갑상선암 환자의 말초혈액 백혈구로부터 수지상세포의 유도분화

이대희 KOSIN UNIVERSITY COLLEGE OF MEDICINE 2006 高神大學校 醫學部 論文集 Vol.21 No.1

Background : Recent studies suggest that immunization with autologous dendritic cells(DCs) result in protective immunity and rejection of established tumors in various human malignancies, It has been reported that a dense infiltration of dendritic cells correlates with a favorable prognosis in several types of cancer. The purpose of this study is to determine whether DCs are generated from peripheral blood monocytes by using cytokines such as Flt-3 ligand, granulocyte macrophage-colony stimulating factor (GM-CSF), IL-4 and TNF-α , and whether cytotoxic T cells activated against the medullary thyroid carcinoma(MTC) tissues by the DCs. Methods : Peripheral blood was obtained from 2 patients with MTC. DCs were established from mononuclear leukocytes by culturing in the presence of Flt-3 ligand, GM-CSF, IL-4, and TNF-α for 14 days. At day 14, the differentiated DCs was analyzed morphologically. The immunophenotypic features of DCs such as CDIa, CD83, and CD86 were analyzed by Immunofluorelescence microscopy. At day 15, DCs were incubated with thyroid cancer tissues and normal thyroid tissues for 7 additional days, respectively. Results : DCs were generated from the peripheral blood mononuclear cells. The generated cells showed the typical morphology of DCs. Activated cytotoxic T lymphocytes (CTLs) were observed. DCs attached to the thyroid cancer tissues and the CTLs were attached to the MTC tissues on scanning electron microscope. Conclusion : We could differentiate DCs from the peripheral blood mononuclear cells. And the DCs activate the CTLs which able to attack the MTC tissues. These results suggest that DCs can be used as adjuvants for immunotherapy of MTC. And this study represent the basis for the develop of new therapeutic strategies not only in MTC but also in the other malignancies.

Human 92-kD type Ⅳ Collagenase (Gelatinase B)의 분리와 특성

이대희 KOSIN UNIVERSITY COLLEGE OF MEDICINE 2006 高神大學校 醫學部 論文集 Vol.21 No.1

Background : Human 92-kD type Ⅳ collagenase (gelatinase B), a family of matrix metalloproteinases (MMP), play important roles in the degradation of the basement membrane and the migration of leukocytes and metastatic tumor cells during inflammation and invasion, respectively. Methods : To investigate the biochemical and enzymatic charicteristics of human neurtrophil type Ⅳ collagenase, the enzyme was extracted from human leukocytes and purified by a combination of Ultrogel AcA 54 and Bi0-Rex 70 chromatographies. Results : The purified enzyme showed a single band of molecular weight: 92-kD on the sodium dodecyl sulfate-polyacryl amide gel electrophoresis (SDS-PAGE). Human neutrophil type Ⅳcollagenase degraded gelatin at the specific cleavage site, but did not affect intact typeⅠcollagen. Human 92-kD type Ⅳ collagenase was inhibited by EGTA, EDTA and tetracycline. These inhibitory effects may be related to the chelation effect of these agents since 92-kD type Ⅳ collagenase is a metalloenzyme. Conclusion : Tetracycline showed the strongest inhibition effect on the gelatinolytic activity of the 92-kD type Ⅳ collagenase, and this strong effect of tetracycline among these agents might be associated with the amide group of tetracycline, besides the chelation effect of tetracycline. The carbonyl group of the amide group may be coordinated to the active site zinc atom as a fourth ligand in a way that blocks the active site.

