• Journal of Microbiology and Biotechnology
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• 제23권9호
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• pp.1308-1321
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• 2013

The emergence of microcarrier technology has brought a renewed interest in anchorage-dependent cell culture for high-yield processes. Well-known in vaccine production, microcarrier culture also has potential for application in other fields. In this work, two types of microcarriers were evaluated for small-scale monoclonal antibody (mAb) production by CHO-K1 cells. Cultures (5 ml) of microporous Cytodex 3 and macroporous CultiSpher-S carriers were performed in vented conical tubes and subsequently scaled-up (20 ml) to shake-flasks, testing combinations of different culture conditions (cell concentration, microcarrier concentration, rocking methodology, rocking speed, and initial culture volume). Culture performance was evaluated by considering the mAb production and cell growth at the phases of initial adhesion and proliferation. The best culture performances were obtained with Cytodex 3, regarding cell proliferation (average $1.85{\pm}0.11{\times}10^6$ cells/ml against $0.60{\pm}0.08{\times}10^6$ cells/ml for CultiSpher-S), mAb production ($2.04{\pm}0.41{\mu}g/ml$ against $0.99{\pm}0.35{\mu}g/ml$ for CultiSpher-S), and culture longevity (30 days against 10-15 days for CultiSpher-S), probably due to the collagen-coated dextran matrix that potentiates adhesion and prevents detachment. The culture conditions of greater influence were rocking mechanism (Cytodex 3, pulse followed by continuous) and initial cell concentration (CultiSpher-S, $4{\times}10^5$ cells/ml). Microcarriers proved to be a viable and favorable alternative to standard adherent and suspended cultures for mAb production by CHO-K1 cells, with simple operation, easy scale-up, and significantly higher levels of mAb production. However, variations of microcarrier culture performance in different vessels reiterate the need for optimization at each step of the scale-up process.

• Nutrition Research and Practice
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• 제8권4호
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• pp.368-376
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• 2014

BACKGROUND/OBJECTIVES: The objectives of this study were to investigate the effects of lycopene on the migration, adhesion, tube formation capacity, and p38 mitogen-activated protein kinase (p38 MAPK) activity of endothelial progenitor cells (EPCs) cultivated with high glucose (HG) and as well as explore the mechanism behind the protective effects of lycopene on peripheral blood EPCs. MATERIALS/METHODS: Mononuclear cells were isolated from human peripheral blood by Ficoll density gradient centrifugation. EPCs were identified after induction of cellular differentiation. Third generation EPCs were incubated with HG (33 mmol/L) or 10, 30, and $50{\mu}g/mL$ of lycopene plus HG. MTT assay and flow cytometry were performed to assess proliferation and apoptosis of EPCs. EPC migration was assessed by MTT assay with a modified boyden chamber. Adhesion assay was performed by replating EPCs on fibronectin-coated dishes, after which adherent cells were counted. In vitro vasculogenesis activity was assayed by Madrigal network formation assay. Western blotting was performed to analyze protein expression of both phosphorylated and non-phosphorylated p38 MAPK. RESULTS: The proliferation, migration, adhesion, and in vitro vasculogenesis capacity of EPCs treated with 10, 30, and $50{\mu}g/mL$ of lycopene plus HG were all significantly higher comapred to the HG group (P < 0.05). Rates of apoptosis were also significantly lower than that of the HG group. Moreover, lycopene blocked phosphorylation of p38 MAPK in EPCs (P < 0.05). To confirm the causal relationship between MAPK inhibition and the protective effects of lycopene against HG-induced cellular injury, we treated cells with SB203580, a phosphorylation inhibitor. The inhibitor significantly inhibited HG-induced EPC injury. CONCLUSIONS: Lycopene promotes proliferation, migration, adhesion, and in vitro vasculogenesis capacity as well as reduces apoptosis of EPCs. Further, the underlying molecular mechanism of the protective effects of lycopene against HG-induced EPC injury may involve the p38 MAPK signal transduction pathway. Specifically, lycopene was shown to inhibit HG-induced EPC injury by inhibiting p38 MAPKs.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제33권5호
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• pp.405-418
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• 2007

