第五章 結論與建議
第二節 建議
可進一步確認環孢靈 A 對於其他種類細胞株是否也有抗病毒之作 用，以及環孢靈A 下游之訊息傳遞，使其分子機制更清楚。同時可以利 用即時定量連鎖反應的方式去確認二維電泳圖譜之結果。
參考文獻
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圖
Fig.1
圖一、利用TE671 細胞表現 NS5 蛋白
A.將 NS5 蛋白序列構築在含有 Flag-tag 的 PCR3.1 上面，並且轉染進 TE671 細胞內表現。B.將空載體對照細胞及 NS5 蛋白表現細胞收下，利 用西方墨點法，Anti-Flag 做為一抗、Anti-AP 為二抗呈色，於 103 kDa 的位置有NS5 蛋白表現。
Fig.2
圖二、利用IFN-β 測試 NS5 蛋白對干擾素的抗性
利用穩定表現空載體對照細胞及NS5 蛋白表現細胞培養至 6 well plate 中，於培養液加入IFN-β 1000 U/ml 處理，經過 0、24、48 小時於顯微 鏡下觀察細胞，發現NS5 蛋白表現的細胞對干擾素有抵抗性。
NS5
PCR3.1 NS5 PCR3.1 PCR3.1 NS5
1000 U 500 U Control
48 hours 24 hours
0 hour
Fig.3
圖三、利用流式細胞儀觀察細胞凋亡現象
利用空載體對照細胞及NS5 蛋白表現細胞培養至 6 well plate，加入 IFN-β 1000 U/ml、48 小時後，進行 Annexin V FITC 與 PI 雙染，利用 Flow cytometry 分析螢光反應。
Annexin V FITC
Mock
PCR3.1 PCR3.1-NS5
PCR3.1+IFN β PCR3.1-NS5+IFN β
PI
TE671 PCR3.1/PCR2.1-NS5 cells treated by IFN beta
0
%Percentage of cell apoptosis
-IFN +IFN
Fig.4
圖四、以西方墨點法觀察Caspase-9 之表現
利用空載體對照細胞及NS5 蛋白表現細胞加入不同濃度干擾素 48 小時 處理後所收集下的蛋白，利用西方墨點法去觀察Caspase-9 之表現。
PCR3.1 PCR3.1-NS5 IFN-β
(u/ml)
0 250 500 1000 0 250 500 1000
Pre-caspase
Activated- caspase9 β-actin
Fig.5
圖五、利用冷光報導測試NS5 蛋白表現細胞經 IFN-β 處理後對 ISRE 活性之影響
利用穩定表現空載體對照細胞及NS5 蛋白表現細胞培養至 6 well plate，
將pISRE-Luc 與 pRunilla-Luc 的冷光報導基因以 9:1 的比例轉染進細胞 中，待48 小時培養之後，於培養液中加入 IFN-β 3000 U/ml、4 小時，
利用冷光儀所得的數值以pISRE-Luc/pRunilla-Luc 呈現。
TE671 ISRE+IFN beta
0.00
Relatived Luciferase activity
-IFN beta +IFN beta
Fig.6
圖六、利用冷光報導測試NS5 蛋白細胞經 IFN-β 處理後對 NF-κB 活 性之影響
利用穩定表現空載體對照細胞及NS5 蛋白表現細胞培養至 6 well plate，
將pNF-κB-Luc 與 pRunilla-Luc 的冷光報導基因以 9:1 的比例轉染進細胞 中，待48 小時培養之後，於培養液中加入 IFN-β 3000 U/ml、4 小時，
利用冷光儀所得的數值以pNF-κB-Luc/pRunilla-Luc 呈現。
TE671 NF kB+IFN beta
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00
PCR3.1-FLAG PCR3.1-FLAG-NS5
Relatived Luciferase activity
-IFN beta +IFN beta
Fig.7
圖七、以即時定量連鎖反應測試NS5 對細胞激素與干擾素下游基因表現 之影響
將空載體對照細胞及NS5 蛋白表現細胞培養在 25 T 培養瓶中，於培養 液中加入IFN-β 1000U/ml、8 小時，將細胞收下並萃取細胞 RNA，並反 轉錄成cDNA 後，利用 Real time PCR 分析後以△△Ct 值(Cycle threshold value)呈現(△△Ct=(Ct. treated-Ct. GAPDH)-(Ct. untreated-Ct.
