由於 ATA 對於細胞低毒性，在醫學方面有廣泛的研究，如可促進細胞增生與
抑制細胞凋亡[Andrew et al., 1999]。先前研究發現低濃度(15 ppm) ATA 可引起懸 浮培養 CHO β-gal SF cell 產生形態改變貼附到培養皿，高濃度(50 ppm) ATA 影 響細胞聚集，造成細胞懸浮不聚集現象[Liu et al., 2001a]。實驗在 DMEM/F12 無 血清培養基中添加低濃度(30 ppm) ATA，亦可看到 ATA 促進 CHO β-gal SF cell 生長之現象，但高濃度(100 ppm) ATA 不會促進細胞生長(圖五)。我們有興趣的是 ATA 可改變細胞型態，添加 30 ppm DMSO 不會改變細胞型態(圖六 B)，30 ppm ATA 使部份懸浮細胞改變型態而變成貼附狀態(圖六 C)，100 ppm ATA 可造成細 胞由聚集變成不聚集之現象(圖六 D)。
5.5 ATA 對 CHO β-gal SF cell 細胞型態之影響
ATA 為無法穿透細胞膜之分子，因此分別偵測位於細胞膜上的 cadherin 和
integrin 表現，以探討 ATA 濃度對細胞造成型態(adhesion 與 aggregation)改變之機 制。Cadherin 與 aggregation 有關，讓細胞與細胞間相連與交互作用，integrin 與 adhesion 有關，可和 extra cellular molecule 結合使細胞貼附[Grodzki et al., 2003;
Koch et al., 2004; Patel et al., 2003; Schlippe et al., 2000] 。圖七 e、h、k、n、q 為 cadherin 免疫螢光染色結果，CHO β-gal SF cell 皆有 cadherin 表現，顯示 ATA 濃度不影響 cadherin 表現。在 integrin 方面，發現 ATA 可引起 integrin 表現(圖八
h、k、n)，integrin 表現程度受到 ATA 濃度影響，30 ppm ATA 使 CHO β-gal SF cell 貼附，其 integrin β1 表現較懸浮細胞強(P < 0.05)。100 ppm ATA 可造成細胞由 聚集狀態變成不聚集，偵測 integrin β1 表現，發現其 integrin β1 表現(圖八 n) 較貼附細胞(圖八 k)強(P < 0.05)，定量結果亦顯示 100 ppm ATA 引起 integrin β1 過度表現(overexpression)，顯示 ATA 濃度會影響 integrin β1 表現，高濃度 ATA 導致細胞由聚集變成不聚集狀態，可能是經由 integrin 所調控。
Integrins 是 adhesion 最主要的角色，可藉由 hetero-type 方式和細胞膜上其他 extra cellular molecule (ECM)結合，讓細胞貼附到基質上[Rosenberger et al., 2004]。Integrins 也可藉由 homo-type 方式和其他 integrins 結合，造成細胞之間 aggregation 的情形[Khyrul et al., 2004]。Integrin cytoplasmic domain 可與 calpain 或 integrin-linked kinase (ILK)結合，進而調控下游 actin cytoskeleton 影響細胞貼附 與細胞間交互作用(如附圖六所示)，影響細胞生長與分化。研究發現 ILK 過度表 現可引起 cyclin D1、Cdk4、cyclin E-associates kinase 活化，及引起 Cdk inhibitors p21 和 p27 表現，並導致細胞從貼附狀態變成懸浮狀態生長[Radeva et al., 1997]。
ILK 過度表現也會抑制 ECM fibronectin polization 與 motility [Hannigan et al., 1996;
Khyrul et al., 2004] ，及抑制細胞內 actin organization，造成 cell spreading 改變與 抑制細胞 adhesion，影響細胞生長 [Etienne-Manneville et al., 2002] 。細胞經由 integrin 與 ECM 作用，調控細胞 shape、motility，刺激細胞生長、分化及基因表
現，因此推測高濃度(100 ppm ) ATA 引起 CHO β-gal SF cell integrin β1 過度表 現時，也可能促進 ILK 過度表現而抑制細胞間 ECM 如 fibronectin 之作用，進而 影響聚集細胞變成不聚集狀態。
320 330 340 350 360 370 380
0 15 30 45
Time course (hours)
Osmolarity (mosmol/kg)
MEM
MEM with 9 g/L glucose MEM with 9 g/L sorbitol
