• 한국발생생물학회지:발생과생식
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• 제20권1호
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• pp.63-71
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• 2016

Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feeder layers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KO-SR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xeno-free conditions for clinical grade hESCs culture will be useful data in future clinical studies.

• 한국축산식품학회지
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• 제43권6호
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• pp.1017-1030
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• 2023

Fetal bovine serum (FBS), which contains various nutrients, comprises 20% of the growth medium for cell-cultivated meat. However, ethical, cost, and scientific issues, necesitates identification of alternatives. In this study, we investigated commercially manufactured serum-free media capable of culturing Hanwoo satellite cells (HWSCs) to identify constituent proliferation enhancing factors. Six different serum-free media were selected, and the HWSC proliferation rates in these serum-free media were compared with that of control medium supplemented with 20% FBS. Among the six media, cell proliferation rates were higher only in StemFlex^TM Medium (SF) and Mesenchymal Stem Cell Growth Medium DXF (MS) than in the control medium. SF and MS contain high fibroblast growth factor 2 (FGF2) concentrations, and we found upregulated FGF2 protein expression in cells cultured in SF or MS. Activation of the fibroblast growth factor receptor 1 (FGFR1)-mediated signaling pathway and stimulation of muscle satellite cell proliferation-related factors were confirmed by the presence of related biomarkers (FGFR1, FRS2, Raf1, ERK, p38, Pax7, and MyoD) as indicated by quantitative polymerase chain reaction, western blotting, and immunocytochemistry. Moreover, PD173074, an FGFR1 inhibitor suppressed cell proliferation in SF and MS and downregulated related biomarkers (FGFR1, FRS2, Raf1, and ERK). The promotion of cell proliferation in SF and MS was therefore attributed to FGF2, which indicates that FGFR1 activation in muscle satellite cells may be a target for improving the efficiency of cell-cultivated meat production.

• 대한한방부인과학회지
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• 제29권1호
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• pp.14-34
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• 2016

목 적: 본 연구에서는 한의학에서 다양한 피부질환과 대사질환에 빈번히 사용되고 있는 온청음 물 추출 동결건조물(수율=13.82%)의 피부 노화 개선 효과 평가의 일환으로 세포독성, 피부재생, 주름개선, 미백 및 보습 효과를 각각 평가하였다.방 법: 본 연구에서는 human normal fibroblast(CRL-2076) 및 B16/F10 murine melanoma(CRL-6475) 세포에 대한 온청음의 세포독성을 MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium Bromide) 방법으로 평가하였으며, 피부 재생 및 주름 개선 효과를 transforming growth factor(TGF)-β1와 비교한 fibroblast의 collagen type I 합성능, phosphoramidon disodium salt(PP)와 비교한 elastase 활성 억제, oleanolic acid(OA)와 비교한 hyaluronidase, collagenase 및 matrix metalloproteinase (MMP)-1 활성 억제를 통해 각각 평가하였고, 미백효과를 B16/F10 murine melanoma cells의 melanin 생성 억제 정도 및 tyrosinase 활성 억제를 통해 arbutin과 비교 평가하였으며, 모든 실험은 OCE의 농도별로 군을 나누어 농도에 따른 효과의 변화를 함께 분석하였다. 보습효과는 흰쥐의 피부 수분함량 변화를 통해 평가하였다.결 과: 본 실험의 결과, 온청음은 human normal fibroblast 및 B16/F10 murine melanoma 세포에 대해 의미 있는 세포독성을 나타내지 않았으며, fibroblast의 collagen type I 합성을 증가시켰고, 세포외 기질의 파괴에 관여한다고 알려진 hyaluronidase, elastase, collagenase 및 MMP-1 활성을 억제하였으며, 피부의 색을 결정하는 melanin 의 생성에 관여하는 tyrosinase의 활성 및 B16/F10 murine melanoma cells의 melanin 생성을 억제하는 것으로 관찰되었다. 이 반응의 효과들은 모두 농도에 비례하여 증가하였고, 이와 함께 정상 매체 대조군에 비해 흰쥐의 피부 수분 함량이 세 용량의 온청음 경구 투여군 모두에서 투여용량 의존적으로 의미 있는 증가를 보였다.결 론: 이상의 결과에서, 온청음은 세포 독성 없이 비교적 우수한 피부 재생, 주름개선, 미백 및 보습 효과를 나타내는 것으로 관찰되어, 차후 피부 노화 억제 개선제 또는 기능성 화장품의 주요 소재로서 그 가치가 매우 높을 것으로 판단되나, 금후 개별 구성 약재 각각에 대한 효능 및 생리활성을 나타내는 화학성분의 검색과 더불어 다양한 방면으로 기전적인 연구와 피부 보호 효과에 대한 in vivo 평가를 체계적으로 수행해야 할 것으로 판단된다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권5호
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• pp.1833-1837
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• 2015