HL-60 세포주에서 myeloblastin mRNA 발현의 하향조절

이대희 고신대학교 의학부 2005 高神大學校 醫學部 論文集 Vol.20 No.1

Background: Recent clinical studies have shown that a high proportion of patients with acute promyelocytic leukemia(APL) achieves complete remission after treatment with all-trans retinoic acid (ATRA). However, most patients who receive continuous treatment with ATRA relapse and develop ATRA-resistant leukmia. In this study, the author investigated the strategies to overcome ATRA resistance of acute promyelocytic leukemia (APL) cells by inducing the differentiation of dendritic cells (DCs) from human leukemic cell lines for the developtment of adoptive immunotherapy. Myeloblastin (mbn) was used as one of the indicators of differentiation in this study. Methods: To study the biochemical and enzymatic charicteristics of the human myeloblastin the enzyme was extracted from human leukocytes and purified by a combination of Ultrogel AcA 54 and Bio-Rex 70 chromatographies. To evaluate the mbn protein expression in cells, anti-mbn antibody was prepared by two-step procedure including ammonium sulfate precipitation and DEAE-cellulose ion exchange chromatography. HL-60 cell differentiation was induced by the addition of all-trans retinoic acid (ATRA), dimethyl sulfoxide (DMSO), phorbol 12-myristate 13-acetate (PMA), cholecalcitriol (VD) to the media for 6 days and the expression of mbn mRNA and mbn protein were determined by RT-PCR method and ELISA, respectively. HL-60 cells, K-562 cells, NC-37 and RPMI 7666 cells were cultured in RMPI 1640 supplemented with 10% fetal calf serum for 7 days, with various agents or ligands such as calcium ionophore (CI), Flt3-ligand (FL) and PMA to generate dendritic cells from the cell lines. A portion of ezch cell lines was havested and the rest of them was cultured in the new RPMI 1640 supplemented with 10% fetal calf serum, with Flt3-ligand for 7 days more. RNA was extracted and gene expression from each cell lines was determined by RT-PCR method. The morphology of the cells was evaluated from cytospin slide preparations with Wright's stain. Result: 3.8 mg of proteinase-3 was isolated from 67 ㎎ of leukocyte extract. 6g of anti-mbn polyclonal antibody was raised. PMA induced a significant inhibition of mbn mRNA expression in HL-60 cells. The cells exposed to ATRA or DMSO or VD show a little change in the mbn mRNA expression. Thus the terminal differention of HL-60 cells and K-562 cells by ATRA, DMSO, VD, and hemin was incomplete and a large fraction of the cells was in undifferentiated or premature states. The promyelocytic leukemic cell line HL-60, B lymphoblast cell lines RPMI 7666 and NC-37 could be induced to dendritic cells in vitro. Treatment of HL-60 with PMA resulted in the expression of myeloid-related DC phenotypes, while treatment of RMPI 7666 with FL and treatment of NC-37 with PMA and FL lead to the expression of lymphoid-related DC phenotypes. Conclusion: In conclusion, myeloid-related DC phenotypes and lymphoid-related DC phenotypes can be generated from HL-60, NC-37 RPMI 7666 cell lines, respectively. These DC phenotypes can potentially be used as a cellular leukemia vaccine in vivo or to generate antileukemic T cells in vitro for adoptive immunotherapy.

Tetracyclines와 단백질분해효소 억제제에 의한 Human 92-kD type IV Collagenase (Gelatinase B)의 활성도 억제

이대희 고신대학교의과대학 2008 고신대학교 의과대학 학술지 Vol.23 No.2

Background : Human 92-kD type IV collagenase(gelatinase B), a family of matrix metalloproteinases(MMP), is involved in the remodeling of connective tissue as well as in disease states associated with acute and chronic inflammation or tumoral metastatic processes. Methods : To investigate the pharmacological and enzymatic characteristics of human neutrophil type IV collagenase, the enzyme was extracted from human neutrophils and purified by a combination of Ultrogel AcA 54 gel filtration and Bio-Rex 70 ion exchange chromatographies. Results : The Purified enzyme showed a single band of molecular weight: 92-kD on the sodium dodecyl sulfate-polyacryl amide gel electrophoresis(SDS-PAGE). Human neutrophil type IV collagenase degraded gelatin at the specific cleavage site, but did not affect intact type I collagen. Human 92-kD type IV collagenase was activated by aminophenylmercuric acetate and molecular conformation was changed at the disulfide bond finger site. Conclusion : Human 92-kD type IV collagenase was inhibited by tetracycline, de-dimethylaminotetracycline and pepstatin A, an inhibitor of aspartic proteinase, E-64, an inhibitor of cysteine proteinase and leupeptin, an inhibitor of cystein and serine proteinase had no inhibitory effect on human 92-kD type IV collagenase activity.

