• Nutrition Research and Practice
• /
• 제12권2호
• /
• pp.129-134
• /
• 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
• /
• 제34권1호
• /
• pp.1-7
• /
• 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
• /
• 제30권6호
• /
• pp.553-558
• /
• 1997

동종동맥판은 심장판막질환, 선천성 심기형 및 대동맥 질환의 치료에 있어서 우수한 판막도관으로 사용 되고 있다. 이 때 동종동맥 판의 장기성적을 좌우하는데 있어서 혈관내피세포의 생육성이 중요한 역활을 할 것이다. 혈관내피세포의 생육성을 평가하기 위하여 현재 임상에서 사용되는 보존방법으로 보존처리된 성돈의 대동맥판 및 대동맥 벽을 collagenase로 분해시켜서 순수한 내퍼세포군을 획득한 뒤, 혈관내피세포에 특이한 친화성을 갖는 GSA-FTTC(Criffonia simplicifolia agglutininfluorescein isothiocyanate)와 반응시켰다. 이 내피세포군을 세척한 다음, 살아있는 세포에는 침착되지 않는 Pl(Ropidium iodide)와 반응시켰다. 이렇게 처리된 내피세포군을 Row Cytometry 로 분석하여 GSA-FTIC(+), Pl(-) 인 세포를 생육성을 유지한 것으로 평가하였다. 동종동맥판은 $4^{\circ}C의$ 멸균용액에 24시간 담궈 멸균처리를 한 후, 2개군으로 나누어 (1군)은 $4^{\circ}C$ RPM 1640 with HEPES buffer cultlue medium with 10% fetal bovine uTm 용액에 1~14일간 보존하였고 (2군)은 냉동보존을 하였다. 조직의 획득과정과 멸균과정에서 각각 22.8%와 24.4%의 생육성이 소\ulcorner되었다. (1군) 에서는 14일의 보존기간 동안 11.9%의 생육성감소가 일어났고 (2군) 에서는 13.7%의 생육성감소가 일어났다. 이 실험의 결과로 동종동맥 판의 보존처리과정 초기에 대부분의 생육성소실이 일어나며, 14일간의 냉장보존이나 냉동보존 후에도 약 40%의 생육성이 보존됨을 알 수 있었다. 또한 혈관내피세포가 판막에서 얻어진 경우나 동맥벽에서 얻어진 경우에서 생육성의 차이는 없었다.

• 한국임상수의학회지
• /
• 제20권4호
• /
• pp.437-442
• /
• 2003

CLA를 급여시킨 돼지에 있어 말초 혈액 세포의 변화와 탐식세포의 탐식반응을 조사하였다. 100일령, 평균체 중 80kg인 거세 수퇘지에게 10t-l2c CLA와 CLA mixture를 각각 사료 중에 5% 첨가하여 총 4주간에 걸쳐 자유급여 시켰다. 급여 전 (0주), 급여 2주후, 4주후 그리고 급여 중지 후인 5주차에 각각 혈액세포 수치(PCV 백혈구수 및 백혈구 감별계산치)의 변화와 탐식세포의 탐식활성을 측정하였다. CLA의 급여 따른 PCV의 수치에 있어서는 아무런 변화가 없었다. 그러나 10t-l2c CLA 또는 CLA mixture가 함유된 사료를 급여한 돼지에 있어서 CLA를 급여하지 않은 돼지에 비해 백혈구수 특히 호중구 수가 현저히 증가하였다. 말초혈액 단핵구세포(mononuclear cells; MNC)와 다형핵백혈구(polymorphonuclear cells; PMN)의 탐식능를 유세포분석기로 분석한 결과, 10t-l2c CLA 또는 CLA mixture 급여와 상관없이 MNC와 단구세포들(monocyte-rich cells)의 탐식능에는 변화가 없었다. 그러나 PMN의 탐식 활성은 CLA 무급여 대조돼지들에 비해 CLA 급여군은 2주에서 5주차에 걸쳐 증가된 탐식능를 나타내었다. 이상의 결과로부터 돼지에 CLA 급여는 호중구수 증가와 호중구 탐식능 증가를 유도할 수 있음을 제시하였으며 향후 생체방어 증강을 위해 CLA를 임상적으로 투여할 수 있을 것으로 보인다.

• 대한한방소아과학회지
• /
• 제22권1호
• /
• pp.69-93
• /
• 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.

• 대한한방내과학회지
• /
• 제24권2호
• /
• pp.290-299
• /
• 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.

