材料與方法

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1 Minimum Essential Medium (MEM) Dulbecco s Modified Eagle Medium (D-MEM) Dulbecco s Modified Eagle Medium Ham s Nutrient Mixture F-12 (D-MEM/F-12, 1:1) B-27 Supplement Minus AO (50 ) N-2 Supplement (100 ) L-Glutamine 200 mm (100 ) MEM Sodium Pyruvate Solution 100 mm (100 ) MEM Non-Essential Amino Acids Solution 10 mm (100 ) Penicillin-Streptomycin Hanks Balanced Salt Solution (HBSS) (fetal bovine sera, FBS) Gibco-BRL (Gaithersburg, MD, USA) (6-,12-,24-well culture plates) (2 ml-, 5 ml-, 10 ml-pipettes) (15 ml-, 50 ml centrifuge tubes) (33 mm-bottle filters) Nalge Nunc International (Rochester, NY, USA) (immunocytochemical staining) neuron specific enolase (NSE) microtubule associated protein-2 (MAP-2) ED1 glial fibrillary acidic protein (GFAP) antibody (antibody diluent) (DAKO LSAB 2 Kit, Peroxidase) DAKO (Carpinteria, CA, USA) OX-42 antibody Serotec Inc. (Raleigh, NC, USA) (3-nitrotyrosine, 3-NT) Upstate Biotechnology, Inc. (Lake Placid, NY, USA) horse anti-mouse and goat anti-rabbit biotinylated IgG (H+L) VECTASTAIN Elite ABC (VECTASHIELD Mounting Medium) (normal goat and horse serum) Vector Laboratories (Burlingame, CA, USA) terminal deoxy-nucleotidyl transferease-mediated dutp nick end labeling (TUNEL) Roche Molecular Biochemicals (Indianapoplis, IN, USA) Annexin-V-FITC Trevigen (Gaithersburg, MD, USA) (western blotting) p38 MAP Kinase 25

2 antibody phospho-p38 MAP Kinase antibody Cell Signaling Technology (Beverly, MA, USA) Caspase-3 antibody Santa Cruz Biotechnology (Santa Cruz, CA, USA) Peroxidase-conjugated AffiniPure IgG (H+L) antibody Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA) (Dye reagent) Bio-Rad Laboratories (Hercules, CA, USA) poly-vinylidene difluoride (PVDF) NEN Life Science Products, Inc. (Boston, MA, USA) Chemiluminescence Reagent Plus PerkinElmer Life Sciences (Boston, MA, USA) (Restore Western Blot Stripping Buffer) (GelCode Blue Stain Reagent) Pierce Biotechnology (Rockford, IL, USA) (Biomax light film) Eastman Kodak Company (Rochester, NY, USA) MAPK SB PD98059 caspase Z-Val-Ala-Asp-CH 2 F (Z-VAD-FMK) Z-Asp(OCH 3 )-Glu(OCH 3 )-Val-Asp (OCH 3 )-FMK (Z-DEVD-FMK) Calbiochem (La Jolla, CA, USA) Dihydrorhodamine-1, 2, 3 (DHR-123) 2,7 -dichlorofluorescin diacetate (DCFH-DA) Molecular Probes (Eugene, OR, USA) Rat TNF-α enzyme-linked immunosorbent assay (ELISA) Genzyme Diagnostics (Cambridge, MA, USA) R&D Systems, Inc. (Minneapolis, MN, USA) Sigma (St Louis, MO, Sprague-Dawley (SD) Fisher ~17 ( 225~325 ) ( 250~350 ) ( 200~300 ) 12 (AM 8:00~PM 8:00) (25±1 ) 26

