• 생명과학회지
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• 제34권3호
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• pp.170-178
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• 2024

지방줄기세포는 다양한 세포로 분화할 수 있어서 세포치료제로 주목받고 있으나 세포 증식 속도가 느리고 세포의 수명이 짧아 임상 적용에 어려움이 있다. 본 연구팀은 선행연구에서 지방줄기세포의 증식속도를 향상시키고 세포 수명을 연장시키기 위해 SV40의 T항원 유전자를 사람의 ADSC에 도입하여 불사화시킨 ADSC 세포주를 제작하였으며 ADSC-T라고 명명하였다. 본 연구에서는 ADSC-T 세포의 분화능력을 검증하고, ADSC-T 세포를 배양하여 획득한 무혈청 배양액의 항염증 효능을 평가하였다. 그 결과, ADSC-T 세포주는 지방세포와 골세포로 분화할 수 있는 능력을 보유하고 있어 줄기세포의 가장 중요한 특징인 분화능력을 유지하고 있음을 알 수 있었다. 또한 ADSC-T의 배양액은 염증을 유발하는 NF-κB의 활성을 억제하였고, NF-κB의 표적 유전자인 COX-2와 iNOS의 발현도 억제하였다. 또한 ADSC-T 배양액은 ERK, JNK, p38의 인산화를 저해하여 세포 내 염증 유발 신호 전달 회로인 MAPKs 회로를 억제하는 것으로 나타났으며, 염증을 유발하는 cytokine인 TGF-β, TNF-α, IL-6, IL-13의 발현도 억제하는 효과를 보여 강한 항염증 효능이 있을 것으로 추정되었다. 실제로, 아토피 모델 쥐인 Nc/Nga 마우스를 DNCB로 처리하여 아토피 피부염을 유발한 뒤 ADSC-T 배양액을 도포한 결과 강한 치료 효과를 나타내었다. 이상의 결과로부터, 불사화된 ADSC-T 세포주는 지방줄기세포의 장점과 효능을 유지하고 있으며 향후 지방줄기세포의 세포 수급 문제를 해결할 수 있을 뿐 아니라 임상 적용이 가능한 배양액의 생산에도 널리 이용될 수 있을 것으로 사료된다.

• 식물조직배양학회지
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• 제28권1호
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• pp.7-13
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• 2001

담배 (Nicotiana tabacum cv. BY4)의 약을 Nakata배지에 치상하여 반수체식물을 유도하였고, 반수체와 이배체 식물의 잎절편으로부터 0.5 mg/L 2,4-D가 첨가된 MS배지에서 캘러 스를 유도하였다. 이배체와 반수체의 캘러스를 배양 4주 후 2.0 mg/L 2,4-D와 0.1 mg/L BAP, 2.0 mg/L Kinetin이 조합 처리된 MS기본배지에서 계대배양 및 현탁배양을 실시하였 다. 현탁배양된 세포들을 100$\mu\textrm{m}$와 65$\mu\textrm{m}$의 나일론 망으로 걸러서 0.8% low melting agarose를 사용하여 카드뮴과 PFP 가 조합된 선발배지에 도말하였다. 도말 배양 30일 후 형성된 저항성 세포주를 선발하여 경화시키기 위해 카드뮴을 500 $\mu$M과 1,000 $\mu$M 을 처리한 다음 500 $\mu$M에서 세포주를 선발하였다. 선발된 세포주를 0.5, 1.5, 2.0 mg/L BAP 단독 및 500$\mu$M과 1,000$\mu$M의 카드뮴이 첨가된 BAP와 auxin의 조합처리구에서 식물체 재생을 유도하였다. 이때 식물체 재생은 카드뮴이 첨가되지 않은 처리구에서 일어났으며, 이배체인 경 우에는 카드뮴 단독처리시 선발된 세포주에서, 반수체인 경우에는 카드뮴과 PFP 조합처리시 선발된 세포주에서 식물체 재생이 일어났다. 세포의 생장이 왕성한 카드뮴 500$\mu$M의 것을 선발하여 식물체를 재생시켰고, 재생된 시물체의 잎, 줄기 및 뿌리의 단백질 유형을 분석하였다. 대조구로서는 스트레스를 받지 아니한 식물체를 사용하였다. Bradford방법으로 정량하였고, 그 함량은 재생된 식물체의 잎에서 6.5188 $\mu\textrm{g}$/mg.fr.wt., 줄기에서 5.3611 $\mu\textrm{g}$/mg.fr.wt. 뿌리에서 3.0213 $\mu\textrm{g}$/mg.fr.wt.이었다. 반면에 대조구의 잎에서는 5.9652 $\mu\textrm{g}$/mg.fr.wt., 줄기에서 3.5974 $\mu\textrm{g}$/mg.fr.wt. 뿌리에서 4.3766 $\mu\textrm{g}$/mg.fr.wt.이었다. 일차원 SDS-PAGE를 Laemmli 방법으로 수행한 경우 잎은 카드뮴 스트레스에 대하여 내성이 있는 것은 198.7 KD등 49개가 나타났다. 스트레스로 인하여 사라진 밴드는 160.5 KD등 4개, 합성된 단백질 밴드는 83.4 KD 등 3개이었다. 줄기에서는 카드뮴 스트레스 내성의 단백질 밴드는 43 KD등 41개가 나타났고, 사라진 밴드는 114.8 KD등 5개이었다. 또한 합성되어진 단백질 밴드는 128.7 KD 등 6개가 나타났다. 뿌리에서는 내성 단백질 밴드는 166.9 KD 등 27개, 사라진 밴드는 198.7 KD 이었고, 83.4 KD 등 5개의 단백질 밴드가 나타났다.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제30권4호
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• pp.323-332
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• 2008

