성장인자를 제외 한 배양액에서의 간암세포주의 구체 형성 배양

민선옥(Seon Ok Min),이상우(Sang Woo Lee),박선영(Seon Young Bak),김경식(Kyung Sik Kim) 대한종양외과학회 2015 Korean Journal of Clinical Oncology Vol.11 No.1

Purpose: Cancer stem cells have the characteristics similar to the normal stem cell but are cancer cells. Sphere forming culture has been adapted as a culture method for cancer stem cells. In this study, we validated to optimal culture condition for sphere formation of hepatocellular carcinoma cell line using three different types of culture media. Methods: Huh7 cells were plated in different culture media allowed for sphere formation. M1, Dulbecco’s Modified Eagle’s medium (DMEM)-high glucose with fetal bovine serum (FBS); M2, DMEM-high glucose without FBS; and M3, DMEM-F12 (Gibco, Grand Island, NY, USA) with B27, epidermal growth factor, basic fibroblast growth factor (bFGF, Invitrogen, Seoul, Korea). Sphere formation was observed by light microscope. Proliferation of the sphere-forming cells was evaluated by Cell counting kit-8 (CCK-8) analysis. Results: Cells in M1 and M3 media were formed sphere. Sphere-forming cells in M1 and M3 were bigger than M2 cells and had similar morphology. Sphere-forming cells in M3 media showed higher level of cell proliferation than M1 and M2 cells on 15 days, while sphere-forming cell in M1 media were exhibited higher level than others on post 20 days. Proliferation of M2 cells did not improve. Conclusion: Results showed that sphere-forming cells in M1 and M3 media had similar character. So, it is assumed that a growth factor- free medium is adaptable or efficient tool for in vitro cultivation of cancer stem cells, because the tool shall be reduced expense for experiments without the adding of growth factors.

Human Adipose Tissue-Derived Mesenchymal Stem Cells 배양 시 효율적인 Extracellular Matrix의 증명

민선옥(Seon Ok Min),이상우(Sang Woo Lee),최새별(Sae Byeol Choi),김경식(Kyung Sik Kim) 한국간담췌외과학회 2009 한국간담췌외과학회지 Vol.13 No.4

Purpose: Human mesenchymal stem cells (hMSCs) have the potency for self-renewal and differentiation into various kinds of cells. The hMSCs are obtained from the various tissues, including adipose tissue, bone marrow and cord blood. The extracellular matrix (ECM) is an important factor that affects cell adherence, growth, migration, apoptosis and differentiation both in vitro and vivo. The adipose-derived mesenchymal stem cells (AD-MSCs) have CD29 (integrin) on the cell surface, which is the receptor for fibronectin. The aim of this study is to validate the efficacy of ECM, and especially fibronectin, for cell expansion. Methods: The AD-MSCs were obtained from the abdominal fat of humans. These cells were seeded onto culture plates coated with fibronectin-Human (FN) and plates without ECM (control). The cells were incubated for 3 passages and the cellular morphology was simultaneously observed with microscopy. CCK-8 assay was performed to compare the proliferation ability in each condition at the same passage. Immunocytochemistry staining for integrin-beta1 was performed to observe the cell to cell interaction. Results: The hAD-MSCs in the FN-coated and non-coated plates exhibited cytoplasm staining for integrin-beta1. In all the cultures, extended fibroblastic-shaped cells that turned into rhomboid cells were most frequently observed. The cell growth rates for the non coated culture plate were lower than those for the FN coated plates. After 72 hour culture under the different coated concentrations of FN and the non coated condition (control), the control group had a lower growth rate. In the culture with a FN coated plate, a significant change was observed as compared with that of the control group. We observed an increase in cell proliferation, with a maximum of 140%, on the FN coated plate by performing CCK-8 assay. In comparison, integrin β1 on the cells was more expressed in the FN-coated plates than that in the non-coated plates. Conclusion: The cell morphology can be changed faster in the FN coated culture plates than that in the non coated culture plates. Because proliferation and adhesion with FN can enhance the expansion, the culture within a FN coated plate is needed to encourage hAD-MSCs to proliferate in vitro.