圖一. 培養時間與培養基滲透壓之關係
收取各培養時間點培養基測量滲透壓，在不同時間下測得高葡萄糖培養基和 山梨醇培養基仍維持高滲透壓趨勢(滲透壓 350 mosmol/kg 以上)。MEM 培養基維 持低滲透壓趨勢(滲透壓 330 mosmol/kg)。
(A)
Specific growth rate (1/day) ＊
＊ P < 0.05
(A)
(A)
(A) ATA 會促進細胞生長，100 ppm ATA 不會促進細胞生長。Control 組比生長速率為 0.81 ± 0.01 /天，添加 30 ppm DMSO 比生長速率為 0.95 ± 0.01 /天，30 ppm ATA 比生長速率為 1.02 ± 0.15 /天，100 ppm ATA 比生長速率為 0.74 ± 0.09 /天。
aggregation A. Control (DMEM/F12)
B. 30 ppm DMSO
aggregation
C. 30 ppm ATA adhesion
D. 100 ppm ATA
non-aggregation
圖六. ATA 對 CHO β-gal SF cell 細胞型態之影響
(A)Control (DMEM/F12 培養基) CHO β-gal SF cell 懸浮聚集型態 (B)添加 30 ppm DMSO，細胞懸浮聚集的型態 (C)添加 30 ppm ATA，部分細胞為懸浮聚集，
部分細胞貼附至培養皿成梭狀之型態 (D)添加 100 ppm ATA，細胞懸浮不聚集的 型態。
A. Negative control
a b c
(比例尺圖示 50μm)
B. Control (suspension)
d e f
C. DMSO (suspension)
g h i
D. 30 ppm ATA (suspension)
j k l
(比例尺圖示 50μm)
E. 30 ppm ATA (adhesion)
m n o
F. 100 ppm ATA (suspension)
p q r
Cadherin 表現程度 Suspension Adhesion control 1011 ± 54 -
30 ppm DMSO 917 ± 36 - 30 ppm ATA 879 ± 170 602 ± 160 100 ppm ATA 954 ± 28 -
圖七. ATA 濃度對 CHO β-gal SF cell cadherin 表現影響
(A)未加入一級 cadherin 抗體 negative control (B)加入 cadherin 抗體之 control (DMEM/F12)，細胞懸浮聚集的型態 (C)添加 30 ppm DMSO，細胞懸浮 聚集的型態 (D)添加 30 ppm ATA，細胞懸浮聚集的型態 (E)添加 30 ppm ATA，
細胞貼附的型態 (F)添加 100 ppm ATA，細胞懸浮不聚集的型態。a、d、g、j、
m、p 圖為 confocal 顯微鏡可見光照相結果。b、e、h、k、n、q 為螢光染色結果。
c、f、i、l、o、r 為可見光與螢光染色重疊結果。以 Fluoview 軟體對於細胞螢光 染色結果定量，將 cadherin 表現強度數值化如上表所示，定量結果顯示不同 ATA 濃度不影響 cadherin 表現。
A. Control (suspension)
a b c
(比例尺圖示 50μm)
B. DMSO (suspension)
d e f
C. 30 ppm ATA (suspension)
g h i
D. 30 ppm ATA (adhesion)
j k l
(比例尺圖示 50μm)
E. 100 ppm ATA (suspension)
m n o
Integrin 表現程度 Suspension Adhesion
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柒. 附表
捌. 附圖
Shaw A et al., 2003
附圖一. 葡萄糖活化 p42 MAPK 之訊息途徑 R 4
R1 R2 R3
glucose
G protein cAMP AC
PKA
Raf
MEK
nucleus gene expression ERK
Cowan et al., 2003
Proliferation Apoptosis Osmoregulation Cellular
Differentiation Tumorigenesis Oxidative stress Responses
Development Inflammation Apoptosis
附圖三. Aurintricarboxylic acid 結構
cadherin
catenin
catenin
actin
附圖四. Cadherin 結構及其訊息途徑
A. Glucose B. Sorbitol
附圖五. (A)葡萄糖結構式 (B)山梨醇結構式
Rosenberger G et al., 2004
附圖六. Integrin 影響細胞貼附之訊息途徑