독성평가를 위한 생체지표 연구 - webbook.me.go.kr

1 NIER-GP2015-157 11-1480523-002434-01 2015- i - 1I. 21. MTT Assay 22. LDH leakage Assay 43. Neutral red Assay 5II. 71. ROS Assay 82.

2 NO Assay 103. GSH/GSSG Assay 12 III. 141. IL-1 / 14 1) IL-1 14 2) IL-1 152. IL-6 163.

3 IL-8 164. IL-10 175. VEGF 176. TNF- 187. TGF- 19- ii - 8. 191) ELISA 202) 22IV.

4 26V. 29- iii - 1. MTT Assay 2 2. 7 3. ROS Assay 8 4. NO Assay 10 5. GSH/GSSG Assay 12 6. 15 7.

5 ELISA 21 8. ELISA 22 9. Microplate layout 23 2015- 1 - 1. (2015)2. - - - In vivo In vitro 3. - MTT, LDH, Neutral Red Assay- ROS, NO, GSH/GSSG Assay- 8 Assay- 2 -I. in vitro . MTT assay, LDH leakage assay, Neutral red assay.

6 3 .1. MTT Assay1) MTT assay Tetrazolium-based colorimetric (MTT) . tetrazoliumsalt[3-(4,5-dimethylthiazol-2 -yl)-2-5-diphenyltetrazolium bromide] (MTT) MTT formazan ( 500 ~ 600nm) .1),2) 1. MTT 2015- 3 -2) -PBS-MTT : MTT 2 ~ 5mg/ml PBS , tube 4 C -DMSO- 96well plate (flat bottom) - 96well plate well 1 104 ~ 1 106 .- well .- 37 C, 5%, CO2 incubator .- plate .- 2 ~ 5mg/ml MTT.

7 (MTT )-37 C, 5%, CO2 incubator 4 .-MTT DMSO .- plate 10 ~ 30 .-Plate reader . ( : 500nm ~ 600nm)- 4 -2. LDH leakage Assay1) LDH (latate dehydrogenase) leakage assay LDH assay , . , ..2),4) 2) - LDH Assay kit - 96well plate (flat bottom) - 96well plate well 50 ~ 200 . ( : , )- Triton X-100 kit .- well .- 37 C, 5%, CO2 incubator .- 400 ~ 600g, 5 . - 96well plate LDH reaction well.

8 - 37 C 30 . ( )-Plate reader . ( : 450nm ~ 490nm) 2015- 5 -3. Neutral red Assay1) Neutral red assay Neutral red . neutral red dye neutral red .2),3),4) 2) - Neutral red Assay kit - 96well plate (flat bottom) - 96well plate well .- 37 C, 5%, CO2 incubator .- 96well plate .- Neutral red well .-2 ~ 4 37 C, 5%, CO2 incubate .- , PBS HBSS . ( )- Neutral red.

9 ( - 6 - )-10 shaker .-Plate reader . ( : 540nm) 2015- 7 -II.. , DNA , , . , , .6),7) , .6),7) 3 .Amira Adly., 2010 2. - 8 -1. ROS Assay1) ROS (Reactive Oxygens Species) assay .. 4 ROS . ROS DCFH-DA (Dichlorofluorescin diacetate) . DCFH-DA , DCFH.

10 DCFH ROS DCF . ROS .7) Gomes, A., et al. 2005 3. ROS assay 2015- 9 -2) - ROS Assay kit - 96well plate (flat bottom) - 96 well clear black plate 37 C, 5%, CO2 incubator .- DPBS HBSS .- .- DPBS HBSS .-DCFH_DA 30 ~ 60 .- DPBS HBSS 2 ~ 3 .- Microplate fluorometer excitation 499 nm, emission 522 nm , .- 10 -2. NO Assay1) NO (Nitric Oxide) assay NO , ROS NO . peroxynitrite (ONOO-).