3 1. (Wang et al., 2000; 2002) 16 ~ 17 SD Ca 2+ /Mg 2+ -free HBSS (ph 7.4) (cerebral cortex) 2 Ca 2+ /Mg 2+ -free HBSS 10 % FBS D-MEM (pipet-aid) 20 5 (500 g, 10 min) 10 % FBS B27 Supplement D-MEM (10 % FBS + D-MEM/B27) pipet-aid (cell aggregate) (hemacytometer) cells/ml poly-l-lysine (0.1 mg/ml) 24 (24-well culture plate) cells/500 µl/well 37 5 % (5%CO2/95%Air) (incubator) 3 5 µm β-cytosine arabinoside (ARA-C µm ARA-C 10 % FBS +D-MEM/B27 4~5 9~10 27

4 N2 Supplement ( ) MEM 2. (Wang et al., 1998; 2000) 24 SD Ca 2+ /Mg 2+ -free HBSS (ph 7.4) % trypsin-ca 2+ /Mg 2+ -free HBSS % trypsin Ca 2+ /Mg 2+ -free HBSS 10 % FBS D-MEM (10 % FBS+D-MEM) pipet-aid 30 5 (500 g, 10 min) 10 % FBS+D-MEM pipet-aid cells/ml cells/500 µl/well 75cm 2 flask cells/20 ml/flask flask 37 5 % (5%CO2/95%Air) 3 (10 % FBS+D-MEM) ~20 D-MEM/F

5 Fisher 344 (Liu et al., 2001; Wang et al., 2001) 75 cm 2 flask 14~16 flask (shaker) 200 rpm 4~5 flask flask (500 g) % FBS+D-MEM pipet-aid 10 % FBS+D-MEM cells/ml cells/500 µl/well 24 24~48 10 % FBS+D-MEM D-MEM/F-12 flask 4 18~20 4. Fisher 344 (Liu et al., 2001) 75 cm 2 flask flask flask Phosphate-Buffered Saline (PBS) 0.25 % trypsin-ca 2+ /Mg 2+ -free HBSS 37 C 5 flask trypsin (500 g) % FBS+D-MEM pipet-aid 10~15 75cm 2 flask 10~14 29

6 (subculture) 2~ cells/ml cells/500 µl/well 24 24~48 10 % FBS+D-MEM D-MEM/F-12 anti-nse anti-map-2 anti-gfap ED-1 OX-42 PBS 4 % paraformaldehyde (PFA, ph 7.4 PBS) 30 PBS 1 % H 2 O 2 10 酶 (peroxidases) PBS 3 10 (non-specific binding) 0.4 % Triton X % (Bovine Serum Albumin, BSA) 0.4 % (blocking buffer) 1 anti-nse (1:200) anti-gfap (1:2000) anti-ox-42 (1:200) anti-ed1 (1:300) 4 (overnight) PBS (Biotinylated secondary antibody) 2 PBS Avidin/Biotinylated peroxidase Complex (VECTASTAIN Elite ABC kit) 1 PBS 3 3 diaminobenzidine tetrahydrochloride dihydrate (DAB) ( ) (inverted 30

7 microscope) 1) NO SNAP 2) H 2 O 2 3) LPS 4) SOD catalase UA TEMPO DMTU NAC 5) MAP Kinase SB203580, PD ) Caspases Z-DEVD-FMK, Z-VAD-FMK 7) (17α-estradiol 17β-estradiol) Tamoxifen LPS (inflammagen) LPS H 2 O 2 SNAP 95% dimethyl sulfoxide (DMSO) 95% TEMPO 17α-estradiol 17β-estradiol Tamoxifen DMSO SNAP SB PD Z-DEVD-FMK Z-VAD-FMK (treatment duration) ( ) ( H 2 O 2 H 2 O 2 +SNAP)

8 ( ) ( ) Bio-Rad (Bio-Rad Protein Assay) (dye-binding assay) (Coomassie Brilliant Blue G-250) 595 nm ml (NaOH, 1N) 150 rpm BSA stock BSA (0, 20, 40, 80, 120, 160, 180, 200 µg/ml) (HCl, 3N) (dye H 2 O = 3 2) 96-well RIA/EIA plate 20 ELISA (ELISA reader, Molecular Devices Corporation, Menlo Park, CA, USA) (endpoint) 595 nm ELISA reader (phase-contrast microscope) 1) 酶 (lactase dehydrogenase, LDH) 2) MTT 32

