首先在存活率的實驗中發現，A10 細胞經由 PM 處理後，細胞生 長情形明顯受到抑制【圖2、3、4、5】，且細胞型態 （ Morphology ） 在給藥後也明顯改變，細胞膜變的不規則、皺縮 （ shrinkage ）【圖 6、7、8】。然後藉由 DNA fragmentation 的電泳分析中，了解 48 小 時作用時間點時，給藥組 （40 及 80 μg/ml ）會較控制組有 DNA 的裂解片段【圖9】。再來，我們為了更進一步確定細胞凋亡的可能，
運用二種細胞凋亡偵測方式，經由流式細胞儀及螢光顯微鏡檢測下，
結果顯示給予 PM 組別（20、40、80 μg/ml）與控制組相比，明顯 會有較多的凋亡細胞被偵測出【圖10、11、12、13】。最後，為了明 白給藥後A10 凋亡相關蛋白之表現情形，藉由西方墨點法，發現 p53
【圖14】、Bax【圖 15】、p21【圖 16】、Fas【圖 17】、p38【圖 18】、
Caspase 3（17 kda）【圖 19】蛋白，於給予 PM 後似乎有較多的表現 量。推測凋亡相關路徑中 p53 蛋白表現增加，進而使得下游的 p21 也 被活化，且與凋亡相關的蛋白如 Fas、Bax 及 p38，也會進而有較多 的蛋白表現量【圖20】。
故根據上述的研究結果顯示，桑黃 Phellinus merrillii（PM）於體 外實驗（in vitro）方式中，具有誘發平滑肌細胞（A10）走向細胞凋 亡（Apoptosis）的情形。
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表 1、細胞凋亡（Apoptosis）與細胞壞死（Necrosis）之比較
表2、1X Phosphate buffer saline（PBS）配製法（PH：7.4）
表 3、蛋白質量化所使用之配方表
細胞凋亡（Apoptosis） 細胞壞死（Necrosis）
起因 缺少生長荷爾蒙
細胞核的變化 濃縮（condensation）
分裂（fragmentation）
－
細胞膜的變化 Phosphatidylserine 翻轉 表面不規則突出
Baradford（μl） 200
總體積（μl） 1000
組成 體積 / 重量
6X Loading buffer 10ul
MQ water 45ul
DNA marker 原液 5ul
表 4、DNA marker 的製作方式
Dose (ug/ml)
% of cell number
0 分比，lane 1（控制組）為 100％、lane 2（10μg/ml）為 116％、
lane 3（20μg/ml）為 97％、lane 4（40μg/ml）為 120％、lane 5
（80μg/ml）為 95％。本實驗為三次重複，結果以平均值呈現，
並加上平均值之standard deviations。
Fig 1.PM didn’t inhibit the growth of A10 vascular smooth muscle cells for 12 hours. A10 cells (20000 at start of experiment) were treated with different concentrations of PM for 12 hours, amount of cell number was assessed by cell count, each group were divison of control group, calculated the percentage. Results show that lane 1 (control) were 100 %, lane 2 (10 μg/ml) were 116 %, lane 3 (20 μg/ml) were 97 %, lane 4 (40 μg/ml) were 120 %, lane 5 (80 μg/ml) were 95 %. Data shown are mean values with bars indicating the standard deviations of the mean (n = 3).
Dose (ug/ml)
% of cell number
0 分比，lane 1（控制組）為 100％、lane 2（10μg/ml）為 92％、
lane 3（20μg/ml）為 64％、lane 4（40μg/ml）為 56％、lane 5
（80μg/ml）為 58％。本實驗為三次重複，結果以平均值呈現，
並加上平均值之standard deviations。＊表示以 One way ANOVA 進行統計，與控制組相較細胞數目有明顯抑制（p＜0.05）。
Fig 2.PM inhibits the growth of A10 vascular smooth muscle cells for 24 hours. A10 cells (20000 at start of experiment) were treated with different concentrations of PM for 24 hours, amount of cell number was assessed by cell count, each group were divison of control group, calculated the percentage. Results show that lane 1 (control) were 100 %, lane 2 (10 μg/ml) were 92 %, lane 3 (20 μg/ml) were 64 %, lane 4 (40 μg/ml) were 56 %, lane 5 (80 μg/ml) were 58 %. Data shown are mean values with bars indicating the standard deviations of the mean (n = 3). (*) Inhibition of PM is statistically significat according to one way ANOVA (p<0.05).
Dose
control 10 20 40 80
% of cell number
0
lane 1（控制組）為 100％、lane 2（10μg/ml）為 96％、lane 3
（20μg/ml）為 57％、lane 4（40μg/ml）為 54％、lane 5（80 μg/ml）為 45％。本實驗為三次重複，結果以平均值呈現，並加 上平均值之standard deviations。＊表示以 One way ANOVA 進行 統計，與控制組相較細胞數目有明顯抑制（p＜0.05）。
Fig 3.PM inhibits the growth of A10 vascular smooth muscle cells for 48 hours. A10 cells (20000 at start of experiment) were treated with different concentrations of PM for 48 hours, amount of cell number was assessed by cell count, each group were divison of control group, calculated the percentage. Results show that lane 1 (control) were 100 %, lane 2 (10 μg/ml) were 96 %, lane 3 (20 μg/ml) were 57 %, lane 4 (40 μg/ml) were 54 %, lane 5 (80 μg/ml) were 45 %. Data shown are mean values with bars indicating the standard deviations of the mean (n = 3). (*) Inhibition of PM is statistically significat according to one way ANOVA (p<0.05).