간섬유화가 유도된 백서에서 이식된 간세포 착상과 증식에 관여하는 인자

이상우(Sang Woo Lee),민선옥(Seon Ok Min),최새별(Sae Byeol Choi),김경식(Kyung Sik Kim) 한국간담췌외과학회 2009 한국간담췌외과학회지 Vol.13 No.4

Purpose: Cell therapy for various diseases has gained wide acceptance. Because most patients with chronic liver failure have mild-to-severe liver cirrhosis, there are many limitations to clinical applications. We analyzed how to increase cell engraftment in rats with liver fibrosis. Methods: We used analbuminemic SD rats (NARs) as recipients of syngeneic CAG-EGFP SD hepatocytes obtained by the 2 perfusion method. Hepatic fibrosis was induced with thioacetamide in drinking water for 6 weeks in the recipient NARs. NARs were pre-treated with gadolinium, doxorubicin, and gliotoxin before hepatocyte transplantation. We evaluated the degree of cell engraftment by RT-PCR and immunofluorescent staining for GFP and albumin. The transplanted cells were detected by immunostaining for albumin, and serum albumin was also measured. Results: Although detection of GFP by RT-PCR was variable, albumin was detected in all groups 4 wks after hepatocyte transplantation. GFP and albumin were also detected by immunofluorescent staining 1 and 4 wks after cell transplantation. In control rats, albumin production was maximal at 3 wks, and after that it rapidly decreased. In the gadolinium and doxorubicin-treated group, albumin production was increased up to 4 wks. Albumin production in the gadolinium-treated group was superior to that of the doxorubicin-treated group. Conclusion: Kupffer cells play the most important role in cell engraftment in hepatic fibrosis. Therefore, perturbation of kupffer cells in hepatic fibrosis is needed to increase cell engraftment.

마이크로와이어 기반 nitric oxide 가스 측정 센서의 개발 및 그 유용성에 대한 기초연구

이상우(Sang Woo Lee),민선옥(Seon Ok Min),이경훈(Kyoung Hoon Lee),권대성(Dae-Sung Kwon),임진홍(Jin Hong Lim),남기창(Ki-Chang Nam),김종백(Jongbaeg Kim),김경식(Kyung Sik Kim) 대한종양외과학회 2013 Korean Journal of Clinical Oncology Vol.9 No.2

Purpose: Although nitric oxide (NO) is considered one of the initial signals that promote postsurgery liver regeneration, NO’s exact role in the liver regeneration mechanism is still not clear. Therefore, developing a practical gas sensor and testing it in a clinical setting through basic research will lay the groundwork for advanced clinical research on the action mechanism of NO. Methods: A thin nano wire NO sensor was made by wrapping a pair of parlyene-coated gold wires around a needle. The NO blood concentration determined by measuring the potential difference across the oscilloscope using electrical conductivity. In order to measure changes in NO level before and after the surgery, the NO sensor was inserted in the hepatic portal vein and a 75 percent partial hepatectomy was performed. The NO blood concentration was measured regularly with both the NO kit and the sensor. Results: One significant challenge was separation of the wire upon insertion into the hepatic portal vein. Despite separation, the constant measurements of the wire-type sensor were similar to those measured by the NO measurement kit. Conclusion: The development of a needle-type sensor allowed for easier insertion. In the future, using difference in electric potential, as used in the NO sensor, may be a more effective method of measuring blood ion concentration.

Activin A를 이용한 지방 유래 간엽줄기세포의 내배엽 분화 유도 증진

이상우(Sang Woo Lee),민선옥(Seon Ok Min),김신영(Shin Young Kim),최새별(Sae Byeol Choi),김현옥(Hyun Ok Kim),김경식(Kyung Sik Kim) 한국간담췌외과학회 2009 한국간담췌외과학회지 Vol.13 No.4

Purpose: The most important consideration for therapy using MSCs is the differentiation of the target organ’s cell type. For in-vitro hepatogenic differentiation of MSCs, the main focus is efficient induction of the MSCs into the endoderm stage. Activin A, which is a signaling molecule that is similar to Nodal, promotes the induction of definitive endoderm from both ESs and MSCs. The protocols for induction into definitive endoderm have shown different efficiency and reproducibility depending on the researchers or the sources of the MSCs. Thus, a study on the various conditions of Activin A is needed to efficiently differentiate MSCs into the definitive endoderm lineage of MSCs. Methods: MSCs were isolated from human adipose tissues and these were cultured in MCM (MSCs Culture Medium) on a human fibronectin coated plate. At 70∼80% confluence, the MSCs were harvested and cultured in MCM supplemented with Activin A, at a 50 ng/mL concentration, and FGF4. The expression of the genes related with MSCs or primitive endoderm were analyzed by RT-PCR. The changes of cell morphology for differentiation were also observed by a light microscope & a SEM. Results: The expression of genes related with primitive foregut endoderm was seen in the groups that were treated with a higher concentration of Activin A. The morphology of the cells that differentiated into definitive endoderm were not different from those of the undifferentiated MSCs. The expression of genes related with functional primitive hepatocytes was seen in the early phase during hepatic differentiation. The cell morphology was changed to a similar cuboidal form in a time-dependent manner. Conclusion: Activin A promotes a more rapid induction of definitive endoderm. It also makes an efficient condition for the differentiation into primitive foregut endoderm at a higher concentration.