9 (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium reduction assay) 1. 酶 bromide Wroblewski LaDue LDH LDH LDH LDH (Koh and Choi, 1987) Pyruvate + NADH LDH Lactate + NAD (High A 340 ) (Low A 340 ) LDH (pyruvate) (lactate) 酶 (reduced nicotinamide adenine dinucleotide, NADH) 酶 (oxidized nicotinamide adenine dinucleotide, NAD) 酶 340 nm 酶酶 (units/ml) 500 units/ml Enzyme Control 2-E (LDH ) LDH ( units/ml) (0.1 M) β-nadh (0.3 µg/µl) (sodium pyruvate solution, 22.7 mm) (50 µl) 96-well RIA/EIA plate β-nadh (100 µl) 30 (100 µl) ELISA reader (kinatics) 340 nm 15 3 ELISA reader ( Abs/min) LDH LDH 1 mmol NADH LDH 2. MTT Mosmann MTT 33

10 (Mosmann, 1983) MTT formazan formazan MTT (5 mg/ml PBS) 0.5 mg/ml 37 C 90 MTT DMSO formazan 150 µl DMSO 96-well RIA/EIA plate ELISA reader 570 nm 630 nm ELISA reader Nitrite NO (NO + O 2 NO 2 + O 2 NO 3 ) nitrite NO NOS griess reagent (0.1 % sulfanilic acid + 1 % N-(1-naphthyl) ethylenediamide Dihydro-chloride % Phosphoric acid) nitrite NO NOS (Green et al., 1982) (colorimetric assay) nitrite nitrite griess reagent 540 nm (NaNO 2 ) MEM D-MEM/F-12 (sodium nitrite solution) ( µm) (50 µl) 96-well RIA/EIA plate griess reagent 10 ELISA reader 540 nm ELISA reader nitrite 34

11 H 2 O 2 3,3 -dimethoxybenzidine H 2 O 2 酶 H 2 O 2 3,3 -dimethoxybenzidine horseradish peroxidase 500 nm 30 % H 2 O 2 (=8.82 M) MEM H 2 O 2 (50 µl) 96-well RIA/EIA plate 3,3 -dimethoxybenzidine (100 µl, 2 mm) horseradish peroxidase (100 µl, 240 IU/ml) ELISA reader 500 nm ELISA reader H 2 O 2 (Desagher et al., 1996) ROS DCFH-DA ROS (Rosenkranz et al., 1992) DCFH-DA ROS NO (Rosenkranz et al., 1992) DCFH-DA ( 100 µm) 485 nm 500 nm fluorometric ELISA reader DCFH-DA 30 ROS 1. DHR-123 DHR

12 ONOO ONOO DHR-123 ONOO (Kooy et al., 1994) DHR-123 ( 20 µm) 485 nm 500 nm fluorometric ELISA reader DHR ONOO 2. 3-NT antibody (protein nitration) ONOO PBS 4 % PFA 30 PBS 1 % H 2 O 2 10 酶 0.3 % Triton X % (normal goat serum) 1 % BSA 1 (rabbit polyclonal anti-3-nt antibody, 1:200) 4 C PBS (biotinylated goat anti-rabbit IgG antibody) 1 酶 (streptavidin horseradish peroxidase) DAB (positive staining) Rat TNF-α DuoSet TM ELISA kit TNF-α (multiple antibody sandwich principle) 96 TNF-α (capture antibody) TNF-α 酶 TNF-α (peroxidase-conjugated polyclonal anti-tnf-α) 36