Aim of the study: Scaffolds are crucial to tissue engineering/regeneration. Biodegradable polymer/ceramic composite scaffolds can overcome the limitations of conventional ceramic bone substitutes such as brittleness and difficulty in shaping. In this study, poly(L-lactide)/hydroxyapatite(PLLA/HA) composite scaffolds were fabricated for in vivo bone tissue engineering. Material & methods: In this study, PLLA/HA composite microspheres were prepared by double emulsion-solvent evaporation method, and were evaluated in vivo bone tissue engineering. Bone marrow mesenchymal stem cell from rat iliac crest was differentiated to osteoblast by adding osteogenic medium, and was mixed with PLLA/HA composite scaffold in fibrin gel and was injected immediately into rat cranial bone critical size defect(CSD:8mm in diameter). At 1. 2, 4, 8 weeks after implantation, histological analysis by H-E staining, histomorphometric analysis and radiolographic analysis were done. Results: BMP-2 loaded PLLA/HA composite scaffolds in fibrin gel delivered with osteoblasts differentiated from bone marrow mesenchymal stem cells showed rapid and much more bone regeneration in rat cranial bone defects than control group. Conclusion: This results suggest the feasibility and usefulness of this type of scaffold in bone tissue engineering.

• 대한수의학회지
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• 제58권4호
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• pp.201-209
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• 2018

Canine mammary tumors are among the most frequently observed cutaneous tumors in female dogs. Cancer stem cells (CSCs), referred to as tumor-initiating cells, are thought to have properties similar to normal stem cells such as the ability to self-renewal and to differentiate into various cell types. Biological understanding of CSCs and the critical pathways involved in their maintenance are important in research and therapy for mammary tumors. We conducted the present study on sphere formation from REM134 cells by using methylcellulose to produce tumorspheres on a large scale and compared the specific markers of the spheres-formed and plating-cultured REM134 cells. The results revealed that the tumorspheres cultured in methylcellulose had higher seeding density and improved morphology compared to those produced in normal sphere formation medium. Expression levels of stemness markers and CSC-related markers were higher in tumorsphere-forming cells than in plating-cultured cells. Subsequently, we transplanted the tumorsphere-forming and plating-cultured cells into female nude mice to examine their tumorigenic potential. Tumor volume increased rapidly in mice transplanted with tumorsphere-derived cells compared to plating-cultured cells. We observed a novel sphere-forming condition for REM134 cells and showed that REM134 cell tumorspheres can exhibit improved CSC properties.

