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客命孙
7 years ago
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1 理類 CNIB 行 92 年年參行 93 年 2 28

2 益見利 RAW264.7 來 LPS 見

3 論 (NO) (NOS ) L-arginine guanidino 類神 NOS 類神 nnos 類 enos 兩 NO 類 i NOS LPS 刺來量 NO (Kilbourn and Griffith, 1992). 量 NO (septic shock) (Rees et al., 1990)(cerebral injury) (Dawason et al., 1993), (Matheis et al., 1992) 糖尿 (Stefanovic-Racic et al., 1993; Connor et al., 1995; Wu and Thiemermann, 1996) inos 不 enos nnos [Southan et al, 1996] 年來不利六葉兩 LPS/IFN-γ NO [Chiou et al., 2000] 裂 IL-2 金金龍 15 [Kuo et al, 1995 ; Kuo et al, 1996; Yang et al, 1999] 來益利尿漏不

4 暈便不利 1 省省痢 2 丹痢尿暈糖尿 3 利尿降 4 痢 5 (Gandarussa vulgaris Nees) 北利痢淋尿便尿路 6 數利淋利咽淋痢 7 理老 RAW264.7 益了理料

5 料益陸 MTT reagentlps( E coli 055:B5) Griess reagent Sigma (USA)Sodium nitrite J.T.Baker (USA) HycloneUSARAW264.7 老 (Taiwan) ~180 不 ( 益 ) 500 ml~1000 ml 不 24 2 濾濾濾 500 ml 24 () 2 濾濾濾濾行 35 ml ~ 50 ml 不量 -20 冷 RAW well 10% fetal calf serum (Hyclone)2mM glutamine1mm pyruvate1% non-essential amino acid 100U/ml penicillin 100g/ml streptomycin Dubach Modified Eagle (DMEM) 37 5% CO 2 48 行 RAW264.7

6 3 RAW264.7 RAW264.7 參 chiou 8 RAW well well 度 30mg/ml10mg/ml 度 LPS(1g/ml) l 量 Griess reagent ( 1% sulfanilamide 0.1% naphthylenediamine 5% phosphoric acid ) 550nm Sodium nitrite 量 4 RAW264.7 RAW well well 度 LPS(1g/ml) RAW l MTT(0.5%) 37 4 MTT tetrazolium bromide mitochrondrial dehydrogenase 不 formazan DMSO ELISA reader A550 論見利量量錄率 1.4 % % 不率 LPS 们 RAW264.7 NO (A) RAW264.7 LPS

7 NO 量 0.07 ± 0.24 µm RAW264.7 NO 量度度 0.1 mg/ml RAW264.7 NO 量 µm 0.3 mg/ml NO 量 µm RAW264.7 NO 利 MTT RAW264.7 率 (B) 異 ( 益 ) RAW264.7 度益度 0.1 mg/ml RAW264.7 率 100 % 降 5.4 ± 0.6 % 13.6 ± 1.9 % 0.3 mg/ml RAW264.7 率 41.2 ± 4.7 %66.6 ± 1.9 % 0.1 mg/ml ( 率 %) 0.3 mg/ml 0.3 mg/ml 度 ( 0.53 mg/ml) ( 率 81.2 ± 2.7 %) 0.3 mg/ml ( 率 %) 度 ( 0.63 mg/ml)( 率 65.7 ± 6.5 %) LPS RAW264.7 NO 量率 RAW264.7 LPS 20 hr NO 量來 0.07 ± 0.24 µm 40.5 ± 1.3 µm ( A) LPS 20 hr NO 度 0.1

8 mg/ml NO 降 µm 0.3 mg/ml NO 降 µm 利 MTT LPS RAW264.7 率 (B) 益 RAW264.7 類度益度不度 ( 0.63 mg/ml)( 率 70.2 ± 6.2 %) NO 量益度 NO 度度 0.3mg/ml NO 不 MTT+LPS Nitrite concentration(µm) cell viability(%) A B C D E F G extract final concentration(mg/ml) NO+LPS A B C D E F G extract final concentration(mg/ml) control 0.1+LPS 0.3+LPS origin concentration+lps control 0.1+LPS 0.3+LPS origin concentration

9 Nitrite concentration(µm) NO-LPS A B C D E F G extract final concentration(mg/ml) control origin concentration MTT-LPS 180 (B) cell viability(%) control origin concentration A B C D E F G extract final concentration(mg/ml) LPS LPS RAW264.7 NO 量 A LPS B LPS (A)control LPS A 益 B C D E F G origin concentration 益度 0.44mg/ml(A). 度 0.53mg/ml(B). 度 0.62mg/ml(C). 度 0.69mg/ml(D). 度 1.27mg/ml(E). 度 0.52mg/ml(F). 度 1.16mg/ml(G) MTT 來率 (A) 來益率降 (B)

10 (A) cell viability(%) (B) LPS RAW264.7 (B) LPS RAW264.7 率 normal 100% 率來益 control origin concentration MTT-LPS A B C D E F G extract final concentration(mg/ml) LPS LPS RAW264.7 MTT assay 來率 A LPS B LPS (A)control LPS A B C D E F G origin concentration 度 0.44mg/ml(A). 度 0.53mg/ml(B). 度 0.62mg/ml(C). 度 0.69mg/ml(D). 度 1.27mg/ml(E). 度 0.52mg/ml(F). 度 1.16mg/ml(G) 兩 LPS RAW264.7 NO 論 : 參理理 9 NO NOS L-arginine guanidino NOS 類 i NOS lipopolysaccharidelpsinterferon-(ifn-) interleukin-1 刺來量 NO lipopolysaccharide (LPS)interferon-(IFN-) interleukin-1 來刺刺力 nitric oxide synthase (inos)

11 RAW264.7 兩兩更 RT-PCR 來 inos Western blot 來 protein 更了兩參 : 1 立李李聯 91 p93 2 立李李聯 91 p66 3 立李李聯 91 p124 4 立李李聯 91 p36 5 立李李聯 91 p136 6 立李李聯 91 p126 7 立李李聯 91 p50

12 利率益量 (g) 量 (ml) 量 (g) 量 (ml) 度 (mg/ml) 率 (%)

國立自然科學博物館館訊第263期

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