showed a strong inhibitory activity with the $IC_{50}$ value of 60 pM against bleb formation on K562 cells induced by PDBu, but it did not inhibit the activity of protein kinase C (PKC) in vitro. The inhibitory activity of CgA might be due to the modulation of actin filaments on the cell membrane. CgA exhibited strong cytotoxicity against various human cancer cell lines.

• Asian-Australasian Journal of Animal Sciences
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• 제22권7호
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• pp.962-968
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• 2009

The aim of this study was to compare the effects of oviductal fluid, porcine zygote medium (PZM)-3, PZM-4 and PZM-5, and modified PZM-5 culture media, and determine the effects of zona pellucida (ZP) removal on the development of nuclear transfer (NT) embryos. There were no significant differences in the rates of fusion and cleavage among the five different oviductal fluid concentrations. However, the rates of blastocyst formation and the cell numbers per blastocyst were high in the embryos at the 14 and 28 $\mu{g}$/ml concentrations of oviductal fluid compared to the 0, 56 and 100 $\mu{g}$/ml concentrations. The rates of cleavage and blastocyst formation, and the cell numbers per blastocyst were higher in the PZM-3, PZM-5 and modified PZM-5 media than in the PZM-4 medium. However, there were no significant differences in the fusion rates of oocytes among the four culture media. The cell numbers per blastocyst in the embryos without ZP were significantly greater than those with ZP. However, there were no significant differences in the rates of fusion, cleavage and blastocyst formation between the embryos with and without ZP. In conclusion, we improved blastocyst development and the quality of NT embryos by replacing PVA with 3 mg/ml of BSA in PZM-5 medium and supplementing the PZM-5 medium with 14 $\mu{g}$/ml oviductal fluid. The NT embryos produced by the zona-free NT method had a high rate of blastocyst formation in the modified PZM-5 medium.