• 한국발생생물학회지:발생과생식
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• 제15권4호
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• pp.281-289
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• 2011

돼지 중간엽 줄기세포를 Dimethyl sulfoxide(DMSO), Ethylene glycol(EG), 그리고 DMSO/EG을 이용하여 세포동결을 유도한 후 적절한 동결보호제를 알아보았다. 2개월 이내 돼지 골수에서 중간엽 줄기세포를 분리하여 colony 형성 및 alkaline phosphatase(AP) 활성을 확인하고, 지방 세포로의 분화 유도에 의한 줄기세포의 능력을 확인하였다. 이들 중간엽 줄기세포의 완만 동결을 위해, DMEM에 각각 10% DMSO, 1.5M EG, 5% DMSO/0.75M EG의 동결보호제를 섞은 후 cryovial에 넣고, cryo-containe를 이용하여 $25^{\circ}C$에서 $-80^{\circ}C$까지 $-1^{\circ}C$/min 속도로 동결하였다. 일주일간 저장 후 세포의 생존률은 미동결 세포는 동결 세포군보다 유의적으로 높음을 확인할 수 있었으나, 동결 처리군 간에는 차이가 없었다. 줄기세포 유지 유전자인 Sox-2와 Nanog 발현은 동결 후 배양 시간에 따라 발현량이 증가하는 경향을 보였으나, 동결처리군 간에는 유의적인 차이가 없었다. 세포사 관련 유전자인 Bax의 발현은 모든 군에서 비슷하였다. 또한 지방, 연골 및 뼈세포 분화와 관련된 유전자의 발현은 동결 전 세포와 동결 후 세포군에서 비슷한 경향을 보였다. 이러한 결과는 돼지중간엽 줄기세포 동결함에 있어서 10% DMSO, 1.5MEG, 5% DMSO/0.75M EG 모두 적절한 동결보호제로 이용할 수 있음을 시사한다.

• Reproductive and Developmental Biology
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• 제35권1호
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• pp.9-15
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• 2011

The functional cardiovascular system is comprised of distinct mesoderm-derived lineages including endothelial cells, vascular smooth muscle cells and other mesenchymal cells. Recent studies in the human embryonic stem cell differentiation model have provided evidence indicating that these cell lineages are developed from the common progenitors such as hemangioblasts and cardiovascular progenitor cells. Also, the studies have suggested that these progenitors have a common primordial progenitor, which expresses KDR (human Flk-1, also known as VEGFR2, CD309). We demonstrate here that sustained activation of BMP4 (bone morphogenetic protein 4) in hESC line, CHA15 hESC results in $KDR^+$ mesoderm specific differentiation. To determine whether the $KDR^+$ population derived from hESCs enhances potential to differentiate along multipotential mesodermal lineages than undifferentiated hESCs, we analyzed the development of the mesodermal cell types in human embryonic stem cell differentiation cultures. In embryoid body (EB) differentiation culture conditions, we identified an increased expression of $KDR^+$ population from BMP4-stimulated hESC-derived EBs. After induction with additional growth factors, the $KDR^+$ population sorted from hESCs-derived EBs displays mesenchymal, endothelial and vascular smooth muscle potential in matrix-coated monolayer culture systems. The populations plated in monolayer cultures expressed increased levels of related markers and exhibit a stable/homologous phenotype in culture terms. In conclusion, we demonstrate that the $KDR^+$ population is stably isolated from CHA15 hESC-derived EBs using BMP4 and growth factors, and sorted $KDR^+$ population can be utilized to generate multipotential mesodermal progenitors in vitro, which can be further differentiated into cardiovascular specific cells.

• Applied Microscopy
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• 제23권1호
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• pp.35-55
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• 1993