• 식물조직배양학회지
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• 제25권3호
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• pp.195-200
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• 1998

기내에서 발아시킨 애기장대(Arabidopsis thaliana)의 잎과 줄기 절편체로부터 캘러스 유도는, 잎절편체의 경우 2mg/L 2,4-D가 첨가된 MS 고체배지에서 유도되었고, 줄기절편체의 경우는 0.5mg/L 2,4-D와 0.1mg/L BAP가 첨가된 CP 고체배지에서 배양 4주 후 다량의 캘러스가 유도되었다. 유도된 캘러스를 잘게 자르고 0.5mg/L 2,4-D와 0.1mg/L BAP가 첨가된 CP 액체배지에서 7일 간격으로 암조건에서 4개월간 배양하였을 때 균일한 shoot-forming(SF)캘러스를 얻을 수 있었다. 액체배지에서 유도된 SF 세포괴는 0.05 mg/L IAA, 7.0mg/L 2-iP, 30g/L sucrose가 첨가된 MS 재분화배지에서 광조건으로 배양하였을 때 캘러스를 거쳐 녹화되기 시작하였으며 배양 4주 후부터는 전체적으로 녹화된 SF캘러스로부터 shoot가 형성되어 식물체 재분화가 가능하였다. 또한 재분화 배지에 옮겼을 때 IAA와 2-iP가 첨가된 배지에서 50% 이상의 shoot 형성률(SF 캘러스당 한 개 이상의 shoot가 형성된 캘러스의 백분율)을 보였다. 절단된 shoot는 호르몬이 첨가되지 않은 배지에서 4주 후 뿌리를 형성하였으며, 재분화된 식물체는 기내에서 6주 후부터 화뢰가 형성되고 꽃이 피기 시작하였다.

• 한국수정란이식학회지
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• 제20권1호
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• pp.35-41
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• 2005

본 연구는 가축유전자원의 효율적 보존을 위해 정조세포를 줄기세포 형태로 장기보관하면서 필요에 따라 증식, 분화를 통해 가축의 복원에 활용하기 위한 연구의 일부로 진행되었다. 정조세포를 분리하여 배양한 결과 배양온도는 다른 세포들과는 달리 $32^{\circ}C$에 세포분열이 활발하였으며, TCM199에 $10\%$ FCS를 첨가한 배양액과 세르톨리세포 공배양으로 정조세포의 배양을 지지하였다. 40일령이 지나면서 정조세포 콜로니 즉 germline stem cells를 형성하였으며, 일부에서는 외형상 ES-like cells를 형성하거나, 세정관 형태로 정조세포들이 재구성되었다. 40일령까지 배양한 상태에서는 정조세포의 정모세포나 정자세포로 분화하는 징후를 관찰할 수 없었으며, 추후 이들 세포로 분화를 유기하는 실험이 진행되어야 할 것이다.

• BMB Reports
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• 제53권11호
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• pp.600-604
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• 2020

Macrophages are re-educated and polarized in response to myocardial infarction (MI). The M2 anti-inflammatory phenotype is a known dominator of late stage MI. Mesenchymal stem cells (MSCs) represent a promising tool for cell therapy, particularly heart related diseases. In general, MSCs induce alteration of the macrophage subtype from M1 to M2, both in vitro and in vivo. We conjectured that hypoxic conditions can promote secretome productivity of MSCs. Hypoxia induces TGF-β1 expression, and TGF-β1 mediates M2 macrophage polarization for anti-inflammation and angiogenesis in infarcted areas. We hypothesized that macrophages undergo advanced M2 polarization after exposure to MSCs in hypoxia. Treatment of MSCs derived hypoxic conditioned medium (hypo-CM) promoted M2 phenotype and neovascularization through the TGF-β1/Smad3 pathway. In addition, hypo-CM derived from MSCs improved restoration of ischemic heart, such as attenuating cell apoptosis and fibrosis, and ameliorating microvessel density. Based on our results, we propose a new therapeutic method for effective MI treatment using regulation of macrophage polarization.

