第三章 結果
五、 魚腥草粗抽物對 A549 細胞之週期的影響
不同濃度的魚腥草粗抽物 ( 0, 0.125, 0.25, 0.5 mg/ml )，經過 24、48、72 小時作用，對 A549 細胞之週期影響，結果顯示細胞週期 之DNA 含量在 G1 期最多，S 期其次，G2/M 期最少，以不給藥組當 標 準 比 較 ， 發 現 較 投 予 高 濃 度 魚 腥 草 粗 抽 物 時 ( 0.25mg/ml 和 0.5mg/ml ) 經過 24、48 小時產生蓄積作用在 G1 期最多，而 72 小時 產生蓄積作用在G1 及 S 期最多，如 Fig.12-14 所示。
六、Annexin V-FITC/PI 試劑雙染呈現 A549 細胞死亡情形
不同濃度的魚腥草粗抽液 ( 0, 0.125, 0.25, 0.5 mg/ml ) 對 A549 細胞分別作用24、48、72 小時，結果如 Fig.15 和 Fig.16 所示，經過 24 小時後，A549 細胞隨著魚腥草粗抽液劑量 0, 0.125, 0.25, 0.5 mg/ml 之細胞死亡百分比分別為 12.38±2.39%, 12.5±2.07%, 15.92±1.41%, 21.43±3.59% ; 經過 48 小時後，A549 細胞隨著魚腥草粗抽液劑量 0, 0.125, 0.25, 0.5 mg/ml 之 細 胞 死 亡 百 分 比 分 別 為 10.50±3.8%,
胞隨著魚腥草粗抽液劑量0, 0.125, 0.25, 0.5 mg/ml 之細胞死亡百分比 分別為10.35±1.27%, 7.05±0.58%, 18.18±2.18%, 35.68±3.67% 。由上 述結果顯示，魚腥草粗抽液隨著劑量與時間的增加，造成A549 細胞 死亡百分比也增加。
七、魚腥草粗抽物對人類 A549 細胞之週期相關及凋亡相關 蛋白質的影響
不同濃度的魚腥草粗抽液 ( 0, 0.125, 0.25, 0.5 mg/ml ) 處理 48 小 時，觀察其對 A549 細胞之週期中的蛋白質及凋亡相關蛋白質的影 響。結果如 Fig.17-26 所示，細胞週期 G1 期相關的 cyclinD1、cyclinE、
cyclinA 和 CDK4 / CDK6 、CDK 2 蛋白質隨著魚腥草粗抽液劑量增 加而遞減，而 p27 蛋白表現量增加；與細胞凋亡相關的 caspase 8、
caspase 3 蛋白質表現量增加。
Time(h) carrageenan-induced paw edema in mice.
Fig.7 The acute toxicity of Hottuynia cordata ( HC ) crude extract in mice.
Fig.8 Effect of HC-crude extract on the cell viability of human lung carcinoma cells (A549) . Cell viability was detected by MTT assay.
control 0.125mg/ml 0.25mg/ml 0.5mg/ml
control 0.125mg/ml 0.25mg/ml 0.5mg/ml
Fig.9 Effect of HC-crude extract on the cell viability of murine lung cancer cells (LLC ). Cell viability was detected by MTT assay.
Fig.10 Effect of HC-crude extract on the cell viability of human lung cancer cells (A549) by Flow Cytometry.
concentrartion(mg/ml)
control 0.125 0.25 0.5
cell viability(%)
Fig.11 Effect of HC-crude extract on the cell morphology of human lung cancer cells ( A549 ).
(B)
Fig.12 Alternation of HC-crude extract on cell cycle distribution of A549 cells for 24h.
G1 S G2/M Apoptosis
% of cell cycle distribution
0 20 40 60 80 100
control
0.125 mg/ml HC-crude 0.25 mg/mlHC-crude 0.5 mg/ml HC-crude
***
**
(B)
Fig.13 Alternation of HC-crude extract on cell cycle distribution of A549 cells for 48h.
G1 S G2/M Apoptosis
% of cell cycle distribution
0 20 40 60 80 100
control
0.125mg/ml HC-crude 0.25mg/ml HC-crude 0.5mg/ml HC-crude
***
**
(B)
Fig.14 Alternation of HC-crude extract on cell cycle distribution of A549 cells for 72h.