• 한국해양바이오학회지
• /
• 제1권2호
• /
• pp.76-83
• /
• 2006

본 연구에서는 다양한 인체암세포의 증식에 미치는 등불가사리 메탄올 추출물(MEGF)의 영향을 조사하였다. MEGF는 처리 농도의존적으로 암세포의 형태적변이 및 증식억제가 효과를 보여주었으며, 특히 백혈병세포에서 가장 높은 감수성을 보여주었다. 따라서 백혈병세포의 증식억제 효과가 apoptosis 유발과 연관성이 있는지를 flow cytometry 분석 및 DAPI staining 법으로 조사한 결과, MEGF 처리에 의한 백혈병세포의 증식억제는 세포주기 교란과 무관한 apoptosis 유발에 의한 것임을 sub-G1기 세포의 빈도 증가 및 apoptotic body 형성의 증가 등으로 확인하였다. 또한 MEGF 처리에 의한 백혈병세포의 증식억제 및 apoptosis 유발은 p53 비의존적인 p21의 발현 증가 및 Fas/FasL system의 발현 증가와 연관성이 있음을 알 수 있었다. 이상의 결과들은 인체 암세포, 특히 백혈병세포에서 MEGF의 항암작용을 이해하는데 중요한 자료가 될 것이고 나아가 MEGF 내 함유된 생리활성 물질의 분리 및 항암적용 연구를 위한 중요한 기초 자료로 활용될 것이다.

• Nutrition Research and Practice
• /
• 제11권6호
• /
• pp.470-478
• /
• 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.

• 대한소아신경학회지
• /
• 제23권2호
• /
• pp.45-50
• /
• 2015

목적: X-ALD는 Xq28에 위치한 ABCD1 유전자의 돌연변이로 긴사슬지방산이 신경 조직과 부신에 축적되어 일어나는 퇴행 뇌질환이며, 소아기 대뇌형의 경우 빠르고 심한 임상 경과를 보인다. 이 과정에서 산화스트레스도 조직 손상에 영향을 주는 것으로 알려져 있다. 골수이식이나 로렌조 기름 등이 치료 방법으로 이용되나 치료의 위험성과 효과에서 한계를 보이는 실정이다. 이에 저자들은 X-ALD 환자에게 채취한 섬유모세포를 이용하여, X-ALD의 치료 가능성을 알아보기 위하여 VPA의 효과를 연구해보고자 하였다. 방법: X-ALD 환자의 피부에서 채취한 섬유모세포와 정상인의 피부에서 채취한 섬유모세포를 배양하였다. 배양된 섬유모세포에 VPA를 처리한 후 RNA발현 정도를 통해 ABCD2 발현을 확인하고 유동세포계측법으로 활성산소종을 측정하였다. 결과: VPA을 처리한 후 정상과 X-ALD 섬유모세포 모두에서 ABCD2의 mRNA 발현이 증가하였다. 특히 X-ALD 섬유모세포에서 ABCD2 유전자 mRNA 발현이 2.22배로 정상의 1.76배보다 더 증가하였다. 유동세포계측법으로 활성산소종을 확인한 결과 대조군에서 13.7, VPA를 처리한 군에서는 각각 0.25 mM에서 8.67, 0.5 mM에서 9.37, 1 mM에서 5.83을 나타내었다. 결론: X-ALD 환자에서 VPA의 항산화능을 이용하여 신경손상을 막을 수 있는 가능성이 있을 것으로 보이며, 이를 실제 환자에 적용하는 연구가 필요할 것이다.

• Allergy, Asthma & Immunology Research
• /
• 제10권6호
• /
• pp.698-715
• /
• 2018

Purpose: Hrd1 has recently emerged as a critical regulator of B-cells in autoimmune diseases. However, its role in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) remains largely unexplored. This study aimed to examine Hrd1 expression and B-cell accumulation and their possible roles in CRSwNP. Methods: Quantitative real-time polymerase chain reaction, immunohistochemistry, enzyme-linked immunosorbent assay and Western blotting were used to assess gene and protein expression in nasal tissue extracts. Cells isolated from nasal tissues and peripheral blood mononuclear cells were characterized by flow cytometry. Local antibody production was measured in tissue extracts with a Bio-Plex assay. Additionally, changes in Hrd1 expression in response to specific inflammatory stimuli were measured in cultured dispersed polyp cells. Results: Nasal polyps (NPs) from patients with eosinophilic CRSwNP (ECRS) had increased levels of Hrd1, B-cells and plasma cells compared with NPs from patients with non-eosinophilic CRSwNP (non-ECRS) or other control subjects (P < 0.05). The average Hrd1 levels in B-cells in NPs from ECRS patients were significantly higher than those from non-ECRS patients and control subjects (P < 0.05). NPs also contained significantly increased levels of several antibody isotypes compared with normal controls (P < 0.05). Interestingly, Hrd1 expression in cultured polyp cells from ECRS patients, but not non-ECRS patients, was significantly increased by interleukin-$1{\beta}$, lipopolysaccharide and Poly(I:C) stimulation, and inhibited by dexamethasone treatment (P < 0.05). Conclusions: Differential Hrd1 expression and B-cell accumulation between the ECRS and non-ECRS subsets suggests that they can exhibit distinct pathogenic mechanisms and play important roles in NP.