Mesenchymal stem cells(MSCs) have been though to be multipotent cells that can replicate that have the potential to differentiate into lineages of mesenchymal tissue including the bone, cartilage, fat, tendon, muscle, and marrow stroma. Especially, scaffolds to support cell-based tissue engineering are critical determinants of clinical efforts to regenerate and repair the body. Selection of a matrix carrier imvolves consideration of the matrix's role as a scaffold for physical support and host tissue integration as well as its ability to support of synergize the osteoinductive program of the implanted mesenchymal stem cell. The aim of this study is to evaluate the effect of autobone and Bio-$Oss^{(R)}$ to adherent mesenchymal stem cells as scaffolds on sinus augmentation with fibrin glue mixture in a rabbit model. 16 New Zealand White rabbits were divided randomly into 4 groups based on their time of sacrifice(1, 2, 4 and 8 weeks). First, mesenchymal stem cells were isolated from iliac crest marrow of rabbits and expanded in vitro. Cell culture was performed in accordance with the technique described by Tsutsumi et al. In the present study, the animals were sacrificed at 1, 2, 4 and 8 weeks after transplantation, and the bone formation ability of each sides was evaluated clinically, radiologically, histologically and histomorphologically. According to the histological observations, autobone scaffolds group showed integrated graft bone with host bone from sinus wall. At 2 and 4 weeks, it showed active newly formed bone and neovascularization. At 8 weeks, lamellae bone was observed in sinus graft material area. Radiologically, autobone with stem cell showed more radiopaque than Bio-$Oss^{(R)}$ scaffolds group. there were significant differences in bone volume between 4 and 8 weeks(p<0.05).

• 한국산학기술학회논문지
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• 제20권8호
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• pp.477-484
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• 2019

포유류에 존재하는 Collectin-Placenta 1 (CL-P1)을 포함한 여러 종류의 scavenger 수용체는 주로 내피 세포, 대식 세포 및 평활근 세포 표면에 발현되는 분자이다. 이들 분자는 산화 된 저밀도 지질 단백질 (oxLDL)에 결합하여 처리 할 수 있는 세포 표면 당 단백질이다. 이들 분자 중 케르세틴이 CL-P1 활성화에 어떤 영향을 미치는가를 확인하였다. 케르세틴은 산화 반응을 담당하는 자유 라디칼의 제거제 역할을 하여 산화를 중지시키는 항산화제로 알려져 있다. 본 논문에서는 마우스 CL-P1 유전자 promoter 부분의 전사 시작 점부터 -500 번째 염기까지의 단편을 DNA 중합효소를 이용하여 클로닝 하였다. 그 후에 대식세포 계열인 RAW264.7 및 섬유아세포계열의 NIH3T3 세포에 도입하여 케르세틴이 CL-P1 유전자 발현에 어떠한 영향을 미치는지에 대한 연구를 하였다. 이 부위에는 세포주기 조절 인자인 E2F 결합부위를 비롯해서 여러 종류의 전사 인자가 결합하는 염기서열이 다수 위치하고 있다. 이러한 500염기 단편을 pGL4.10 기본 벡터 및 프로모터에 연결시킨 후 세포에 도입시켰다. 그리고 배양 중에 케르세틴을 처리하여 유전자 발현양을 형광 색소 발현기법으로 측정하였다. 그 결과 유전자 발현이 시작되는 앞쪽 부분의 -250에서 -350사이의 염기들이 CL-P1 단백질을 만드는데 중요하다는 것을 확인하였다. 그 중에서도 E2F결합 부위가 결정적인 것 이라는 것을 DNA 돌연변이 실험을 통해 확인 하였다. 또한 부착 세포인 RAW264.7 배양액에 케르세틴을 첨가 한 결과, 배양용기 표면에서 탈락하는 현상을 확인하였다. 즉 발현된 CL-P1단백질이 케르세틴에 의해 세표 표면의 부착 분자에도 영향을 주는 것을 확인하였다.