海成粘土와 다짐粘土의 透水係數 豫測에 관한 硏究

임희대,이상헌 忠南大學校 産業技術硏究所 1997 산업기술연구논문집 Vol.12 No.2

It is very important for geotechnical engineers to understand properties of coefficient of permeability. The Kozeny-Carman equation is generally used at the present for estimating the coefficient of permeability. Even though the equation proposed by Kozeny-Carman gives fairly good results in estimating of sandy soils. But it is not appropriate for fine-grained soils such as clay. In the case of clayey soils, structure and distribution of particles play and important role in the coefficient of permeability. These structure and distribution are also closely related to specific surface. Large specific surface means that the grain size is very small. Soils possessing large amount of fine grains have a long flow of water. The study comes to the conclusion that the mostly related factor between specific surface and permeability appears to be #200 passing percentage in the case of compacted clays and clay content for marine clay. So new equation is proposed with the two factor in this study. But the developed equation can be used for estimating rough values in the field. That is, it is skeptical to calculate an accurate coefficient of permeability of soils. The other equations proposed until now were considered. But the results of the equations developed by plasticity index or effective grain size are far from the actual value.

중앙심벽형댐의 응력전이에 관한 연구

임희대,임찬수,이성용 忠南大學校 産業技術硏究所 1995 산업기술연구논문집 Vol.10 No.2

Various modes of load transfer may exist in rockfill dams. If the core is softer than the shells, load is transferred from the core onto the shells as a result of greater downward displacement of the core with respect to the shells. But if the core is stiffer than the shells, load transfer occurs from the shells onto the core. In this study, the mechanisms of load transfer in rockfill dams have been outlined and load transfer of Juam main and regulation dam with central core are evaluated by load transfer ratio. The load transfer ratio is defined as the ratio of computed values of major principal stresses in the core to the core overburden stresses. The vertical stresses are determined by the FEM, which use hyperbolic model and incremental method to simulate placement of successive layers of fill. The hyperbolic paramaters for the core was estimated from a series of conventional triaxial tests carried out during construction. From the results of this analyses and its comparison with the values measured by soil pressure meters, it has been shown that significant load transfer from the core to the shells was observed in two dams and that the load transfer ratios predicted are higher than the values measured.

국내 해안 연약지반의 점토광물 분포 및 공학적 특성

임희대,이상규 忠南大學校 産業技術硏究所 2002 산업기술연구논문집 Vol.17 No.2

These studies were carried out to investigate the characteristics of alluvial clay deposits distributed in the main coastal areas. Also we can use their outcomes which were analyzed from the soil test results surveyed during the latest for the purpose of design, construction and maintenance of civil engineering structures to be built in these areas in the future. Samples of clay minerals were analyzed using an X-ray diffraction test. Also, the geotechnical properties of the distribution of clay minerals were evaluated after collection physical and mechanical soil data. The results of Siroquant analysis though X-ray diffraction analysis show that natural water content, liquid limit., initial void ratio, compression index, and compression ratio are positively proportional th the amount of montmorillonite, whereas they are inversely proportional to those of illite and kaolinite. In addition, dry unit weight, unconfined compressive strength, and cohesion are inversely proportional to the amount of montmorillonite, and positively proportional to the amount of illite and kaolinite. In particular, the amount of montmorillonite is closely related to geotechnical characteirstics. However, it is considered that the relative composition ratio, physical and chemical characteristics of other clay minerals have influence on marine soil characteristics with montmorillonite, since the amount of montmorillonite is less than other clay minerals.