G1 S G2/M Apoptosis
% of cell cycle distribution
0 20 40 60 80 100 120
control
0.125 mg/ml HC-crude 0.25 mg/ml HC-crude 0.5 mg/ml HC-crude
**
***
*
*
Fig.15 Effects of HC-crude extract on the cell vaibility of A549 cells.
A549 cells were treated with HC-crude extract and were stained with Annexin V conjugated FITC and PI. Cell viability was measured by using Flow Cytometer.
percent (%)
late apoptotic or necrosis (Anexin +/ PI+ )
late apoptotic or necrosis
(Annexin +/ PI+ ) early apoptotic cells
late apoptotic or necrosis (Annexin +/ PI+ )
Fig.16 Effect of HC-crude extract on the cell viability of A549 cells.
A549 cells were treated with HC-crude extract for different time (A) 24h (B) 48h (C) 72h and were stained with Anexin V conjugated with FITC and PI.
control 0.125 0.25 0.5 (mg/ml)
Fig.17 Suppression of CDKs and cyclins and up-regulation of p27, caspase 8, caspase 3 by HC-crude extract treatment. A549 cells were treated with various concentration of HC-crude extract for 48h.
control 0.125 0.25 0.5 (mg/ml)
concentration (mg/ml)
The ratio of cyclinD1 protein level (%)
0 20 40 60 80 100 120
0 0.125 0.25 0.5
Fig.18 Suppression of cyclinD1 protein expression by HC-crude extract treatment on A549 cells for 48h by immunoblotting.
control 0.125 0.25 0.5 (mg/ml)
concentration (mg/ml)
The ratio of cyclin E protein level (%)
0 20 40 60 80 100 120
0 0.125 0.25 0.5
Fig.19 Suppression of cyclinE protein expression by HC-crude extract treatment on A549 cells for 48h by immunoblotting.
control 0.125 0.25 0.5 (mg/ml)
concentration (mg/ml)
The ratio of cyclin A protein level (%)
0 20 40 60 80 100 120
0 0.125 0.25 0.5
*
**
Fig.20 Suppression of cyclinA protein expression by HC-crude extract treatment on A549 cells for 48h by immunoblotting.
control 0.125 0.25 0.5 (mg/ml)
concentration (mg/ml)
The r a ti o of C D K 4 pr ot ei n le v e l( % )
0 20 40 60 80 100 120
0 0.125 0.25 0.5
*
Fig.21 Suppression of CDK4 protein expression by HC-crude extract treatment on A549 cells for 48h by immunoblotting.
control 0.125 0. 25 0.5 (mg/ml)
concentration (mg/ml)
The ratio of CDK6 protein level (%)
0 20 40 60 80 100 120
0 0.125 0.25 0.5
Fig.22 Suppression of CDK6 protein expression by HC-crude extract treatment on A549 cells for 48h by immunoblotting.
control 0.125 0.25 0.5 (mg/ml)
concentration (mg/ml)
The ratio of CDK2 protein level (%)
0 20 40 60 80 100 120
0 0.125 0.25 0.5
**
**
***
Fig.23 Suppression of CDK2 protein expression by HC-crude extract treatment on A549 cells for 48h by immunoblotting.
control 0.125 0.25 0.5 (mg/ml)
concentration (mg/ml)
The ratio of caspase 8 protein level (%)
0 20 40 60 80 100 120
0 0.125 0.25 0.5
*
Fig.24 Up-regulation of caspase 8 protein expression by HC-crude extract treatment on A549 cells for 48h by immunoblotting.
control 0.125 0.25 0.5 (mg/ml)
concentration (mg/ml)
The ratio of caspase 3 protein level (%)
0 20 40 60 80 100 120 140
0 0.125 0.25 0.5
Fig.25 Up-regulation of caspase 3 protein expression by HC-crude extract treatment on A549 cells for 48h by immunoblotting.
control 0.125 0.25 0.5 (mg/ml)
concentration (mg/ml)
The ratio of p27 protein levle (%)
0 20 40 60 80 100 120 140
0 0.125 0.25 0.5
Fig.26 Up-regulation of p27 protein expression by HC-crude extract treatment on A549 cells for 48h by immunoblotting.