GAPDH))，數值越高代表細胞內表現量越低。
0 0.5 1 1.5 2 2.5
IL-6 IRF-3 PKR OAS
Relative RNA Expression
PCR3.1 PCR3.1-NS5
Fig.8
Fig.9
Fig.10
Fig.11
Fig.12
圖十二、Spot 24. Cyclophilin A
利用MALDI-TOF 鑑定蛋白再經由 MASCOT database 分析比對。
PCR3.1 (-) IFN beta
PCR3.1 (+) IFN beta
PCR3.1-NS5 (-) IFN beta
PCR3.1-NS5 (+) IFN beta
Fig.13
圖十三、確認Cyclophilin A 在空載體對照細胞及 NS5 蛋白表現細胞的 表現
將空載體對照細胞及NS5 蛋白表現細胞於培養液中加入 IFN-β 0、250、
500、1000 U/ml，經過 24、48 小時之後，把細胞收下並處理蛋白，利用 西方墨點法以及壓X 光片呈色方式呈現。
Cyclophilin A GAPDH Beta actin
PCR3.1 PCR3.1-NS5
0 250 500 1000 0 250 500 1000
24hrs
Cyclophilin A GAPDH Beta actin
48hrs
IFN-β (U/ml)
Fig.14
圖十四、利用Cyclosporin A 做為抑制劑測試 ISRE 啟動子活性
利用穩定表現空載體對照細胞及NS5 蛋白表現細胞培養至 6 well plate，
將pISRE-Luc 與 pRunilla-Luc 的冷光報導基因以 9:1 的比例轉染進細胞 中，待48 小時培養之後，於培養液中分別加入四組濃度：IFN-β 0 U/ml+
CsA 0 ng/ml、IFN-β 3000 U/ml+ CsA 0 ng/ml、IFN-β 0 U/ml+ CsA 2000 ng/ml、IFN-β 3000 U/ml+ CsA 2000 ng/ml，4 小時，利用冷光儀所得的 數值以pISRE-Luc/pRunilla-Luc 呈現。
TE671 PCR3.1/PCR3.1-NS5 cells ISRE treated with IFN beta and Cyclosporin A
0
Relatived Luciferase activity
IFN 0U/ml+CsA 0ng/ml
IFN 3000U/ml+CsA 0ng/ml
IFN 0U/ml+CsA 2000ng/ml
IFN 3000U/ml+CsA 2000ng/ml
Fig.15
圖十五、Cyclosporin A 對於 Cyclophilin A 下游 ERK 分子之影響
利用空載體對照細胞及NS5 蛋白表現細胞分別加入 0 U/ml IFN-β+0 ng/ml CsA、3000 U/ml IFN-β+ 0 ng/ml CsA、0 U/ml IFN-β+2000 ng/ml CsA、3000 U/ml IFN-β+ 2000 ng/ml CsA 的藥物濃度，經過 1 小時後將 細胞收下，利用西方墨點法以及壓X 光片呈色方式呈現。
Phosphate-ERK2 Phosphate-ERK1
ERK1/2 Beta-actin
PCR3.1 PCR3.1-NS5 IFN-beta
Cyclosporin A
－ ＋ －
＋ － － ＋ ＋
－ ＋ － ＋ － － ＋ ＋
Fig.16
圖十六、測試Cyclosporin A 於 TE671 細胞的抗病毒能力
將TE671 細胞培養至 25 T 培養瓶中，於培養液中加入四組濃度: (1) 0 U/ml IFN-β+ 0 ng/ml CsA、(2) 500 U/ml IFN-β+0 ng/ml CsA、(3) 0 U/ml IFN-β+ 1000 ng/ml CsA 以及(4) 500 U/ml IFN-β+ 1000 ng/ml CsA，並且
同時加入JEV T1P1 strain (m.o.i=0.5)，24 小時之後觀察細胞病變情形 (Cytopathic effect)。
24 hours
Virus Virus+IFN beta
Virus+
Cyclosporin A
Virus+IFN beta+
Cyclosporin A A B
C D
Fig.17
圖十七、利用即時定量連鎖反應測試Cyclosporin A 抗病毒複製之能力 將TE671 細胞培養至 25 T 培養瓶中，於培養液中加入四組濃度: (1) 0 U/ml IFN-β+ 0 ng/ml CsA、(2) 500 U/ml IFN-β+0 ng/ml CsA、(3) 0 U/ml IFN-β+ 1000 ng/ml CsA 以及(4) 500 U/ml IFN-β+ 1000 ng/ml CsA，並且 同時加入JEV T1P1 strain (m.o.i=0.5)，24 小時之後收取上清病毒液後，
