1R&D동향.PDF

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1 30 / 2001 [R&D ] 1. 75%. 20, 20.,., (biological response modifier, BRM). (immune system),. screening,,, in vivo. mechanism, BRM,.

2 R &D ) (pathogenic bacteria, virus ), (nonself) (transformed self cells, malignant cells) (immune system) (homeostasis).,,, (innate immunity, nonspecific immunity) (adaptive immunity, nonspecific immunity) ( 1 ). macrophage, natural killer cell (NK-cell) complement, interferon, tumor necrosis factor (TNF- ), T-lymphocyte B-lymphocyte. cytokine (cytokine network),. 2) (Compleme nt syste m activation) (complement system) C1-C9 9 20,,. classical pathway alternative pathway lectin pathway ( 2 ), classical pathway IgM, IgG (immune complex) C1qrs, C4, C2 C3. alternative pathway activator C3b B, D factor 1

3 32 / 2001 lectin pathway mannose binding lectin(mbl), MBL associated serine protease C4 C2 C3. pathway C3b C5b C6 - C9 membrane attack complex (MAC) lipid bilayer ( 2. MAC ). macrophage lymphocyte,. Mayer. pathway (inhibition of total complement hemolysis 50%, ITCH50), (inhibition of alternative complement hemolysis 50%, IACH50) lectin (inhibition of lectin complement hemolysis 50%, ILCH50). NHS, NHS, IgM (EA cell) hemoglobin. Mayer, EA cell. Lectin EA cell mannan MBL label cell Ca++.,,,,, pectic polysaccharide Yamada (Angelica acutiloba). 4 pectin(ar-2iia, IIb, IIc, 2IId) 4 pectin 90% - (1 4) galacturonan Rha C-4 arabinan, galactan arabinogalactan rhamnogalacturonan (ramified region, RG) 2 mechanis m membrane attack complex

4 R &D 33. pectin endo-pgase RG (PG-I),, PG- 1 RG. 3) (Macrophage activation) ( 3 ). IgG Fc, complement, IFN, TNF bacteria receptor,. lysosomal enzyme, reactive oxygen intermediates (ROI, superoxide anion, H2O2 ) lactoferrin, lysozyme, TNF-, NO, ROI. T-cell cytokine(il- 1, IL-6, IL-8, IL- 12 ). ( 3). ( 1), mouse macrophage J774.4, Raw264.7, U C-labelled bacteria method, Candidacidal method, FITC-zymosan method, FITC-E. coli method, , 4. FITC-E. coli method FITC labelled E. coli. Macrophage lysosomal enzyme PNP-phosphate Phagocytosis : Attachment via scarvenger R, IgG FcR and CR Intracellular and extracellular killing - Lysosomal enzyme production - NO production - Reactive oxygen intermediate - Lysozyme, lactoferrin - Cytokines - TNF- Antigen processing and presentation for specific immune response 3

5 34 / Macrophage stimulation Phagocytosis Lysosomal enzyme production NO production Reactive oxygen intermediates production TNF- production IL-1 production Cytotoxicity Methods Macrophages Taget cells/ reagents Measure ment 14 C-labelled bacteria method Candidacidal method FITC-zymosan method FITC-E. coli method Acid-phosphatase method Preparation of macrophage monolayer 14 C-labelled bacteria Candida parapsilosis FITC labelled zymosan FITC labelled E. coli (S. aureus) PNP-phosphate Liquid scintillation counter Viable cell counting (agar plating) Fluorescence microplate reader (FMR) Ex 490, Em 530 FMR (Ex 490, Em 530) Microplate reader (MR) ( 405 nm) Griess method Griess reagent, IFN MR ( 540 nm) : Peritoneal Phenol red, glucose, PMA Phenol red method MR ( 610 nm) horseradish peroxidase macrophages Scopoletin, PMA, glucose, Scopoletin method FMR (Ex 365, Em 450) horseradish peroxidase : Macrophage NBT method cell lines NBT, PMA MR ( 572 nm) Chemiluminescence method - J774.4 Luminol FMR (Ex 365, Em 400) - Raw264.7 L927 fibrosarcoma, crystal Bioassay method - U937 MR ( 490 nm) violet - U-M Anti-TNF, ELISA method (sandwich) biotin-anti-tnf MR ( 405 nm) AP-streptoavidin,PNPP Bioassay method C3H/HeJ thymus cells, [ 3 H] thymidine Liquid scintillation counter ELISA method (sandwich) Anti-IL-1, biotin-anti-il-1 MR ( 405 nm) 5 1 Cr release method 5 1 Cr labelled FBL-3 -Counter MTT method MCF-7, MTT MR ( 540 nm) acid-phosphatase. Nitric oxide Griess, IFN NO,. Superoxide ROI,. TNF-, IL- 1 cytokine cytokine cell line bioassay cytokine antibody ELISA. monoclonal antiboby ELISA.. casein peptide

