第三章 評估台灣紫芝多醣體之抗腫瘤活性
4. 台灣紫芝多醣體抗 C26 腫瘤之活性
前面結果顯示 PS-F2 對 S180 及 B16 腫瘤細胞均有顯著性的抑制效果，為了 評估PS-F2 對各種腫瘤細胞生長抑制之廣度，另外以 BALB/c 小鼠皮下接種 C26
腫瘤細胞之同種腫瘤移植動物模式，評估台灣紫芝多醣體PS-F2 之抗腫瘤活性。
0
Days after tumor implantation
Body weight (g)
Time after inoculation (day)
Tumor size (mm3 ) PBS PS-F2
*
Figure 3-13. Antitumor effect of PS-F2 in B16 tumor-bearing mice. The body weight (A) and tumor size(B) were monitored.
B16 (1×106 cells) was subcutaneously implanted into C57BL/6 mice. PS-F2 (50mg/kg) was i.p. administered to mice for 3 weeks starting from 24 h before inoculation. Control mice were treated with equal amounts of PBS alone on the same schedule. Data are the means ± SE (n=10). *P<0.05; **P<0.01 compared to the PBS group.
(A)
0 2 4 6 8 10 12 14
Tumor
W ei ght ( g)
PBS PS-F2
*
(B)
圖3-14. PS-F2 對 B16 腫瘤生長之影響
Figure 3-14. Anti-tumor effects of PS-F2 in B16 tumor-bearing mice. The change of tumor weight(A) was determined on day 21. (B) is tumor picture after cut off.
B16 (1×106 cells) was subcutaneously implanted into C57BL/6 mice. PS-F2 (50mg/kg) was i.p. administered to mice for 3 weeks starting from 24 h before inoculation. Control mice were treated with equal amounts of PBS alone on the same
PBS
PS-F2
0.0 0.1 0.2 0.3 0.4 0.5 0.6
Spleen
W ei ght ( g)
PBS PS-F2
**
圖3-15. PS-F2 對接種 B16 小鼠脾臟重量之影響
Figure 3-15. Effect of PS-F2 on spleen weights in B16 tumor-bearing mice.
Data are the means ± SE (n=10).
(A)
Pe rc en ta ge o f p os itiv e c ell s
PBS PS-F2*
P er cen ta ge o f p os iti ve cel ls
PBS PS-F2圖3-16. PS-F2 對接種 B16 小鼠脾臟細胞族群比例之影響
Figure 3-16. Effect of PS-F2 on the populations of T and B cells (A) and CD4 and CD8 T cells (B) in splenocytes of B16 melanoma-bearing mice.
The date are the means ± SE for each group (n = 10). *P<0.05 compared to the PBS group.
(A)
P er cen ta ge o f p os iti ve cel ls
PBS PS-F2(B)
NK cell activity (%)
PBS PS-F2
圖3-17. PS-F2 對接種 B16 小鼠脾臟 NK 細胞之影響
Figure 3-17. Effect of PS-F2 on the CD49b population (A) and NK cells activities (B) in B16 tumor-bearing mice.
Cytotoxicity of NK cells were assessed by measuring the lysis of the target YAC-1cells mediated by splenocytes from tumor implanted mice. Data are the means
± SE (n=10).
0 500 1000 1500 2000 2500 3000
IgM IgG
A nt ibody ( µ g/ m l)
PBS PS-F2
**
圖3-18. PS-F2 對接種 B16 小鼠血清抗體濃度之影響
Figure 3-18. Effect of PS-F2 on serum IgM and IgG in B16 tumor-bearing mice.
The antibody levels were determined by ELISA. Data are the means ± SE (n=10).