萃取出病毒RNA，並利用 Real time PCR 以及設計好之 JEV T1P1 ED3 引子去測試病毒的表現量，此結果以Ct 值(Cycle thereshold value)呈現，
數值越高表示表現量越低。
TE671_JEV T1P1_treated IFN-beta and Cyclosporin A
20 21 22 23 24 25 26
virus virus+IFN Virus+CsA Virus+IFN+CsA
Ct value
24 hours
Fig.18
圖十八、利用流式細胞儀觀察Cyclosporin A 抑制病毒誘導之細胞凋亡 現象
將TE671 細胞培養至 25 T 培養瓶中，於培養液中加入四組濃度: (1) 0 U/ml IFN-β+ 0 ng/ml CsA、(2) 500 U/ml IFN-β+0 ng/ml CsA、(3) 0 U/ml IFN-β+ 1000 ng/ml CsA 以及(4) 500 U/ml IFN-β+ 1000 ng/ml CsA，並且
同時加入JEV T1P1 strain (m.o.i=0.5)，24 小時之後將細胞收下，經由 Annexin V FITC-PI 雙染色處理後利用流式細胞儀分析細胞凋亡情形。
control
Virus Virus+IFN-β Virus+CsA Virus+IFN-β+CsA
Annexin V FITC
PI
3.2%
28.7% 13.2% 11.1% 6.5%
Fig.19
圖十九、藉由病毒蝕斑觀察Cyclosporin A 於 BHK-21 細胞抗病毒之情 形
將 BHK-21 細胞培養至六孔盤中，分別加入以下藥物濃度：(1) 500 U/ml IFN-β，(2) 500 ng/ml、1000 ng/ml CsA，(3) 500 U/ml IFN-β + 500ng/ml CsA、500 U/ml IFN-β + 1000ng/ml CsA，感染病毒 100 PFU/ml，48 小時 後觀察病毒蝕斑的情形。
virus IFN-β Virus+CsA Virus+IFN+CsA BHK-21 plaque assay
0
virus titers (1.E+02 PFU/ml)
virus
BHK-21 plaque assay
0
virus titers (1.E+02 PFU/ml)
virus
Fig.20
Plaque inhibtion (%)
表
表 1-1 Western blot 1.5M Tris-base (pH8.8)
45.4g tris-base add ddH2O 250ml pH=8.8
0.5M Tris-HCl (pH6.8)
7.88gtris-HCl,dd ddH2O 100ml,pH=6.8
10% SDS
10g SDS sodium dodecyl sulfate ,add ddH2O to 100ml
APS 10% ammonium persulfate 1g/10ml ddH2O
Stacking gel(4%)
ddH2O 1.35ml ,30%Acy/Bis 0.27ml,0.5M
Tris-HCl (pH6.8)0.55ml, APS10% 22.5μl,10%
SDS 22.5μl,TEMED 3.5μl
separating gel (10%)
ddH2O 4.8ml ,30 % Acy/Bis 4.2ml,1.5M
Tris-base (pH8.8)3.0ml, APS 10% 120μl,10%
SDS 120μl,TEMED 6.5μl
2x sample
loading buffer
glyceol 2.5 ml , 2-mercaptoethanol 100μl, 10% SDS 2 ml, 0.5 M Tris-HCl 1.25 ml, pH6.8, 0.5% (W/V)bromophenyl blue 0.2ml, ddH2O
3.55 ml , glycerol 2.5ml
running 或 separation buffer
10X : 30g tris-base,144g glycine,10%
SDS100ml add ddH2O to 1L
1X :25mM tris-base,250mM glycine,0.1%SDS Coommassie
brilliant blue 染色液
40%methanol ,10% acetic acid, 0.1%
Coommassie brilliant bluer-250 Destain solution 40%methanol ,10% acetic acid
Transfer buffer
8.72g tris-base,4.4g glycine, add ddH2O to 1.5L, pH 8.4,add methanol 300ml,10% SDS 5.6ml store at 4℃.