cis-Dichlorodiammineplatinum (II) (cis-Platin), a metallic compound, has widely been used as an effective anticancer chemotherapeutic agent. The precise mechanism of action of this agent is still unknown, but it is postulated that cis-Platin may act on the cancer cell like bifunctional alkylating agents. Although this agent is very beneficial to the patients with cervical cancer, germinoma of testis, neuroblastoma and others, it may also damage to the normal cell so that many side effects; severe hemorrhagic enterocolitis, bone marrow depression, renal damage and liver damage will develope. This experiment has been undertaken to pursue the cytotoxic effects of the cis-Platin on the ultrastructures of the interalveolar septum in the mouse lung. A total of 55 healthy male mice of ICR strain were used as experimental animals and divided into 5 mice of normal control group and 50 mice of cis-Platin treated group. The mice of cis-Platin treated group were sacrificed by carotid exsanguination at 6, 12, 24 hours, 3 days and 7 days after intraperitoneal injection of 6.0 mg of cis-Platin ($Abiplatin^R$ Abic Co. Ltd.) per kg of mouse body weight. The specimen obtained from the lower lobe of left lung were sliced into $1mm^3$ and prefixed with 2% glutaraldehyde -2.5% paraformaldehyde solution prepared with Millonig's phosphatae buffer solution (pH 7.4) at $4^{\circ}C$ for 3-4 hours. After postfixation with 1% osmium tetroxide solution all specimens were embedded in Epon 812. Ultrathin sections about $600-800{\AA}$ in thickness were stained with uranyl acetate and lead citrate and observed with Hitachi-600 electron microscope. The results obtained were as follows: 1. Local swellings with increase of electron density and number of pinocytic vesicles in the cytoplasms of the type I pneumocyte and endothelial cell of the blood air barrier in interalveolar septum of cis-platin treated mice were observed. 2. Cisternae of rough endoplasmic reticulum were dilated and sacculated in association with detachment of membrane bound ribosomes of the type II pneumocyte in interalveolar septum of cis-Platin treated mice. 3. Swollon mitochondria with uneven electron density of their matrix were observed in the type II pneumocyte of interalveolar septum in the cis-Platin treated mice. 4. The lamellae of lammelar bodies in type II pneumocyte of interalveolar septum in cis-Platin treated mice were devoided or transformed into homogeneous electron dense material. It is consequently suggested that cis-Platin would induce the cellular edema of type I pneumocyte and endothelial cell, and degenerative changes of cytoplasmic organelles of the type II pneumocyte in the interalveolar septum of the mouse lung.

• 대한소아치과학회지
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• 제35권1호
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• pp.30-38
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• 2008

치아 치수 세포는 치아 손상에 따르는 병리적인 상황에서 골과 상아질 기질을 형성하는 능력을 가진 것으로 생각된다. 본 연구에서는 MTA가 사람 치수세포의 성장에 미치는 영향과 상아질 형성에 관여하는 유전자의 발현을 유도하는지를 알아보고자 하였다. 또한 상아질 형성의 잠재적 지표인 alkaline phosphatase(ALP) activity에 미치는 영향을 평가하였다. 유전자 발현 검사를 위해 glyceraldehyde-3-phosphate dehydrogenase, type I collagen, alkaline phosphatase, osteonectin(SPARC), and dentin sialoprotein primer set을 이용하여 MTA 처리 2일과 4일 후 reverse transcriptase polymerase chain reaction(RT-PCR)을 시행하였다. cell viability assay(세포 생존력 측정) 에서 5일간 MTA에 노출된 치수 세포의 비율이 대조군보다 높았다. 대조군에 비해 MTA를 처리한 군에서 ALP와 SPARC가 증가되었다. 이상의 결과를 종합하여 보면, 이 연구에 사용한 dental pulp culture system은 MTA를 포함한 치과재료의 처리 후 치수세포의 성장과 분화 그리고 상아질 형성 유도 기전을 연구하는 데 유용한 모델로 사용할 수 있다. MTA 처리는 사람 치수세포에 세포독성을 유도하지 않으며, ALP 활성도와 유전자 발현 그리고 osteonectin (SPARC) 유전자 발현을 증가시켜 수복상아질을 형성할 것으로 사료된다.

• Journal of Periodontal and Implant Science
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• 제30권2호
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• pp.375-390
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• 2000

The purpose of this study is to evaluate the expression ofIGF-I, considered as the mediator of action of estrogen, and IGF-IA and IGF-IB, alternative slicing form of IGF-I, using $17{\beta}-estradiol$ in MC3T3-E1 cells. We observed the effect on type I collagen and osteopontin gene expression and DNA synthetic activity of MC3T3-E1 cells, added by estrogen, IGF-I and combination and the interactionon proliferation and differentiation of MC3T3-E1 cells. The results were as follows :RT-PCR experiment for observing timedependantIGF-I gene expression patternshowed IGF-IA and IB gene expression in both of control and test group. In these IGF-IA gene expression was appeared predominantly. In control, IGF-I geneexpression level was maintained until 24hr and then decreased gradually. In testgroup, IGF-I gene expression level increased as time goes by. Experiment measuring DNA synthetic activity, as it is added by $17{\beta}-estradiol$, IGF-I and combination, showed that first day , there was the tendency of more increase of synthetic activity in all test group than control but no statical significance(P>0.05), and third day, there was more increase of DNA synthetic activity in $17{\beta}-estradiol$ group and combination group and it was statically significant. (P<0.005) Experiment for observing type I collagen gene expression pattern showed more increase of expression in $17{\beta}-estradiol$ group than control and no significant difference in IGF-I group and combination group. Experiment for observing osteopontin gene expression pattern showed no significant difference in control and test group. In conclusion, $17{\beta}-estradiol$ in MC3T3- E1 cells increased IGF-I gene and DNA synthetic activity simultaneously, therefore it appeared that IGF-I is related to the action of estrogen. Combination treatment of IGF-I and $17{\beta}-estradiol$ has effect on cell proliferation but this effect is lower than IGF-I or $17{\beta}-estradiol$ alone. However, combination treatment has not great effect on type I collagen or osteopontin gene expression thus little effect of cell differentiation.

