孔雀石綠（malachite green）是一種三苯甲烷染劑，廣泛使用於水產業，作為抗黴菌抑制劑，malachite green會被代謝還原成還原態孔雀石綠（leucomalachite green），存在於魚類組織中，消費者容易暴露於malachite green 與leucomalachite green污染的食物中，且有證據顯示，在老鼠肝臟能促進癌症形成，因結構上與致癌物質相似，而被懷疑具有致癌性，而許多肝臟毒性物與肝臟致癌物會藉由干擾細胞間縫隙連接相互交流（GJIC），而改變細胞與細胞之間訊息傳遞，本篇研究主要經由malachite green 與leucomalachite green處理老鼠肝臟表皮細胞後，是否會阻斷細胞與細胞之間交互作用。利用SL/DT(scrape loadind/dye transfer)來偵測加入malachite green 與leucomalachite green處理1小時後，細胞間縫隙連接相互交流（GJIC）情形，利用MTT與細胞株形成效率實驗，來偵測細胞存活率與細胞生長繁殖情形，且利用西方墨點法與免疫螢光染色法，來測定connexin43、E-cadherin、β-catenin、α-tubulin、ZO-1與 occludin蛋白質在細胞內的分布與表現。老鼠肝臟表皮細胞用malachite green（0~5μg/ml）或leucomalachite green（0~6μg/ml）分別處理1小後，GJIC隨濃度增加明顯下降，malachite green 與leucomalachite green隨濃度增加，也降低了細胞存活率與細胞繁殖複製能力，而細胞經由蛋白磷酸酶C（protein kinase C）抑制劑先處理20分鐘後，加入5μg/ml malachite green暴露1小時，發現GJIC有明顯增加，以相同方式，將細胞以MEK 1抑制劑處理後，不論暴露於5μg/ml malachite green或5μg/ml leucomalachite green中，GJIC均有明顯增加。再以PI3 kinase抑制劑對細胞進行處理，對leucomalachite green造成細胞間GJIC明顯增加，且減少細胞外鈣離子的濃度，以轉錄層面而言，以10μg/ml孔雀石綠或10μg/ml孔雀石綠代謝物處理1小時後，connexin43 mRNA量無改變，在轉譯層次上，以不同濃度及時間處理細胞後可見connexin43、E-cadherin、β-catenin、α-tubulin、 ZO-1 蛋白質有減少趨勢，而occludin 蛋白質以螢光免疫染色觀察，occludin擴散分佈於細胞質中。因此，由本研究實驗結果推測，藉由減少connexin蛋白質量與改變connexin結合蛋白分佈表現，會干擾細胞間GJIC，而阻斷細胞與細胞間訊息傳遞，有助於了解孔雀石綠與孔雀石綠代謝物對細胞造成毒性、致癌性與細胞凋亡有關之機制。