An aristolactam-type alkaloid, isolated from Orophea enterocarpa, is enterocarpam-III (10-amino-2,3,4,6-tetramethoxyphenanthrene-1-carboxylic acid lactam). It is cytotoxic to various human and murine cancer cell lines; however, the molecular mechanisms remain unclear. The aims of this study were to investigate cytotoxic effects on and mechanism (s) of human cancer cell death in human hepatocellular carcinoma HepG2 and human invasive breast cancer MDA-MB-231 cells compared to normal murine fibroblast NIH3T3 cells. Cell viability was determined by MTT assay to determine $IC_{10}$, $IC_{20}$ and $IC_{50}$ levels, reactive oxygen species (ROS) production with 2',7'-dichlorohydrofluorescein diacetate and the caspase-3, -8 and -9 activities using specific chromogenic (p-nitroaniline) tetrapeptide substrates, viz., DEVD-NA, IETD-NA and LEHD-NA and employing a microplate reader. Mitochondrial transmembrane potential (MTP) was measured by staining with 3, 3'-dihexyloxacarbocyanine iodide ($DiOC_6$) and using flow cytometry. The compound was cytotoxic to HepG2 and MDA-MB-231 cells with the $IC_{50}$ levels of $26.0{\pm}4.45$ and $51.3{\pm}2.05{\mu}M$, respectively. For murine normal fibroblast NIH3T3 cells, the $IC_{50}$ concentration was $81.3{\pm}10.1{\mu}M$. ROS production was reduced in a dose-response manner in HepG2 cells. The caspase-9 and -3 activities increased in a concentration-dependent manner, whereas caspase-8 activity did not alter, indicating the intrinsic pathway activation. Enterocarpam-III decreased the mitochondrial transmembrane potential (MTP) dose-dependently in HepG2 cells, suggesting that the compound induced HepG2 cell apoptosis via the mitochondrial pathway. In conclusion, enterocarpam-III inhibited HepG2 and MDA-MB-231 cell proliferation and induced human HepG2 cells to undergo apoptosis via the intrinsic (mitochondrial) pathway and induction of caspase-9 activity.

• 한국잠사곤충학회지
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• 제46권2호
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• pp.72-76
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• 2004

가잠과 천잠 및 작잠의 실크 단백질 분획물로부터 피부섬유아세포에 대한 증식 효과를 알아본 결과 다음과 같은 결과를 얻을 수 있었다. 1. 가잠 및 천${\cdot}$작잠 단백질로부터 다양한 분자량대의 실크 단백질을 순수 분리 분획화 하여 사람 피부세포(human skin fibroblast; CCD-986sk)에 기계적 상처를 준 후 처리한 결과, 평균분자량(Mw)이 300-500 및 950-1500 분획군이 세포 증식에 뛰어난 효과가 있음을 확인${\cdot}$선발하였다. 2. 가잠의 경우 평균 분자량 590(BM-1), 1400(BM-2)에서, 천잠은 340(AY-1), 940(AY-2), 작잠에서는 300(AP-1), 1440(AP-2)에서 $10\;{\mu}g/ml$의 농도로 처리했을 경우 피부섬유 아세포의 증식이 무처리구에 비하여 40% 이상의 증식 효과를 갖았다. 또한 유효 농도 범위에서 대식세포인 RAW 264.7 세포에 대해서는 세포독성을 지니지 않았다. 3. 아미노산 조성에 있어 저분자량 AY-1 분획의 전아미노산 및 유리아미노산은 Tyr 함량이 매우 높게(25-48 g/100g)나타났으며, 작잠의 저분자 분획인 AP-1에서는 Lys 함량이 높게(5.2 g/100g) 나타남을 확인하였다. 4. 동일한 실크 단백질일 경우 저분자량 분획물에서 피부 세포 증식에 좀 더 효과적이였다.

• Bulletin of the Korean Chemical Society
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• 제33권8호
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• pp.2567-2573
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• 2012

We successfully synthesized $Fe_3O_4@SiO_2$ nanoparticles with ultrathin silica layer of $1.0{\pm}0.5$ nm that polyethyleneimine (PEI) with low molecular weight of 2.0-4.0 kDa was covalently conjugated with the resulting $Fe_3O_4@SiO_2$ nanoparticles by silane coupling reaction. The PEI-$Fe_3O_4@SiO_2$ nanoparticles were further used as gene delivery vector for a human fibroblast cell (IMR-90) line. Gene transfection efficiency of the PEI-$Fe_3O_4@SiO_2$ complexes did not increase remarkably after magnetofection; however, the addition of Lipofectamine 2000 significantly increased the transfection efficiency of the PEI-$Fe_3O_4@SiO_2$ complexes. We believe that the present approach could be utilized for magnetofection as alternative to $Fe_3O_4$ nanoparticles conjugated with the PEI of high molecular weight thanks to its relatively low cytotoxicity and high transfection efficiency.