비영리조직 종사자의 다문화수용성에 영향을 미치는 요인 연구 : 지역아동센터 종사자를 중심으로

이진선(Lee, Jin Seon),민선옥(Min, Sun Ok),김재남(Kim, Jae Nam),강영숙(Kang, Young Sook) 한국비영리학회 2020 한국비영리연구 Vol.19 No.2

다문화사회에 접어들면서 지역아동센터에 다문화가정 이용아동수가 늘어나는 추세이다. 지역아동센터에 종사하는 종사자들의 다문화수용성 제고가 중요하다고 여기며, 지역아동센터 종사자들의 다문화수용성의 수준은 어느 정도인지, 또한 다문화수용성에 영향을 미치는 요인들이 무엇이 있는지 통계를 통하여 살펴보았다. 그 결과 지역아동센터 종사자들의 다문화 수용성은 52.4점으로 나타났으며, 이들의 다문화수용성에 영향을 미치는 변인은 다문화가정아동이 지역아동센터를 이용하는지의 여부와 외국여행경험여부로 나타났다. 연구 결과를 바탕으로 다음과 같은 실천적·정책적 제언을 한다. 첫째, 지역아동센터의 종사자들은 다문화아동들의 지역아동센터 이용을 확대하거나 다양한 프로그램을 만들 필요가 있다. 다문화아동과의 접촉을 통해서 다문화수용성을 높일 필요가 있다. 둘째, 다문화사회와 다문화아동을 이해할 수 있는 다문화사회전문가를 교육하고 양성할 필요가 있다. 훈련받는 다문화사회전문가는 지역아동센터에서 일상생활지도의 부분이나 학습의 부분, 한국어 교육, 학교생활 적응 등과 관련한 지원서비스를 제공하여 다문화가정의 아동들의 학교와 지역사회 적응을 도울 것이다. As we enter a multicultural society, the number of children and multicultural families going to the local children s centers are increasing. It is important to enhance multicultural acceptance of social workers at local children s centers, and we looked at the level of multicultural acceptability of the local children s centers through statistics and what factors affect multicultural acceptability. As a result, the multicultural acceptability of the local children s centers was 52.4 points, and the variables affecting their multicultural acceptability were whether children from multicultural families were used and whether they had foreign travel experience at the local children s centers. Based on the research results, the following practical and policy suggestions are made: First, social workers at the local children s centers need to expand the usage of the local children s centers or create various programs for multicultural children. It is necessary to increase multicultural acceptability through contacts with multicultural children. Second, it is necessary to educate and train multicultural social experts who can understand multicultural societies and multicultural children. The trained multicultural social experts will help the children from multicultural families adapt to schools and communities by providing support services related to parts of daily life guidance or learning, Korean language education, and school life adaptation at local children s centers.

저산소 조건에 의한 HepG2 세포주의 성장에서 HIF-1α 비의존적 기전의 확인

이상우 ( Sang Woo Lee ),정재욱 ( Jae Uk Chong ),민선옥 ( Seon Ok Min ),김경식 ( Kyung Sik Kim ) 대한간암학회 2016 대한간암학회지 Vol.16 No.1

Background/Aims: When hepatocellular carcinoma (HCC) is exposed to hypoxic condition, HIF-1α is activated and results in angiogenesis and increased tumor burden. Although inhibition of HIF-1α may reduce tumor growth, there are some limitations to control tumor growth completely. For a more effective therapy for HCC, we investigated HIF-1α independent pathway related tumor growth with angiogenesis. Methods: We cultured HepG2 cells (HCC cell line) in both normoxia and hypoxia conditions. These cells were divided into three groups: a echinomycin treated group, a echinomycin and quinazoline treated group and a control group without any treatments. Growth morphologies of cells were observed with a microscope after 24 hours. Immunocytochemistry assay was done to detect the angiogenesis during inhibition of HIF-1α and/or NF-κB in hypoxia condition, and compared with results in normoxia condition. Results: In normoxia, the expression of HIF-1α on tumor growth was not found. In hypoxia, inhibition of HIF-1α reduced the tumor growth compared to the control group. But, inhibition of both HIF-1α and NF-κB did not show apparent reduction of tumor growth as shown in HIF- 1α only group. Conclusions: Signaling pathways related to cancer cell growth exist through a vast network. Inhibition of one target molecule may result in over-expression of other molecules related to the tumor growth. For an effective therapy in blocking of the tumor growth, more comprehensive understanding of the network related to signaling pathways on tumor growth is necessary. (J Liver Cancer 2016;16:31-37)