結論 - 嘉南藥理科技大學生物科技系碩士論文

1. 經長期處理 ANE 或 ANE 30-100K 的 Jurkat T 和 OECM-1 細胞，對 cisplatin 和 5-FU 的抗藥性以及對低氧的耐受性均增強。

2. 經長期處理ANE或ANE 30-100K的Jurkat T細胞，其peroxiredoxin-1的分泌量增加。

3. 經短期處理 ANE 或 ANE 30-100K 的正常口腔纖維母細胞，其 peroxiredoxin-1的表現量與分泌量增加。

4. 正常組織peroxiredoxin-1的表現集中在上皮細胞，但卻表現在絕大部分的腫瘤細胞中。

5. 嚼食檳榔者的OSCC比未嚼食者表現更高量的peroxiredoxin-1。

6. Arecoline 與 ANE 30-100K 同時會在 Jurkat T 細胞中誘導 IL-6，而後者則誘導 IL-12, IL-13, IL17A, GM-CSF 的表現，可能因而促成口腔中的發炎反應。

7. 經長期處理 ANE 或 ANE 30-100K 的 Jurkat T 細胞，B7 與 FOXP3 蛋白的表現無顯著的增加。

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附表與附圖

活率。(c) Jurkat T 與篩選的細胞經不同濃度的 5-FU ( 0、6.7、18.3、40 mM ) 刺激 24 小時後以 LDH 來測定細胞毒性。(d) OECM-1 與篩選的細胞經不同濃度的 5-FU ( 0、5.6、12、40 mM ) 刺激 24 小時後以 XTT 來測定細胞存活率。＊：P < 0.05，＊＊：P < 0.01。

(a) (b)

1.5 ^{Jurkat T}

ANE-s

1.5 ^{Jurkat T}

ANE-s

0.01，＊＊＊：P < 0.001。

-actin 的 LC3-II/actin 之平均比值除以各自 Jurkat T 組的 LC3-II/actin 之平均比值±

SD 之柱狀圖。＊＊：P < 0.01，＊＊＊：P < 0.001。

(a) Supernatant

(b)

圖 4. Jurkat T 經 ANE 和 ANE 30-100K 長期篩選後，其條件培養液的蛋白質表現圖譜

細胞培養於無血清的培養基下，之後收其條件培養液，利用二維電泳分析。

(a) 條件培養液( supernatant )的圖譜。取 ANE-s 和 30-100K-s 與 Jurkat T 比較，選出表現較強的點。(b) 經由成大貴重儀器中心比對、定序得知此蛋白質為 peroxidoxin-1。

(a)

(b) AN users

9926 9929 10031 10045 10048 0

(a) CMT-415 中不同濃度的 ANE 30-100K 對 PRDX1 的誘導情形。(b) PRDX1 於檳榔使用者正常口腔與腫瘤組織中分布位置與表現區域(%)之統計結果。(c) PRDX1 於非檳榔使用者口腔腫瘤組織分布的情況。(d)非檳榔使用者與檳榔使用者口腔腫瘤組織 PRDX1 表現區域(%)之統計結果。＊：P <

0.05，＊＊：P < 0.01。Bar=200m。

Ju rk a t T A N E -s si -A N E -s -5 1 1 si -A N E -s -5 1 2 3 0 -1 0 0 K -s si -3 0 -1 0 0 K -s -5 1 1 si -3 0 -1 0 0 K -s -5 1 2

PRDX1

-actin

Ju rk a t T A N E -s si -A N E -s -5 1 1 si -A N E -s -5 1 2 3 0 -1 0 0 K -s si -3 0 -1 0 0 K -s -5 1 1 si -3 0 -1 0 0 K -s -5 1 2

PRDX1

-actin

圖 6. Jurkat T 長期篩選後，經不同 PRDX1 RNAi 構築的 lentivirus 感染後，

其 PRDX1 蛋白的表現情形

Jurkat T 經 ANE 和 ANE 30-100K 長期篩選後的細胞株，經不同序列 PRDX1 RNAi（PRDX1-511 及 PRDX1-512）構築的 lentivirus 感染後，以西方墨點法偵測其 PRDX1 的蛋白表現。

圖 7. Jurkat T 經不同的檳榔子成分刺激所誘導之細胞激素

將 Jurkat T(control)以無血清培養基培養 24 小時後，再以 ANE 30-100K (30-100K)與arecoline(Are) IC25 的劑量以及利用 Con A (25μg/ml)處理細胞 24 小時，取其培養液進行細胞激素套組分析。Con A：正控制組。

(a) (b)

Jurkat T ANE-s 30-100K-s FOXP3

GAPDH

Jurkat T ANE-s 30-100K-s B7-H1

GAPDH

Jurkat T ANE-s 30-100K-s 0.0

0.5 1.0 1.5

FOXP3(fold)

Jurkat T ANE-s 30-100K-s 0.0

表一、ANE 及 ANE 30-100K 對不同細胞的篩選濃度

0.033 0.016

30-100K

0.08 0.023

ANE

OECM-1 Jurkat T

0.033 0.016

30-100K

0.08 0.023

ANE

OECM-1 Jurkat T

單位: µg/ml

GATGAGACTTTGAGACTAGTT PRDX1-512 shRNA GCTTTCAGTGATAGGGCAGAA

PRDX1-511 shRNA

or

附圖 1. PRDX1 RNAi 構築的 lentivirus 圖譜

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