• 공업화학
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• 제16권6호
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• pp.725-730
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• 2005

경조직 대체용 생체재료의 개발에 있어서 재료의 생체적합성 및 골전도성은 가장 중요한 고려사항 중의 하나이다. 인산칼슘 박막은 경조직과 화학적 성분이 매우 유사하기 때문에 재료의 표면에 인산칼슘 박막을 형성함으로써 재료의 생물학적 성질을 매우 증진시킬 수 있다. 생체재료의 성공여부는 재료의 표면에 대한 부착단백질의 작용에 의해 결정되는데, 이는 부착단백질들이 세포의 부착 및 부착된 세포의 생물학적 반응을 유도하기 때문이다. 재료의 표면에 대한 부착단백질들의 흡착기전과 흡착된 입체구조에 따라 조골세포의 부착, 증식, 그리고 분화와 같은 세포반응이 향상되거나 또는 억제된다. 인산칼슘 박막에 의해 개질된 생체재료의 경우 골전도성이 높은 것으로 알려져 있다. 조골세포의 분화 정도가 증가함은 물론 뼈 결절들이 신속하게 형성되는 것이 관찰되었다.

• 고려인삼학회:학술대회논문집
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• 고려인삼학회 2002년도 학술대회지
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• pp.317-334
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• 2002

Ginsenosides are metabolized (deglycosylated) by intestinal bacteria to active forms after oral administration. 20(S)-Protopanaxadiol $20-O-{\beta}-D-glucopyranoside$ (M1) and 20(S)-protopanaxatriol (M4) are the main intestinal bacterial metabolites (IBMs) of protopanaxadiol- and protopanaxatriol-type glycosides. M1 was selectively accumulated into the liver soon after its intravenous (i.v.) administration to mice, and mostly excreted as bile; however, some M1 was transformed to fatty acid ester (EMl) in the liver. EM1 was isolated from rats in a recovery dose of approximately $24mol\%.$ Structural analysis indicated that EM1 comprised a family of fatty acid mono-esters of M1. Because EM1 was not excreted as bile as Ml was, it was accumulated in the liver longer than M1. The in vitro cytotoxicity of M1 was attenuated by fatty acid esterification, implying that esterification is a detoxification reaction. However, esterified M1 (EM1) inhibited the growth of B16 melanoma more than Ml in vivo. The in vivo antitumor activity paralleled with the pharmacokinetic behavior. In the case of M4, orally administered M4 was absorbed from the small intestine into the mesenteric lymphatics followed by the rapid esterification of M4 with fatty acids and its spreading to other organs in the body and excretion as bile. The administration of M4 prior to tumor injection abrogated the enhanced lung metastasis in the mice pretreated with 2-chloroadenosine more effectively than in those pretreated with anti-asialo GMl. Both EM1 and EM4 did not directly affect tumor growth in vitro, whereas EM1 promoted tumor cell lysis by lymphocytes, particularly non-adherent splenocytes, and EM4 stimulated splenic NK cells to become cytotoxic to tumor cells. Thus, the esterification of IBM with fatty acids potentiated the antitumor activity of parental IBM through delay of the clearance and through immunostimulation. These results suggest that the fatty acid conjugates of IBMs may be the real active principles of ginsenosides in the body.