• International Journal of Oral Biology
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• 제41권3호
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• pp.119-123
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• 2016

Acetylcholine receptors (AChR) including muscarinic and nicotinic AChR are widely expressed and mediate a variety of physiological cellular responses in neuronal and non-neuronal cells. Notably, a functional cholinergic system exists in oral epithelial cells, and nicotinic AChR (nAChR) mediates cholinergic anti-inflammatory responses. However, the pathophysiological roles of AChR in periodontitis are unclear. Here, we show that activation of AChR elicits increased cytosolic $Ca^{2+}([Ca^{2+}]_i)$, transient cytotoxicity, and induction of receptor activator of nuclear factor kappa-B ligand (RANKL) expression. Intracellular $Ca^{2+}$ mobilization in human gingival fibroblast-1 (hGF-1) cells was measured using the fluorescent $Ca^{2+}$ indicator, fura-2/AM. Cytotoxicity and induction of gene expression were evaluated by measuring the release of glucose-6-phosphate dehydrogenase and RT-PCR. Activation of AChR in hGF-1 cells by carbachol (Cch) induced $[Ca^{2+}]_i$ increase in a dose-dependent manner. Treatment with a high concentration of Cch on hGF-1 cells caused transient cytotoxicity. Notably, treatment of hGF-1 cells with Cch resulted in upregulated RANKL expression. The findings may indicate potential roles of AChR in gingival fibroblast cells in bone remodeling.

• Archives of Plastic Surgery
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• 제34권4호
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• pp.426-431
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• 2007

Purpose: The aim of this study is to compare the effects of bone marrow stromal cells(BSCs) and fibroblasts on wound healing activity in vivo, especially on epithelization. Methods: The fibroblasts and BSCs were harvested from patients and cultured. Ten Spague-Dawley white rats were used. A 5 mm punches were made to excise skin and subcutaneous tissue in a round fashion at six sites on the back area of each rat. Four hundred thousand cells suspended in 0.05 ml fibrinogen were applied to the created wounds. The cells in group I, II, and III were no cells, fibroblasts and BSCs. The lengths of epithelial gap at the widest wound site were compared with autopsy specimens obtained on the 6th day after cell therapy under light microscope. Statistical comparisons were performed using the Mann-Whitney U-test, and the p value < 0.05 was considered statistically significant. Results: The best epithelization was also seen in the BSC group, followed by fibroblast and no cell groups.Conclusion: These results demonstrate that BSC has superior effect on stimulating wound healing than fibroblast, which is currently used for wound healing.

• Journal of Microbiology
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• 제38권1호
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• pp.24-30
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• 2000

US3 gene is a member of the human cytomegalovirus (HCMV) immediate early gene. Although the precise functions of the US3 gene in HCMV replication and pathogenesis are not known, it has been reported to play a role in inhibiting major histocompatibility class I antigen presentation. For further knowledge of US3 gene expression, rabbit polyclonal antiserum of the US3 gene product was used for indirect immunofluorescence assay. In permissive human foreskin fibroblast (HFF) cells, US3 gene expression was detectable as crescent or half-moon shape in the perinuclear region at immediate early times after virus infection. HFF cells infected with mutant HCMV lacking US3 open reading frames were negative for US3 immunofluorescence assay. Double immunofluorescence assay using monoclonal antibody to gamma adaptin (specific for the Golgi complex) and rabbit anti-US3 antiserum revealed that US3 gene product could be localized to the Golgi complex. At later time after HCMV infection, US3 gene products were detected as globular aggregates in the cytosol. These aggregates were positive for gamma adaptin and stained with preimmune serum, suggesting a nonspecific reaction to the Golgi complex. Northern blot analysis revealed that transcription of US3 was observed only during immediate early times after virus infection (until 6 h postinfection). Therefore US3 gene expression appears to be confined to immediate early time and its gene products are localized to the Golgi complex as crescent shaped forms in the perinuclear cytoplasm.

• 한국미생물·생명공학회지
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• 제16권6호
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• pp.522-525
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• 1988

무혈청 배지에 생산촉진제로 30$\mu$g/m$\ell$의 Heparin 을 첨가해 정상인의 섬유 세포로부터 상업적으로 tPA를 생산할 수 있는 방법의 개발과, 효과적인 tPA 생산을 위해 대량 배양에 적합한 무혈청 배지의 조성을 확립했다. 이 방법으로 연속배양 공법하에서 매일 1.1gram의 tPA가 생산될 수 있으며, 이 생산성은 tPA 생산 단가를 크게 낮출 분만 아니라 무혈청 배지의 사용으로 tPA의 순수 정제 과정을 크게 단축시킬 수 있다. 또한 이 세포에서 생산되는 tPA 는 fibrin lysis 시험결과 섬유질 분해능력이 높음이 입증되었으며, ELISA결과와도 상충했다.