10x TBS（Tris
buffer saline） 200 mM Tris-HCl, pH 7.5, 5 M NaCl 1x TBST
（wash solution）1 X TBS cotaining 0.1% Tween-20 5% skim milk 2.5g skim milk/50ml TBST
表 1-2 Protein concentration detection
Protein Standard bovine serum albumin（1 g/ul）
Protein assay dye
Bio-RAD Protein assay dye reagent oncentrate, 450 ml
表 2-1 蛋白質體學分析之差異性蛋白
Spot
ID Protein Identification Mascot
score MW / pI
2 Inosine-5'-monophosphate
dehydrogenase 2 451 55.8/6.44 11 35 1 3.7 4.16 0.88
3 D-3-phosphoglycerate
dehydrogenase 424 56.6/6.29 13 33 1 3.69 0.01 0.01
4 Heterogeneous nuclear
ribonucleoprotein H3 94 36.9/6.37 3 13 1 1.67 0.72 0.01
5 60 kDa heat shock protein,
mitochondrial precursor 1145 61/5.7 15 52 1 2.21 1.71 1.91
6 Heterogeneous nuclear
ribonucleoprotein D0 96 38.4/7.62 2 6 1 0.56 1.37 0.62
7 Heat shock protein beta-1 458 22.8/5.98 5 66 1 1.57 1.16 0.74
8 ATP synthase subunit beta,
mitochondrial precursor 302 56.5/5.26 7 31 1 1.72 0.91 0.31
9 Heterogeneous nuclear
ribonucleoprotein K 110 50.9/5.39 3 7 1 2.07 1.13 0.91
10 Protein DJ-1 141 19.9/6.33 4 24 1 1.76 0.49 0.63
11 Cofilin-1 107 18.5/8.22 2 15 1 1.49 1.22 1.17
12 T-complex protein 1 subunit beta 129 57.5/6.01 7 20 1 6.33 0.6 0.72 13 Phosphoglycerate kinase 1 361 44.6/8.30 12 40 1 1.65 1.26 1.14
14 Peroxiredoxin-1 192 22.1/8.27 4 13 1 1.59 3.2 4.68
15 Heterogeneous nuclear
ribonucleoprotein L 138 60.1/6.65 4 15 1 0.16 0.01 0.02
16 Superoxide dismutase [Mn],
mitochondrial precursor 91 24.7/8.35 2 12 1 0.23 0.4 0.11
17 Dermcidin precursor 94 11.3/6.08 2 20 1 0.27 0.01 0.01
18 Stress-induced-phosphoprotein 1 70 62.6/6.40 6 11 1 0.94 1.15 0.01
19 Fumarate hydratase, mitochondrial
precursor 81 54.6/8.85 5 10 1 0.01 0.01 0.02
20 Thioredoxin 86 11.7/4.82 1 19 1 0.59 0.27 0,01
21 Elongation factor 1-beta 254 24.7/4.50 4 32 1 1.4 0.5 0.74
22 Prohibitin 72 29.8/5.57 7 37 1 1.96 0.12 0.01
23 Fascin 79 4.75/7.71 7 79 1 3.81 11.45 2.51
24 Chain A, Cyclophilin A Complexed 119 18.09/7.82 11 68 1 1.11 1.27 3.4
25 Calreticulin precursor 101 46.89/4.3 8 24 1 1.49 5.09 0.01
26 EDAR-associated death domain 66 46.11/5.89 5 24 1 0.01 0.14 0.03 27 Triosephosphate isomerase 1 174 31.05/5.65 17 58 1 0.87 0.66 0.4
28 CDK5 regulatory subunit 58 56.88/4.68 8 18 1 0.01 0.03 0.04
29 Cyclin-I 57 42.53/8.23 6 15 1 1.08 0.22 0.07
附錄
http://pathmicro.med.sc.eduvirolflavi1.jpg
附圖 1. 日本腦炎基因體全長與 NS5 非結構蛋白
日本腦炎基因體全長為 10~11 kb，其中含有結構與非結構蛋白。而其中 NS5 蛋白佔有 905 個胺基酸，N＇端具有甲基轉移酶活性，C＇端則為 RNA dependent RNA polymerase domain，與病毒複製有關。
Journal of General Virology (2008), 89, 1–47
附圖 2. 產生干擾素的分子傳遞過程
以 IFN-β為例，病毒引發細胞產生干擾素之過程。
附圖 3. 第一型干擾素刺激細胞產生下游抗病毒蛋白之訊息傳遞