• Journal of Embryo Transfer
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• v.32 no.3
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• pp.73-79
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• 2017

Transplantation is considered to be a very useful approach to improve human welfare and to prolong life-span. Heterologous organ transplantation using pig organs which are similar to human beings and easy to make mass-production has known as one of the alternatives. To ensure potential usage of the pig organ for transplantation application, it is essentially required to generate transgenic pig modifying immuno-related genes. Previously, we reported production of heterozygous ${\alpha}1,3$-galactosyltransferase (GalT) knock-out and human membrane cofactor protein (MCP) expressing pig ($GalT^{-MCP/+}$), which is enforced for suppression of hyperacute and acute immunological rejection. In this study, we reported generation of homozygous pig ($GalT^{-MCP/-MCP}$) by crossbreeding $GalT^{-MCP/+}$ pigs. Two female founders gave birth to six of $GalT^{-MCP/-MCP}$, and seven $GalT^{-MCP/+}$ pigs. We performed quantitative real-time PCR, western blot, and flow cytometry analyses to confirm GalT and MCP expression. We showed that fibroblasts of the $GalT^{-MCP/-MCP}$ pig do not express GalT and its product Gal antigen, while efficiently express MCP. We also showed no expression of GalT, otherwise expression of MCP at heart, kidney, liver and pancreas of transgenic pig. Taken together, we suggest that the $GalT^{-MCP/-MCP}$ pig is a useful candidate to apply xenotransplantation study.

• Nutrition Research and Practice
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• v.12 no.2
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• pp.129-134
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• 2018

BACKGROUND/OBJECTIVES: Although several recent studies have reported the anti-cancer effects of extracts or components of Citrus unshiu peel, which has been used for various purposes in traditional medicine, the molecular mechanisms for their effects remain unclear. In the present study, the anti-cancer activity of a water-soluble extract of C. unshiu peel (WECU) in MDA-MB-231 human breast carcinoma cells at the level of apoptosis induction was investigated. MATERIALS/METHODS: Cytotoxicity was evaluated using the MTT assay. Apoptosis was detected using DAPI staining and flow cytometry analyses. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, caspase activity and Western blotting were used to confirm the basis of apoptosis. RESULTS: The results indicated that WECU-induced apoptosis was related to the activation of caspase-8, and -9, representative initiator caspases of extrinsic and intrinsic apoptosis pathways, respectively, and caspase-3 accompanied by proteolytic degradation of poly(ADP-ribose) polymerase and down-regulation of the inhibitors of apoptosis protein family members. WECU also increased the pro-apoptotic BAX to anti-apoptotic BCL-2 ratio, loss of mitochondrial membrane potential and cytochrome c release from mitochondria to cytoplasm. Furthermore, WECU provoked the generation of ROS, but the reduction of cell viability and induction of apoptosis by WECU were prevented when ROS production was blocked by antioxidant N-acetyl cysteine. CONCLUSIONS: These results suggest that WECU suppressed proliferation of MDA-MB-231 cells by activating extrinsic and intrinsic apoptosis pathways in a ROS-dependent manner.

• Archives of Plastic Surgery
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• v.34 no.1
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• pp.1-7
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• 2007

Purpose: Human adipose tissue-derived stromal cells(hADSCs) can be expanded in vitro and induced to differentiate into multiple mesenchymal cell types. In this study we have examined various neuronal phenotypes and gene expression profiles of the hADSCs in the neuronal induction. Methods: The hADSCs were isolated from human adipose tissue and they were characterized by the flow cytometry analysis using CD13, CD29, CD34, CD45, CD49d, CD90, CD105 and HLA-DR cell surface markers. We differentiated the hADSCs into the neuronal lineage by using chemical induction medium and observed the cells with contrast microscopy. The immunocytochemistry and western blotting were performed using the NSE, NeuN, Trk-A, Vimentin, N-CAM, S-100 and ${\beta}$-Tubulin III antibodies. Results: The hADSCs were positive for CD13($90.3{\pm}4%$), CD29($98.9{\pm}0.7%$), CD49d($13.6{\pm}6%$), CD90 ($99.4{\pm}0.1%$), CD105($96%{\pm}2.8%$) but negative for CD34, CD45 and HLA-DR. The untreated cultures of hADSCs predominately consisted of spindle shaped cells and a few large, flat cells. Three hours after the addition of induction medium, the hADSCs had changed morphology and adopted neuronal-like phenotypes. The result of immunocytochemistry and western blotting showed that NSE, NeuN, Trk-A, Vimentin, N-CAM, S-100 and ${\beta}$-Tubulin III were expressed. However, NSE, NeuN, Vimentin were weakly expressed in the control. Conclusion: Theses results indicate that hADSCs have the capabillity of differentiating into neuronal lineage in a specialized culture medium. hADSCs may be useful in the treatment of a wide variety of neurological disorders.