13 TNF-α 酶 450 nm TNF-α TNF-α (capture antibody) 酶 TNF-α (HRP-conjugated anti-tnf-α second antibody) Rat TNF-α (coating solution) (blocking buffer) (standard diluent) (antibody diluent) (wash buffer) 3,3,5,5 -tetramethyl-benzidine (TMB) H 2 O 2 (stop solution) kit 96 wash buffer 2~3 37 C µl C 1 wash buffer 4~5 100 µl TNF-α 37 C 1 wash buffer 4~5 100 µl 10~ µl 30 ELISA reader 450 nm ELISA reader TNF-α DAPI 4, 6-Diamidino-2-phenylindole (DAPI) DNA (fluorescent DNA-binding probe) (glass coverslips) 37

14 glass coverslips DAPI PBS 4 % PFA 30 PBS 1 % Triton X PBS DAPI (1 µg/ml 0.9 % NaCl) 30 glass coverslips mounting medium 359 nm SPOT (inverted fluorescence microscope, Diagnostic Instruments, Sterling Heights, MI, USA) TUNEL TUNEL DNA PBS 4 % PFA % Triton X-100 (in 0.1 % sodium citrate) 5 PBS PBS kit fluorescein TUNEL reagent mixture 37 C 60 PBS mounting medium fluorescein isothiocyanate (FITC) SPOT 25 (brightfield photos) (fluorescent photos) TUNEL-positive cells TUNEL-positive cells 38

15 Annexin-V-FITC PS (inner leaflet) DNA PS (outer leaflet) Annexin-V PS Annexin-V FITC PBS kit Annexin-V incubation reagent PBS 30 (binding buffer) mounting medium FITC SPOT 25 brightfield photo fluorescent photo brightfield photo fluorescent photo Annexin-V-positive cells Annexin-V-positive cells DNA (total genomic DNA) (agarose gel electrophoresis) PBS lysis buffer ( 10 mm EDTA, 50 mm Tris-HCl ph 8.0, 0.5 mg/ml proteinase K, 0.5 % N-Lauroylsarcosine) 1.5 ml 50 C 3 DNAase-free RNase (20 mg/ml) 50 C 1.5 phenol/chloroform (1 1) 4 C (8000 g) 20 DNA 39

16 1.5 ml DNA 2 % 50 TAE 40~50 ethidium bromide UV DNA (DNA laddering) p38 MAPK caspase-3 PBS 酶 lysis buffer ( 50 mm Tris-HCl (ph 7.5), 1 mm ethyleneglycol bis(aminoethylether)tetraacetate (EGTA), 1 % Triton X-100, 1 mm sodium fluoride, 5 mm benzamidine, 10 mm sodium orthovenadate, 20 mm p-nitrophenyl phosphate and protease inhibitor cocktail) 1.5 ml (sonicator) (8000 g) ml Bio-Rad Protein Assay BSA 80 C sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) 10 % β-mercaptoethanol sample buffer (2 ) 95 C 5 12 % Tris-glycine gel 80~100 PVDF PVDF BSA 1 (rabbit anti-rat polyclonal antibodies against phospho-p38 MAPK [1 : 40

17 1000] and caspase-3 [1 : 1500]) 3 4 C PVDF phospho-p38 MAPK activated caspase-3 wash buffer (goat anti-rat horse-radish peroxidase) 1 wash buffer PVDF phospho-p38 activated MAPK caspase-3pvdf stripping buffer total phospho-p38 p38 MAPK MAPK (band) (ONE-Dscan Version 1.31, Scanalytics, Billerica, MA, USA) activated caspase-3 ± (mean SEM) ± (statistical differences) one-way analysis of variance (one-way ANOVA) (multiple comparison method) Student-Newman-Keuls Test Dunnett s Test Bonferroni t-test P<0.05 SigmaStat program (Jandel Scientific, Inc., San Rafael, CA, USA) 41

材料與方法

材料與方法 10-12 250-350 Sprague-Dawley rat ( SD ) 12 ( AM 8 00-PM 8 00 ) (Control group) (HS) (Heat shock group) 15 0 4 6 16 24 (PAE) ( Pulmonary air embolism group) 20 40 (25 l/min) 40 17 20 (HS&PAE) 15 4 16 20