• 대한임상검사과학회지
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• 제47권4호
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• pp.273-278
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• 2015

이 연구의 목적은 임상 Candida albicans의 바이오필름억제을 위한 Rhadachlorin과 적색발광다이오드를 이용한 광역학치료의 체외적 효과를 평가하고자 하였다. Candida albicans 부유액을 $9{\times}10^8CFU/mL$을 5% 당이 함유된 YNB배지에 준비하였다. Candida albicans의 바이오필름은 96웰 플레이트에 100 uL씩 분주후 3시간 배양하고 상층액을 제거하였다. 각 웰에 $50{\mu}g/mL$ 부터 $0.39{\mu}g/mL$ 농도로 Rhadachlorin을 부착된 세포위에 처리하였다. 30분간 배양 후 빛을 30, 60, 90분을 630 nm 광원의 LED를 이용하여 조사하였고, 이때 각각의 에너지밀도는 14, 29, $43J/cm^2$이였다. 모든 상층액을 제거후에 건조시켰다. 부착된 세포를 safranin O로 염색하였다. 490 nm 파장으로 마이크로플레이트 리더로 흡광도를 측정하였다. 또한, 광감작제포화에 의해 Candida albicans의 형광시그널을 관찰하였다. 결론적으로 확연한 억제는 Rhadachlorin의 농도 $50{\mu}g/mL$과 630 nm LED치료에서 Candida albicans의 바이오필름의 72.5%가 감소되었다. 또한 광감작제 Rhadachlorin은 Candida albicans의 30분에서 충분한 축적을 보였다. 전체적으로 이 결과들은 바이오필름형성의 억제는 Rhadachlorin의 농도에 의존되었다. 이 결과들은 Rhadachlorin과 630 nm LED의 광역학치료는 Candida albicans의 바이오필름 억제가 가능한 것으로 제의된다.

• Journal of Microbiology and Biotechnology
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• 제27권11호
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• pp.1971-1982
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• 2017

Piliated Lactobacillus rhamnosus (pLR) strains possess higher adherent capacity than non-piliated strains. The objective of this study was to isolate and characterize probiotic pLR strains in human fecal samples. To this end, mouse polyclonal antiserum (anti-SpaA) against the recombinant pilus protein (SpaA) of L. rhamnosus strain GG (LGG) was prepared and tested for its reactivity and specificity. With the anti-SpaA, a method combining the de Man, Rogosa, and Sharpe (MRS) agar plating separation and colony immunoblotting (CIB) was developed to isolate pLR from 124 human fecal samples. The genetic and phenotypic characteristics of the resultant pLR isolates were compared by randomly amplified polymorphic DNA (RAPD) fingerprinting, and examination of adhesion to Caco-2 cells, hydrophobicity, autoaggregation, and in vitro gastrointestinal tolerance. Anti-SpaA specifically reacted with three pLR strains of 25 test strains, as assessed by western blotting, immunofluorescence flow cytometry, and immunoelectron microscopy (IEM) assays. The optimized MRS agar separation plus anti-SpaA-based CIB procedure could quantitatively detect $2.5{\times}10^3CFU/ml$ of pLR colonies spiked in $10^6CFU/ml$ of background bacteria. Eight pLR strains were identified in 124 human fecal samples, and were confirmed by 16S RNA gene sequencing and IEM identification. RAPD fingerprinting of the pLR strains revealed seven different patterns, of which only two isolates from infants showed the same RAPD profiles with LGG. Strain PLR06 was obtained with high adhesion and autoaggregation activities, hydrophobicity, and gastrointestinal tolerance. Anti-SpaA-based CIB is a rapid and inexpensive method for the preliminary screening of novel adherent L. rhamnosus strains for commercial purposes.

• 한국해양바이오학회지
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• 제2권4호
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• pp.250-256
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• 2007