• Journal of Chest Surgery
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• v.30 no.6
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• pp.553-558
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• 1997

Arterial allografts have known advantages over prosthetic vascular conduit for treatment of heart valvular disease, congenital heart disease and aortic disease. Cell viability may play a role in determining the longterm outcome of allografts. Endothelial cell is one important part in determining the allograft viability. To evaluate the viability of endothelial cells using current allograft preservation technique, porcine heart valve leaflets and arterial wall were subjected to collagenase digestion. Single endothelial cell suspension was labeled with GSA-PITC(Griffonia simplicifolia agglutininfluorescein isothiocyan te), a vascular, endothelial cell specific marker. The cell suspension was washed and incubated with Pl(Propidium iodide), which does not bind with viable cells, Endothelial cell viability was evaluated by calculating the percentage of GSA-FITC(+) and Pl(-) group using flowcytometric analysis. Allografts were treated with $4^{\circ}C$ antibiotic solo!ion for 24 hours for sterilization. After this, half of allografts were stored in $4^{\circ}C$ RPMI 1640 with HEPES buffer culture medium with 10% fetal bovine serum for 1 to 14 days(Group I). Another half of allografts were cryopreserved with a currently used technique (Group II). During the procurement and sterilization of arterial allografts, 22.8% and 24.4% of endothelial cell viability declined, respectively. In Group I, 11.9% of endothelial cell viability declined further steadily during 14 days of storage. In Group II, 13.7% of endothelial cell viability declined. These results show that largest loss of endothelial cell viability occurs during the nitial process. After 14 days of arterial allograft storage under $4^{\circ}C$ nutrient medium or cryopreservation, about 40% of endothelial cell viability is maintained. There were no differences between the endothelial cell viability from aortic valve leaflet, pulmonic valve leaflets, aortic wall and pulmonic wall.

• Journal of Veterinary Clinics
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• v.20 no.4
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• pp.437-442
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• 2003

To examine the in vivo immunostimulating effect of conjugated linoleic acid (CLA) in pigs, the change of peripheral blood cells and the phagocytic response of phagocytes were evaluated. Spayed male pigs, 80 kg of average body weight, fed a diet containing either 0.5% 10t-12c CLA or 0.5% CLA mixture (mostly 9c-11t CLA and 10t-12c CLA) for 4 weeks. The change of blood cell values (PCV, WBC, differential count of WBC) and the phagocytic activities of phagocytes were evaluated on week 0, 2, 4, and 5, respectively. There were no change in the PCV values regardless of CLA supplement. The number of WBC, especially neutrophils, in pigs fed a diet with CLA was significantly increased (p<0.05 to 0.01) when compared with control pigs fed a diet without CLA. The phagocytosis of peripheral blood mononuclear cell (MNC) and peripheral blood polymorphonuclear cells (PMN) were analyzed by a flow cytometry system. There was no change in the phagocytic activity of MNC and monocyte-rich cells regardless of CLA supplement. However, the phagocytic activity of PMN composed by approximately 95% neutrophils was remarkably increased (p < 0.05 to 0.01) on week 2, 4, and 5 as compared wth control pigs. These results suggested that supplement of CLA into pigs induces the increase of neutrophil number and the enhancement of neutrophil phagocytosis.

• The Journal of Pediatrics of Korean Medicine
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• v.22 no.1
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• pp.69-93
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• 2008