• 한국발생생물학회지:발생과생식
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• 제18권4호
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• pp.197-212
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• 2014

Osteosarcoma (OS) is one of the most common malignant primary bone tumors and NF-${\kappa}B$ appears to play a causative role, but the mechanisms are poorly understood. OS is one of the pleomorphic, highly metastasized and invasive neoplasm which is capable to generate osteoid, osteoclast and osteoblast matrix. Its high incidence has been reported in adolescent and children. Cell signal cascade is the pivotal functional mechanism acquired during the differentiation, proliferation, growth and survival of the cells in neoplasm including OS. The major limitation to the success of chemotherapy in OS is the development of multidrug resistance (MDR). Answers to all such queries might come from the knock-in experiments in which the combined approach of miRNAs with NF-${\kappa}B$ pathway is put into use. Abnormal miRNAs can modulate several epigenetical switching as a hallmark of number of diseases via different cell signaling. Studies on miRNAs have opened up the new avenues for both the diagnosis and treatment of cancers including OS. Collectively, through the present study an attempt has been made to establish a new systematic approach for the investigation of microRNAs, bio-physiological factors and their target pairs with NF-${\kappa}B$ to ameliorate oncogenesis with the "bridge between miRNAs and NF-${\kappa}B$". The application of NF-${\kappa}B$ inhibitors in combination with miRNAs is expected to result in a more efficient killing of the cancer stem cells and a slower or less likely recurrence of cancer.

• Imaging Science in Dentistry
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• 제38권3호
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• pp.125-132
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• 2008

Purpose : To investigate the effects of irradiation on transforming growth factor ${\beta}_1$ (TGF-${\beta}_1$) mRNA expression and calcific nodule formation in MC3T3-E1 osteoblastic cell line. Materials and Methods : Cells were cultured in alpha-minimum essential medium ($\alpha$-MEM) supplemented with 10% fetal bovine serum and antibiotics. When the cells reached the level of 70-80% confluence, culture media were changed with $\alpha$-MEM supplemented with 10% FBS, 5 mM $\beta$-glycerol phosphate, and $50\;{\mu}g/mL$ ascorbic acid. Thereafter the cells were irradiated with a single dose of 2, 4, 6, 8 Gy at a dose rate of 1.5 Gy/min. The expression pattern of TGF-${\beta}_1$ mRNA, calcium content and calcific nodule formation were examined on day 3, 7, 14, 21, 28, respectively, after the irradiation. Results : The amount of TGF-${\beta}_1$ mRNA expression decreased significantly on day 7 after irradiation of 4, 6, 8 Gy. It also decreased on day 14 after irradiation of 6, 8 Gy. and decreased on day 21 after irradiation of 8 Gy. The amount of calcium deposition decreased significantly on day 7 after irradiation of 4, 8 Gy (P < 0.01) and showed a decreased tendency on day 14, 21 after irradiation of 4, 6, 8 Gy. The number of calcific nodules was decreased on day 7 after irradiation of 4, 8 Gy. Conclusion: Irradiation with a single dose of 4, 6, 8 Gy influences negatively the bone formation at the molecular level by affecting the TGF-${\beta}_1$ mRNA expression that was associated with proliferation and the production of extracellular matrix in MC3T3-E1 osteoblastic cell line.