無償增資 情報에 대한 株價의 反應硏究

兪大根,李熙敦 又石大學校 1988 論文集 Vol.10 No.-

급성 골수성 백혈병 환자의 말초혈액 단핵구로부터 수지상세포로의 분화 유도

손상희,이대희,박재선,어완규 고신대학교 의학부 2002 高神大學校 醫學部 論文集 Vol.17 No.1

Background Dendritic Cells (DCs) are the most potent naturally occuring antigen presenting cells and play an important role in T-cell activation. DCs may be suited for in vivo immunotherapy for its capability to stimulate naive T cell. Effective presentation of tumor antigens is fundamental to strategies aimed at enrolling the immune system in eradiation of residual disease after conventional treatments. Myeloid malignancies provide a unique opportunity to derive dendritic cells (DCs), functioning antigen presenting cells, from the malignant cells themselves. These may then co-express leukemic antigens together with appropriate secondary signals and be used to generate a specific, antileukemic immune response. Methods In this study, peripheral blood mononuclear cells (PBMCs) from 5 patients with acute myeloid leukemia (AML) were cultured with combinations of Flt-3 Ligand (FL), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4(IL-4), and tumor necrosis factor-α(TNF-α), and development to DCs was assessed. Peripheral blood mononuclear cells (PBMCs) were obtained from 40-60㎖ of peripheral blood of patients with acute myeloid leukemia (AML) by Ficolling. Cells were resuspended in X VIVO-20 medium supplemented with FL (100ng/㎖), GM-CSF(100ng/㎖), IL-4(50ng/㎖), and TNF-α(20ng-㎖) and seeded into T75 culture flasks at 3×107/50㎖. Results After 12 days in culture, cells from 5 samples exhibited morphological and immunophenotypic features of DCs, including expression of CDla, CD83, and CD86. Conclusion This study indicates that cells with enhanced antigen-presenting ability can be generated from PBMCs of patients with acute myeloid leukemia, that these cells can effectively prime autologous cytotoxic T cells in vitro, and that they may be used as potential vaccines in the immunotherapy of AML.

위암 환자의 말초혈액 조혈모세포로부터 수지상세포로의 분화 유도

최지연,이대희,정현기,조성래 고신대학교 의학부 2002 高神大學校 醫學部 論文集 Vol.17 No.1

Background Throughout the body. Dendritic Cells (DCs) capture the antigens, migrate to draining lymphoid organs and mature to present the processed antigens to naive T cells to generate the effector cytotoxic T cells (CTLs) or helper T cells. The granulocyte-macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor-α(TNF-α) cooperate in vitro generation of DCs from Periperal Blood Stem Cells(PBSCs). Interleukin-4 (IL-4) is an another important factor in promoting DCs outgrowth and expression of CD1a with costimulatory molecules and in blocking monocytic differentiation. The aim of this study was induction of differentiation and maturation of dendritic cells from PBSCs in patient with stomach cancer. Methods The CD34+ PBSCs were obtained from the patients with stomach cancer and divided the samples into 6 groups. Flt-3 Ligand (FL) (F group), GM-CSF (G group), IL-4 (I group), TNF-α(T group), GM-CSF and IL-4(GI group), FL, GM-CSF, IL-4 and TNF-α(FGIT group) were added to X-VIVO culturing media. The cells were cultured for 2 weeks, then. examined the morphology of and functions the cells with phase contrast and fluorescence microscopes. Results The generated cells with FL, GM-CSF, IL-4 and TNF-α showed typical morphology of DCs including multiple dendrites and profuse cytoplasm. The cells stained positively with CD1a, CD83 and CD86, FL, GM-CSF, IL-4 and TNF-α. Conclusion Large quantities of mature DCs from PBSCs in a patient with stomach cancer was possible using FL, GM-CSF, IL-4 and TNF-α. Further studies are needed with various types of diseases and culture environments.