Objectives The purpose of this study is to investigate the effect of KKHS on atopic dermatitis in an in-vivo experiment using an NC/Nga atopic dermatitis mouse, which has histological and clinical similarities to this condition in humans. Methods To investigate the effect of KKHS on atopic dermatitis (AD), we evaluated atopic dermatitis-like skin lesions by clinical skin index and analyzed immunological parameters in peripheral blood mononuclear cells(PBMCs), splenocytes, draining lymph node(DLN) and performed skin histology in ears and dorsal skin of atopic dermatitis of NC/Nga mouse in vivo. Results In vivo, clinical skin severity score was significantly lower in the KKHS group than in the control group. IgE, IL-6, TNF-${\alpha}$, IgM, IgG2a and IgG2b levels in serum decreased remarkably in the KKHS group than in the control group, and the level of IFN-${\gamma}$ production which is secreted from Th1 cell was increased by KKHS. After this experiment we analyzed immunological cells ($CD3^+$, $CD19^+$, $CD4^+$, $CD8^+$, $CD3^+CD69^+$, $CD4^+CD25^+$ and $CD49b^+$) by flow cytometry. It results that the total absolute number of $CD3^+$, $CD19^+$, $CD4^+$ and $CD8^+$ cells were recovered as much as normal state, and the level of $CD3^+CD69^+$ in isolated DLN and PBMCs were significantly decreased, and total absolute number of $Gr-1^+$, $CD11b^+$ and $CD3^+$ in dorsal skin of NC/Nga mouse were decreased by KKHS. We analyzed ear, DLN, and neck-back skin after biopsy and dyeing by hematoxyline/eosin(H&E), toluidine staining (mast cells marker). KKHS were very effective to the histological symptoms which are in dermal and epidermal thickening, hyperkeratosis and inflammatory cell infiltration. Ear thickness was significantly decreased compared with the control group and the size of inflammatory lymphocytes cells (ILC) and plasma cells (PC) in DLN were also decreased. Conclusions KKHS on atopic dermatitis in an in-vivo experiment using an NC/Nga atopic dermatitis mouse was very effectiveness to the atopy dermatitis treatment.

• The Journal of Internal Korean Medicine
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• v.24 no.2
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• pp.290-299
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• 2003

Background : Nowadays many researches about it s cure are going on world widely since cancer is one of the most human health threatening diseases. In Chinese and North Korean medicine, Duchesnea india(Audra.) Foche. is practically used to treat many kinds of cancer, but in Korea it is rarely used. So, we need to scientifically identify anti-tumor effects of Duchesnea india(Audra.) Foche. Objective : We are aimed to identify anti-tumor effects of Duchesnea india(Audra.) Foche. on the stomach cancer cells through molecular biologic methods. Material & Methods : We used AGS as stomach cancer cells from American Type Culture Collection. We added the boiled extract of Duchesnea india(Audra.) Foche. $5{\mu}l$(Sample I), $10{\mu}l$(Sample II) to cultural media(ml)for 0,6, 12, 24, 48 hours. We measured the killing effect on stomach cancer cells through Tryphan blue exclusion test and the suppressive effect on viability of stomach cancer cells via MTT assay. the quantitative RT-PCR was used to examine their effect on the revelation of Bcl-2, Bcl-XL, and Bax, which are genes related to apoptosis. We measured change of mitochondria membrane permeability and membrane potential via flow cytometry. Result : 1. The killing effect on stomach cancer cells showed that each test groups killed more stomach cancer cells than the control group with a time(6 hours later) and density dependent manner, which was statistical significance. 2. The suppressive effect on viability of stomach cancer cells showed that each test groups had more suppressive effects on viability of stomach cancer cells than the control group with a time(6 hours later), which was statistical significance. 3. In the test about the revelation of genes related to apoptosis, the revelation of Bcl-2 and Bcl-XL decreased with a density manner which was statistical significance. but the revelation of Bax was not changed with statistical significance. 4. As a result of this test, Duchesnea india(Audra.) Foche. caused apoptosis by decreasing the absorbance of mitochondria with statistical significance. and also induced apoptosis by decreasing the membrane potential of mitochondria. Conclusion : This experiment showed that Duchesnea india(Audra.) Foche. has anti-tumor effect with statistical significance. This is in vitro experiment and basic experiment on Duchesnea india(Audra.) Foche. We hope more progressive researchs on Duchesnea india(Audra.) Foche. will go on and its anti-tumor effects will be more practically identified.