Dose (ug/ml)
% of cell number
0
lane 1（控制組）為 100％、lane 2（10μg/ml）為 47％、lane 3
（20μg/ml）為 49％、lane 4（40μg/ml）為 24％、lane 5（80 μg/ml）為 29％。本實驗為三次重複，結果以平均值呈現，並加 上平均值之standard deviations。＊表示以 One way ANOVA 進行 統計，與控制組相較細胞數目有明顯抑制（p＜0.05）。
Fig 4.PM inhibits the growth of A10 vascular smooth muscle cells for 72 hours. A10 cells (20000 at start of experiment) were treated with different concentrations of PM for 72 hours, amount of cell number was assessed by cell count, each group were divison of control group, calculated the percentage. Results show that lane 1 (control) were 100 %, lane 2 (10 μg/ml) were 47 %, lane 3 (20 μg/ml) were 49 %, lane 4 (40 μg/ml) were 24 %, lane 5 (80 μg/ml) were 29 %. Data shown are mean values with bars indicating the standard deviations of the mean (n = 3). (*) Inhibition of PM is statistically significat according to one way ANOVA (p<0.05).
hours
0 20 40 60 80
% of viable cells
0
Fig5. PM inhibits the growth of A10 vascular smooth muscle cells for 24, 48, 72 hours.The datas from fig1 to fig4, shows that PM didn’t inhibit the growth of A10 vascular smooth muscle cells for 12 hours.
But PM can inhibits the growth of A10 vascular smooth muscle cells for 24, 48,72 hours. Data shown are mean values with bars indicating the standard deviations of the mean (n = 3).
圖6、A10 細胞型態於給予 PM 後 24 小時。不同濃度的 PM 給予 A10 細胞，置於培養箱培養24 小時，以倒立光學顯微鏡倍率 40 倍拍 攝，圖中 A 為控制組，B 、 C 、D 分別為給予 20、40 及 80 μ g/ml 的 PM，結果顯示，不同濃度的 PM（20、40、80 μg/ml）
造成細胞型態上呈現細胞膜較不規則。
Fig6. After treated with PM in SMC for 24 hours, induced A10 cells morphology changed. A10 cells (20000 at start of experiment) were treated with different concentrations of PM for 24 hours. A, control.
B, 20 μg/ml. C, 40 μg/ml. D, 80 μg/ml.
A B
C D
圖7、A10 細胞型態於給予 PM 後 48 小時。不同濃度的 PM 給予 A10 細胞，置於培養箱培養48 小時，以倒立光學顯微鏡倍率 40 倍拍 攝，圖中 A 為控制組，B 、C 、 D 分別為給予 20、40 及 80 μ g/ml 的 PM，結果顯示，不同濃度的 PM（20、40、80 μg/ml）
造成細胞型態上呈現細胞膜較不規則。
Fig7. After treated with PM in SMC for 48 hours, induced A10 cells morphology changed. A10 cells (20000 at start of experiment) were treated with different concentrations of PM for 48 hours. A, control.
B, 20 μg/ml. C, 40 μg/ml. D, 80 μg/ml.
A B
C D
圖8、A10 細胞型態於給予 PM 後 72 小時。不同濃度的 PM 給予 A10 細胞，置於培養箱培養72 小時，以倒立光學顯微鏡倍率 40 倍拍 攝，圖中 A 為控制組，B 、 C、 D 分別為給予 20、40 及 80 μ g/ml 的 PM，結果顯示，不同濃度的 PM（20、40、80 μg/ml）
造成細胞型態上呈現細胞膜較不規則。
Fig8. After treated with PM in SMC for 72 hours, induced A10 cells morphology changed. A10 cells (20000 at start of experiment) were treated with different concentrations of PM for 72 hours. A, control.
B, 20 μg/ml. C, 40 μg/ml. D, 80 μg/ml.
A B
C D
圖9、給予 A10 細胞 PIM 後 48 小時 DNA fragmentation 的影響。上 圖中lane 1 為 DNA Marker，lane 2 為控制組，lane 3 為只有給 予溶媒DMSO 組，lane 4 為 20μg/ml，lane 5 為 40μg/ml，lane 6 為 80μg/ml， 結果顯示在給予 A10 細胞 PM （40 及 80μ
圖9、給予 A10 細胞 PIM 後 48 小時 DNA fragmentation 的影響。上 圖中lane 1 為 DNA Marker，lane 2 為控制組，lane 3 為只有給 予溶媒DMSO 組，lane 4 為 20μg/ml，lane 5 為 40μg/ml，lane 6 為 80μg/ml， 結果顯示在給予 A10 細胞 PM （40 及 80μ