**P<0.01 compared to the PBS group.
BALB/c 小鼠接種 C26 腫瘤細胞 24 小時後，進行第一次腹腔注射 PS-F2 (50 mg/kg)，每兩天進行腹腔注射 PS-F2 一次，試驗期間紀錄體重變化及測量腫瘤生
長。於試驗3 週後犧牲動物，採血分析血清抗體含量，取脾臟及腫瘤組織秤重，
並分析脾臟其T 細胞及 B 細胞族群比例之變化。結果顯示以腹腔注射 PS-F2 對 於體重變化無顯著影響 (圖 3-19A)；在接種 B16 腫瘤細胞後第十三天，腹腔注 射PS-F2 對 C26 腫瘤生長已有顯著抑制之效果 (圖 3-19B)，而於第二十四天犧牲 小鼠，以腫瘤重量計算抑制率，在兩次個別的C26 腫瘤試驗中，給予小鼠 PS-F2 抑制C26 生長達 43.3 %（P = 0.034）和 53.2 %（P = 0.049） (圖 3-20A)，PS-F2 對C26 腫瘤細胞也具有良好之抑制效果。
觀察犧牲後小鼠脾臟，給予小鼠 PS-F2 的脾臟重量也顯著變大（圖 3-21）。
分析脾臟細胞族群比例之變化，CD3 細胞比例從 15.13 ± 1.30%增加至 19.27 ± 1.74%（naïve 小鼠 21.79 ± 1.40%），CD19 細胞比例從 21.82 ± 1.26%增加至 28.71
± 0.02%（naïve 小鼠 41.42 ± 1.20%），結果顯示腹腔注射 PS-F2 使 T 細胞和 B 細 胞族群比例均有增加（圖3-22A）。CD4 細胞比例從 11.27 ± 0.66%增加至 13.55 ± 0.10%（naïve 小鼠 15.72 ± 0.40%），CD8 細胞比例從 4.51 ± 0.28%增加至 4.95 ± 0.46%（naïve 小鼠 5.71 ± 0.26%），T 細胞中的 CD4 和 CD8 T 細胞比例也同時都 有增加之情形(圖 3-22B)。對 NK 細胞毒殺活性影響上，腹腔注射 PS-F2 跟給予 PBS 的控制組沒有明顯的增減，只與沒有接種 C26 腫瘤的 naïve 小鼠比有較高的 毒殺活性 (圖 3-23)。為了觀察給予 PS-F2 試驗期間小鼠血清中抗體的變化情形，
本次試驗另在第十二天以眼窩採血分析血清中抗體濃度。在第十二天時，給予 PS-F2 的小鼠血清 IgM 濃度已高於 PBS 控制組和 naïve 小鼠；在第二十四天時，
0
Days after tumor implantation
Body weight (g)
Time after inoculation (day)
Tumor size (mm3 ) PBS PS-F2
*
*
* *
圖3-19. PS-F2 對 C26 抗腫瘤之效果
Figure 3-19. Antitumor effect of PS-F2 in C26 tumor-bearing mice. The body weight (A) and tumor size (B) were monitored.
C26 (1×105 cells) was subcutaneously implanted into BALB/c mice. PS-F2 (50mg/kg) was i.p. administered to mice for 3 weeks starting from 24 h before inoculation.
Control mice were treated with equal amounts of PBS alone on the same schedule.
Data are the means ± SE (n=10). *P<0.05; **P<0.01 compared to the PBS group.
(A)
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Tumor
W ei ght ( g)
PBS PS-F2
*
(B)
圖3-20. PS-F2 對 C26 腫瘤生長之影響
Figure 3-20. Anti-tumor effects of PS-F2 in C26 tumor-bearing mice. The change of tumor weight (A) was determined on day24. (B) is tumor picture after cut off.
C26 (1×105 cells) was subcutaneously implanted into BALB/c mice. PS-F2 (50mg/kg) was i.p. administered to mice for 3 weeks starting from 24 h before inoculation.
Control mice were treated with equal amounts of PBS alone on the same schedule.
Data are the means ± SE (n=10). *P<0.05 compared to the control group.
PBS
PS-F2
0.0 0.1 0.2 0.3 0.4 0.5 0.6
Spleen
W ei ght ( g)
PBS PS-F2
*
圖3-21. PS-F2 對接種 C26 小鼠脾臟重量之影響
Figure 3-21. Effect of PS-F2 on spleen weights in C26 tumor-bearing mice.
Data are the means ± SE (n=10). *P<0.05 compared to the control group.
(A)
0 10 20 30 40
CD3 CD19
P er cen ta ge o f p os iti ve cel ls PBS PS-F2
*
(B)
0 5 10 15 20
CD4 CD8
Pe rc en ta ge o f p os itiv e c ell s
PBS PS-F2
圖3-22. PS-F2 對接種 C26 小鼠脾臟細胞族群比例之影響
Figure 3-22. Effect of PS-F2 on the populations of T and B cells (A) and CD4 and CD8 T cells (B) in splenocytes of C26-bearing mice.
The date are the means ± SE for each group (n = 10).
0 5 10 15 20 25 30 35
100:1 50:1
NK cell avtivity (%)
PBS PS-F2 Naive
圖3-23. PS-F2 對接種 C26 小鼠脾臟 NK 細胞毒殺活性之影響
Figure 3-23. Effect of PS-F2 on NK cells activities in C26 tumor-bearing mice.
Cytotoxicity of NK cells were assessed by measuring the lysis of the target YAC-1cells mediated by splenocytes. Data are the means ± SE (n = 10).
給予PS-F2 的小鼠血清 IgM 濃度則顯著高於 PBS 控制組和 naïve 小鼠，其中 PBS 組比naïve 小鼠低(圖 3-24)。
綜合三種不同的腫瘤模式的結果均顯示 PS-F2 之抗腫瘤活性可能是透過增 加脾臟 T 細胞的比例，活化對腫瘤抗原具專一性之 CD4 與 CD8 T 細胞，藉由 CD8 T 細胞達到毒殺腫瘤細胞之效果，而 CD4 T 細胞的活化能進一歩刺激 B 細
胞活化產生針對腫瘤抗原之抗體，在血清中的 IgM 抗體濃度同時也檢測到顯著
性增加，符合假設。這中間的抗腫瘤機制含括了細胞介導免疫反應和體液免疫。