1. 천연자원으로부터 ICAM-1과 LFA-1 매개성 세포접착 저해제를 탐색한 결과, 초두구 에탄올 추출물은 이에 대한 저해 활성을 나타내었다. 2. 초두구로부터 세포 접착 인자 활성에 관한 물질을 분리 정제하기 위하여, 각종 용매추출과 컬럼 크로마토그래피를 이용하여 얻은 화합물 1과 2로 세포접착인자 저해활성을 확인하였다. 3. 분리된 화합물 1과 2의 sICAM-1과 THP-1 세포와의 결합저해활성 측정 결과, $IC_{50}$ 값은 각각 $7.59{\mu}g/m{\ell}$ 및 $6.98{\mu}g/m{\ell}$로 측정되었다. 실험에 사용된 농도에서는 세포독성을 보이지 않았다. 4. 분리된 화합물 1과 2의 CHO-ICAM-1과 THP-1 세포간의 결합저해활성을 측정한 결과, $IC_{50}$ 값이 각각 $6.7{\mu}g/m{\ell}$ 및 $5.5{\mu}g/m{\ell}$로 측정되었다. 5. 이상의 실험 결과로 미루어 보아, 초두구로부터 분리한 비환식 트리테르페노이드계 화합물들은 세포 접착인자에 대한 저해활성을 가지고 있다고 볼 수 있으며 이 기전을 통하여 생길 수 있는 질환에 대한 치료제 개발에 유효할 것이다. 특히 동맥경화에 대해서는 새로운 기전으로 접근하고 있으므로 기존의 치료제들의 작용기작과는 다른 약품의 개발이 기대된다. 또한 ICAM-1은 염증반응 및 암의 전이에도 관련되어 있으므로, 동맥경화 관련 질환이외에도 다양한 질환에 대한 의약품으로의 응용도 기대된다.

• Journal of Periodontal and Implant Science
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• 제32권3호
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• pp.665-683
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• 2002

A novel glucanhydrolase from a mutant of Lipomyces starkeyi KSM 22 has additional amylase activity besides mutanolytic activity and has been suggested as promising anti-plaque agent. It has been shown effective in hydrolysis of mutan, reduction of mutan formation by Streptococcus mutans and removal pre-formed sucrose-dependent adherent microbial film and has been strongly bound to hydroxyapatitie. These in vitro properties of Lipomyces starkeyi KSM 22 glucanhydrolase are desirable for its application as a dental plaque control agent. In human experimental gingivitis　model and 6 month clinical trial, mouthrinsing with Lipomyces　starkeyi KSM 22 dextranase was comparable to 0.12% chlorhexidine mouthwash in inhibition of plaque accumulation and gingival inflammation and local side effect was negligible. This study was aimed to evaluate the cytotoxic effect of Lipomyces starkeyi KSM 22 glucanhydrolase on human gingival fibroblasts. Primary culture of human gingival fibroblasts at the 4th to 6th passages were used. Glucanhydrolase solution was made from lyophilized glucanhydrolase powder from a mutant of Lipomyces stakeyi KSM 22 solved in PBS and added to DMEM medium to the final concentration of 0.5, 1, and 2 unit. Cells were exposed to glucanhydrolase solution or 0.1 % chlorhexidine and the cells cultured in DMEM with 10% FBS and 1% antibiotics as control. After exposure, the morphological change, cell attachment, and cell activity by MTT assay were evaluated in 0.5, 1.5, 3, 6, 24 hours after treatment. The cell proliferation and cell activity was also evaluated at 2 and 7 days after 1 minute exposure, twice a day. The cell morphology was similar between the Lipomyces smkeyi KSM 22 glucanhydrolase groups and control group during the incubation periods, while most fibroblasts remained as round cell regardless of incubation time in the chlorhexidine group. The numbers of the attached cells in the glucanhydrolase groups were comparable to that of control and significantly higher than the chlorhexidine group. The numbers of the proliferated cells in the glucanhydrolase groups at 7 days of incubation were comparable to the control group and higher than the chlorhexidine group. The cell activity in glucanhydrolase groups paralleled with the increased cell number by attachment and proliferation. According to these results, Lipomyces starkeyj KSM 22 glucanhydrolase has little harmful effect on attachment and proliferation of human gingival fibroblasts, in contrast to 0.1% chlorhexidine which was cytotoxic to human gingival fibroblasts. Therefore this glucanhydrolase preparation is considered as a safe and promising agent for new mouthwash formula in the near future.