• The Plant Pathology Journal
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• 제27권1호
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• pp.68-77
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• 2011

Fifty five species of medicinal plant materials were tested for their antifungal activity in vitro against Rhizoctonia solani AG 2-1 and Trichoderma harzianum to select plant species that can be used to improve the biocontrol efficacy of T. harzianum. Six species were effective against R. solani AG 2-1 but were also antagonistic to T. harzianum, except for Cinnamomum loureirii stem bark (CSB). CSB inhibited mycelial growth of R. solani AG 2-1 by 73.7% but showed an inhibitory effect on mycelial growth of T. harzianum by only 2.2%. Scanning electron microscophs showed that the CSB treatment resulted in deformed R. solani AG 2-1 hyphal cells, and transmission electron microscophs revealed degenerated cell structures such as degenerated cytoplasm and disentangled cell wall and the accumulation of electron-dense inclusions (asterisks) in the CSB treatment. The biocontrol efficacy of radish damping-off increased greatly following the combined treatments of T. harzianum and CSB and the combined treatment increased efficacy from 6.4-23.1% to 37.1-87.3% compared with either treatment alone. CSB did not affect T. harzianum population growth, as it was almost the same in rice-bran peat medium (culture) amended with 0.1% and 1.0% CSB powder as in non-amended medium. The formulation of T. harzianum in rice-bran peat medium amended with CSB powder reduced the severity of radish damping-off by 80.6%, suggesting that T. harzianum and CSB can be formulated as a biocontrol product for the control of R. solani AG 2-1.

• Asian-Australasian Journal of Animal Sciences
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• 제30권6호
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• pp.878-885
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• 2017

Objective: Glucose is an essential fuel in the energy metabolism and synthesis pathways of all mammalian cells. In lactating animals, glucose is the major precursor for lactose and is a substrate for the synthesis of milk proteins and fat in mammary secretory (alveolar) epithelial cells. However, clear utilization of glucose in mammary cells during lactogenesis is still unknown, due to the lack of in vitro analyzing models. Therefore, the objective of this study was to test the reliability of the mammary alveolar (MAC-T) cell as an in vitro study model for glucose metabolism and lactating system. Methods: Undifferentiated MAC-T cells were cultured in three types of Dulbecco's modified Eagle's medium with varying levels of glucose (no-glucose: 0 g/L, low-glucose: 1 g/L, and high-glucose: 4.5 g/L) for 8 d, after which differentiation to casein secretion was induced. Cell proliferation and expression levels of apoptotic genes, Insulin like growth factor-1 (IGF1) receptor, oxytocin receptor, ${\alpha}S1$, ${\alpha}S2$, and ${\beta}$ casein genes were analyzed at 1, 2, 4, and 8 d after differentiation. Results: The proliferation of MAC-T cells with high-glucose treatment was seen to be significantly higher. Expression of apoptotic genes was not affected in any group. However, expression levels of the mammary development related gene (IGF1 receptor) and lactation related gene (oxytocin receptor) were significantly higher in the low-glucose group. Expressions of ${\alpha}S1-casein$, ${\alpha}S2-casein$, and ${\beta}-casein$ were also higher in the low-glucose treated group as compared to that in the no-glucose and high-glucose groups. Conclusion: The results demonstrated that although a high-glucose environment increases cell proliferation in MAC-T cells, a low-glucose treatment to MAC-T cells induces higher expression of casein genes. Our results suggest that the MAC-T cells may be used as an in vitro model to analyze mammary cell development and lactation connected with precise biological effects.

• International Journal of Stem Cells
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• 제16권2호
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• pp.244-249
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• 2023

Background and Objectives: To examine whether ischemic retinal ganglion cells (RGCs) will be salvaged from cell death by human adipose-derived mesenchymal stem cells (ADSCs) in an organotypic retina model. Methods and Results: Deprived of arterial oxygen supply, whole mice retinas were cultured as an ex vivo organotypic cultures on an insert membrane in a 24-well plate. The therapeutic potential of ADSCs was examined by co-culture with organotypic retinas. ADSCs were seeded on top of the RGCs allowing direct contact, or at the bottom of the well, sharing the same culture media and allowing a paracrine activity. The number of surviving RGCs was assessed using Brn3a staining and confocal microscopy. Cytokine secretion of ADSCs to medium was analyzed by cytokine array. When co-cultured with ADSCs, the number of surviving RGCs was similarly significantly higher in both treatment groups compared to controls. Analysis of ADSCs cytokines secretion profile, showed secretion of anti-apoptotic and pro-proliferative cytokines (threshold>1.4). Transplantation of ADSCs in a co-culture system with organotypic ischemic retinas resulted in RGCs recovery. Since there was no advantage to direct contact of ADSCs with RGCs, the beneficial effect seen may be related to paracrine activity of ADSCs. Conclusions: These data correlated with secretion profile of ADSCs' anti-apoptotic and pro-proliferative cytokines.