6 R &D 35 peptide, glycinin (11S) globulin conglycinin (7S) globulin soymetide (MITLAIPVNKPGR).. - 1,3- glucan macrophage, (Lentinus edodes) - glucan lentinan. Lentinan in vitro macrophage, TNF g/ml macrophage, 2.5 mg/kg CBA mouse (antibody- dependent macrophage mediated cytotoxicity, ADMC) mg lentinan, 70% IL- 1 50%. 4) (Natura l killer ce ll activation) NK-cell virus lymphocyte perforin ( 4 ). NK-cell sugar-lectin,, IL-2 IFN receptor NK-cell IL- 2 IFN lymphokine activated killer (LAK) cell (tranformed cell, ). killer cell (K-cell) IgG Fc receptor,. NK-cell, LAK-cell K-cell ( 5) 51Cr release method. NK- 4

7 36 / cell mouse ficoll paque lymphocyte, 51Cr-labelled YAC- 1 cell line., -counter lactate dehydrogenase (LDH). LAKell NK-cell LAK-cell NK IFN IL-2 IL- 2. K-cell IgG 51Crabelled P815 cell line anti-p815 serum NK-cell.. (Viscum album) NK- cell. rhamnogalacturonan NK-cell bridge. 5) (T a nd B- lymphocyte activation) T B-lymphocyte. (macrophage, B-cell) T-cell (interleukines) T cell B-cell (plasma cell). T-cell B-celll memory cell,., cytokine, mediator

8 R &D 37. ( 2) lymphocyte T-cell B-cell. lymphocyte mitogenic activity. DNA [3H] thymidine incorporation mitochondrial MTT[3- (4,5-dimethyl- 2-thiazol- 2-yl) -2,5,-diphenyl-tetrazolium bromide] formazan MTT assay. MTT assay formazan Alamar Blue method. lymphocyte cytokine ELISA, anti-lymphocyte surface maker flow cytometry lymphocyte. T-cell 2 T and B- cell activation T-cell activation B-cell activation Lymphocyte activation Mitogenic activity Cytokine network Individual cell population Proliferation activity Cytotoxic T-lymphocyte activity Antibody(Ig) production enhancing activity Methods Effector cells Reagents Measurement [ 3 H] thymidine incorporation method MTT assay Alamar Blue assay ELISA method (sandwich) Flow cytometer [ 3 H] thymidine incorporation method LDH method Rossette forming cell assay Plaque forming cell assay Hybridoma method Human lymphocyte method Splenic exudate cells Splenic T-cell from immunized mouse Splenic exudate cells Splenic cells from immunized mouse Splenic cells from immunized mouse IgM-hybridoma HB4C5 Peropheral blood lymphocyte prep. [ 3 H] thymidine MTT Alamar Blue Anti-IL, biotin-anti-il, streptoavidin-ap, PNPP Fluorochrome labelled anti-lymphocyte surface maker Immunogen: s1-case in, [ 3 H] thymidine Immunogen and target cell : P815, LDH kit Immunogen : sheep RBC, Immunogen : sheep RBC, complement, Cunningham chamber Human IgMELISA kit Human IgMand IgG ELISA kit Liquid scintillation counter MR ( 540 nm) FMR Ex544, Em590 MR ( 405 nm) Flow cytometer Liquid scintillation counter MR ( 610 nm) Microscopic counting Microscopic counting MR ( 405 nm) MR ( 405 nm)

9 38 / 2001 s1-casein mouse T-cell T-cell. B-cell (RBC) anti-rbc immunoglobulin rossette (rossette forming cell assay) (plaque forming cell assay), human IgM-hybridoma, HB4C5 cell line anti-igm ELISA kit hybridoma. mitogenic activity.,. B-cell human-ig hybridoma Yamada, lactoferrin, casein, lipoprotein (YLP) royal jelly. assay, hybridoma lymphocyte, YLP. YLP royal jelly, 10 synergy. 6) (Intestinal immune syste m activation) 1/3 barrier. GALT(gut associated lymphoid tissue) IgA. GALT Peyer's patch IgA inductive site, lumen dome specialized M cell flattered epithelium. M cell lumen, pinocytosis phagocytosis engulfment., Peyer's patch M cell, germinal center, MLN (Mesenteric Lymph Node, ) Peyer's patch. siga,

10 R &D 39 Peyer's patch cell ( 6 ). antisiga ELISA. C3H/HeJ (5-7, ) Peyer's patch, (,, macrophage ), Alamar Blue T M,.,, (Atractylodes lancea DC)., ALR- 5IIa- 1-1 arabino-3,6- galactan, ALR-5IIc-3-1 pectic polysaccharide rhamnogalacturonan II (RG-II). pectic polysaccharide flavone glycoside methanol. 7) (Anti- metastas is activity),. 50%. 1 6

11 40 / 2001 basement membrane, 2 colony. galectin ( 7 ). galctosebinding protein D- galactose oligosaccharide. Platt Raz B16-F1 melanoma ( 7 ) citrus pectin(cp) pectin alkali modified citrus pectin(mcp) B16-F1 melanoma lung colonization 90%. MCP rat adenocarcinoma MAT-LyLu colony. MCP,,. MCP galactose-rich ramified region(rg-i) galectin , (BRM). 7 mecha nis m

12 R &D 41. mechanism..,,.,., group.,,,,,.,. BT. group,.

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