• 대한골관절종양학회지
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• 제13권2호
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• pp.88-95
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• 2007

목적: wild-type p53 단백질을 가지고 있는 U2OS 인간 골육종 세포주의 세포자멸사에서 MMP억제제와 doxorubicin의 작용을 비교하고자 하였다. 대상 및 방법: 배양한U2OS 세포주에 Doxorubicin, MMP억제제(MMPI III)를 따로 투여한 것과 두 약물을 동시에 투여한 것을 비교하였다. 또한 Doxorubicin의 작용이 Fas/FasL 경로를 통해 유발되는지 알아보기 위해 Fas 중화항체를 doxorubicin과 함께 투여하였다. 약물을 처리하여 배양한 세포에서 발생한 세포자멸사와 세포괴사를 확인하기 위해서 유세포 검사를 시행하였다. 결과: Doxorubicin을 처리한 세포가 처리하지 않은 세포보다 약물 농도에서 더 많은 세포괴사(p=0.000)를 보였다. 이에 반해 MMPI III를 처리한 세포는 약물 농도와 반응시간에 따른 세포자멸사 및 세포괴사에서 차이를 보이지 않았다. 두 약물을 동시에 투여한 군은 그렇지 않은 군에 비해 세포 자멸사 및 세포괴사에서 차이가 없었다. Doxorubicin에 Fas 중화항체를 추가한 군과 doxorubicin 단독 투여 군 사이에 세포자멸사 및 세포괴사에서 차이가 없었다. 결론: Doxorubicin과 같이 MMP 억제제를 골육종 치료에 사용하려면 wild-type p53 뿐만 아니라 wild-type p14를 가지고 있는 골육종 세포에 대한 검사가 필요할 것으로 사료된다.

• Journal of Periodontal and Implant Science
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• 제37권4호
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• pp.655-669
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• 2007

Reactive oxygen species (ROS) have been implicated in the pathogenesis of various diseases. And vitamin C has shown a protective effect for the tissues. The aim of this study was to evaluate the effects of $H_2O_2$ and ascorbic acid on matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase (TIMP: TIMP-1, TIMP-2), Type 1 collagen, fibronectin, and PDLs22 level in human periodontal ligament fibroblasts (hPDLF) via reverse transcription-polymerase chain reaction (RT-PCR). hPDLF was obtained from a healthy periodontium and cultured in Dulbecco's modified Eagles's medium plus 10% fetal bone serum. The concentration of ascorbic acid in hPDLF was $50{\mu}g/ml$, and that of $H_2O_2$ in hPDLF was 0.03% and 0.00003%. Ascorbic acid only, $H_2O_2$ only and mixture of ascorbic acid and $H_2O_2$ were applied with hPDLF for 1-, 3-, and 30-min. respectively. The gene expression of MMP-1-, TIMP-1-, TIMP-2-, Type 1 collagen-, fibronectin-, and PDLs22-mRNA in hPDLF was analysed via RT-PCR. The results were as follows; 1. hPDLF in response to 30-min. incubation with 0.03% $H_2O_2$ did not show any gene expression. 2. In all the experimental groups, the gene expression of fibronectin mRNA showed the decreased tendency compared to control. 3. In all the experimental groups, the gene expression of TIMP-1 mRNA showed the tendency similar to control. 4. hPDLF in response to 30-min. incubation with 0.03% $H_2O_2$ and ascorbic acid increased mRNA induction for MMP-1. 5. In all the experimental groups, hPDLF increased mRNA induction for PDLs22, collagen type 1, and TIMP-2 compared to control. Within the limited experiments, $H_2O_2$ and ascorbic acid increased mRNA induction for PDLs22, collagen type 1, TIMP-2 in hPDLF. More research will be needed in order to confirm the relative importance of the different roles of ROS and antioxidants in hPDLF from a periodontal regeneration or repair standpoint.

• 한국식품영양과학회지
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• 제35권1호
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• pp.28-34
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• 2006

갈근(Puerariae Radix)의 $70\%$ ethanol 추출물 중 ethyl acetate 분획물(EPR)은 염증성 cytokine을 처리한 마우스 대식세포 및 토기 연골조직세포에서 염증의 발현과 관련된 NO 생성 저해효과를 보였고, 관절조직의 주요 성분 중 하나인 proteoglycan의 분해 억제효과와 관절조직 분해효소인 MMP-9의 활성이 억제되었다. 또한, 통증유발물질인 프로스타글란딘의 유의성 있는 감소를 보여 통증억제 효과가 있음을 확인하였을 뿐만 아니라 초산 유발 진통 효과테스트인 동물 모델에서도 효과적으로 통증을 억제함을 확인하였다. 즉, 갈근의 $70\%$ ethanol 추출물 중 ethyl acetate 분획물(EPR)은 독성의 문제뿐만 아니라, 소염, 진통 효과 및 연골 조직세포의 분해를 억제하는 다양한 효과를 나타내는 장점을 지니고 있어 관절염 치료제의 훌륭한 후보약재가 될 것으로 기대된다.