• Journal of Marine Bioscience and Biotechnology
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• v.1 no.2
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• pp.76-83
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• 2006

Epidemiological studies have indicated that the ubiquitous consumption of seaweeds is a protective factor against some types of cancer. Previous results showed that the administration of seaweed powder or extract reduced the incidence rate of chemically induced tumorigenesis using in vivo animal model. Recently, we reported that the extracts of Gloiopeltis furcata, a kind of Korean edible seaweed, caused he cell growth inhibition of various human cancer cell lines, among them methanol extract exhibited a relatively strong antiproliferative activity. However, the molecular mechanisms of this seaweed in malignant cells have been poorly studied until now. To elucidate this problem, we investigated the effects of methanol extract of G. furcata (MEGF) on the growth inhibition in several human cancer cell lines, and further we analyzed the effects of this extract were tested on the activity of apoptosis induction in human leukemic cells. The results demonstrated that MEGF treatment resulted in the morphological changes and the growth inhibition in a dose-dependent manner. Furthermore, MEGF potently suppresses the growth of human leukemic U937 cells by induction of apoptosis, which was associated with induction of cyclin-dependent kinase inhibitor p21(WAF1/CIP1) in a tumor suppressor p53-independent fashion and up-regulation of Fas/FasL system. Further studies will be needed to identify the active compounds that confer the anticancer activity of MEGF. Once such compounds are identified, the mechanisms by which they exert their effects can begin to be characterized.

• Nutrition Research and Practice
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• v.11 no.6
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• pp.470-478
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• 2017

BACKGROUND/OBJECTIVE: Orostachys japonicus A. Berger (Crassulaceae) has been used in traditional herbal medicines in Korea and other Asian countries to treat various diseases, including liver disorders. In the present study, the anti-fibrotic effects of O. japonicus extract (OJE) in cellular and experimental hepatofibrotic rat models were investigated. MATERIALS/METHODS: An in vitro hepatic stellate cells (HSCs) system was used to estimate cell viability, cell cycle and apoptosis by MTT assay, flow cytometry, and Annexin V-FITC/PI staining techniques, respectively. In addition, thioacetamide (TAA)-induced liver fibrosis was established in Sprague Dawley rats. Briefly, animals were divided into five groups (n = 8): Control, TAA, OJE 10 (TAA with OJE 10 mg/kg), OJE 100 (TAA with OJE 100 mg/kg) and silymarin (TAA with Silymarin 50 mg/kg). Fibrosis was induced by treatment with TAA (200 mg/kg, i.p.) twice per week for 13 weeks, while OJE and silymarin were administered orally two times per week from week 7 to 13. The fibrotic related gene expression serum biomarkers glutathione and hydroxyproline were estimated by RT-PCR and spectrophotometry, respectively, using commercial kits. RESULTS: OJE (0.5 and 0.1 mg/ mL) and silymarin (0.05 mg/mL) treatment significantly (P < 0.01 and P < 0.001) induced apoptosis (16.95% and 27.48% for OJE and 25.87% for silymarin, respectively) in HSC-T6 cells when compared with the control group (9.09%). Further, rat primary HSCs showed changes in morphology in response to OJE 0.1 mg/mL treatment. In in vivo studies, OJE (10 and 100 mg/kg) treatment significantly ameliorated TAA-induced alterations in levels of serum biomarkers, fibrotic related gene expression, glutathione, and hydroxyproline (P < 0.05-P < 0.001) and rescued the histopathological changes. CONCLUSIONS: OJE can be developed as a potential agent for the treatment of hepatofibrosis.

• Journal of the Korean Child Neurology Society
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• v.23 no.2
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• pp.45-50
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• 2015

Purpose: X-linked adrenoleukodystrophy (X-ALD) is a fatal, axonal demyelinating, neurodegenerative disease, and is caused by mutations the in ABCD1 (ATP-binding cassette transporter subfamily D member 1). Oxidative damage of proteins caused by very long chain fatty acid accumulating in X-ALD, is an early event in the neurodegenerative cascade. We evaluated valproic acid (VPA) as a possible option for oxidative damage in X-ALD. Method: We generated fibroblast of the childhood cerebral ALD from patient. We evaluated mRNA (ribonucleic acid) level of ABCD2 by real-time polymerase chain reaction, and reactive oxygen species (ROS) levels by flow cytometry. Results: VPA increased expression of ABCD2 in both control and ALD fibroblast. ABCD2 gene mRNA expression was increased 1.76 fold in normal fibroblasts, and 2.22 fold in the X-ALD fibroblasts. ROS levels were decreased in VPA treated X-ALD fibroblast, especially in treated with 1 mM of VPA. ROS levels revealed 13.7 in control fibroblast, on the other hand, 5.83 in X-ALD fibroblast treated with 1 mM of VPA. Conclusion: We propose VPA as a promising novel therapeutic approach in oxidant damage that warrants further clinical investigation in X-ALD.