Malachite green(MG), an N-methylated diaminotriphenylmethane dye, has been widely used as an antifungal agent in aquaculture. Malachite green is reduced to and persists as leucomalachite green(LMG) in the tisssues of fish. Concern over MG and LMG are due to the potential for consumer exposure, suggestive evidence of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. Several hepatotxicants and liver carcinogens have been shown to alter cell-cell signaling by interference with gap junction intercellular communication (GJIC).This study wanted to determine if disruption of cell-cell interactions occurs in rat liver epithelial cells in response to MG and LMG treatment. Rat liver epithelial cells(WB) were treated with LMG(0~6μg/ml) or MG.(0~5μg/ml) for one hour and gap junction was analyzed using the scrape- loading/dye transfer assay. The viability and proliferation of rat liver epithelial cells treated with LMG or MG were determined by MTT and colony forming efficiency. In addition, expression and intracellular localization of connexin43, E-cadherin,β-catenin,α-tubulin, ZO-1 and occludin were determined by immunoblot and immunostain analysis. A clear decrease in the distance of dye transfer was evident following treatment with MG(0~5μg/ml) or LMG(0~6μg/ml). Treatment with LMG and MG at different concentrations resulted in a decrease in cell viability and cell proliferation. Preincubation of cells with protein kinase C(PKC) inhibitor decreased the inhibition of GJIC by 5ug/ml MG and the specific MEK 1 inhibitor decreased substantially the inhibition of GJIC by 5μg/ml MG and 5μg/ml LMG. On the other hand, the specific PI3 kinase inhibitor decreased the inhibition of GJIC only by 3μ/mlLMG and we treated WB cells with EDTA to chelate extracellular calcium ion. The decrease of free calcium ion caused the expression of GJIC. At the transcriptional level, 10μg/ml LMG and 10μg/ml MG after treatment for one hour caused no change in the level of connexin43 mRNA. At the translational level, the different concentrations of MG or LMG after treatment for one hour or 24 hours caused a decrease in the level of the concentrations of connexin43 protein, E-cadherin protein,β-catenin protein,α-tubulin protein, ZO-1 protein and changed the distribution of occludin and ZO-1. Therefore, these data speculated the hypothesis that disruption of cell-cell signaling by interference with GJIC may contribute to LMG and MG toxicity, carcinogenicity and apoptosis.

目錄
英文摘要……………………………………………………1

中文摘要……………………………………………………2

第一章、 前言………………………………………………3

第二章、實驗材料與實驗方法……………………………6
第一節 實驗材料……………………………………………6
第二節 實驗方法……………………………………………7
一、細胞培養（cell culture）……………………………………………7
二、細胞存活率實驗（cell viability assay）……………………………7
三、細胞間縫隙連接相互交流生物評估實驗 （GJIC bioassay）……8
四、免疫螢光染色（immunofluorescence stain）………………………8
五、細胞株形成效率（colony forming efficiency）……………………9
六、蛋白質表現分析（Western blotting）………………………………9
七、RNA表現分析 ………………………………………………………10

第三章、實驗結果 …………………………………………11

第四章、討論 ………………………………………………19

第五章、結論 ………………………………………………25

參考文獻……………………………………………………27

圖表…………………………………………………………31
Fig.1 The structures of malachite green（MG）and leucomalachite green（LMG）…………………………….………………………………...31
Fig.2 Gap junctional intercellular communication（GJIC） in WB cell line
as measured by the Lucifer yellow scrape-loading/dye transfer technique.……………………………………………………………….32
Fig.3 Concentration dependent inhibition of GJIC by malachite green in WB cells after the 1hr exposure period.…………………………….………..33
Fig.4 Time course dependent inhibition of GJIC by 5 μg/ml malachite green
in WB cells.……………………………………………………………...34
Fig.5 Gap junctional intercellular communication（GJIC）in WB cell line as measured by the Lucifer yellow scrape-loading/dye transfer technique.………………………………………………………………..35
Fig.6 Concentration dependant inhibition of GJIC by leucomalachite green in WB cells after the 1hr exposure period.…………………………………36
Fig.7 Time course dependent inhibition of GJIC by 3μg/ml leucomalachite green in WB cells..………………………………………………………37
Fig.8 Inhibition of the viability of WB-F344 cells by malachite green.…………………………………………………………………….38
Fig.9 Inhibition of the viability of WB-F344 cells by leucomalachite green.…………………………………………………………………….39
Fig.10 Colony-forming efficiency by WB cells exposured to malachite green for 7 days.…………………………………………………………………...40
Fig.11 Colony-forming efficiency by WB cells exposured to leucomalachite green for 7 days..……………………………………………………….41
Fig.12 Effects of 5μg/ml malachite green (MG), 5μg/ml leucomalachite green (LMG) and 10 nM TPA alone (1h exposure ) or in combination with 10μM GF109203X (PKC inhibitor ) or 50μM PD98059 (MEK 1 inhibitor ) on GJIC in WB cells.………………………………………...42
Fig.13 Effects of 3μg/ mlmalachite green(MG) , 3μg/ml leucomalachite
green (LMG) and in combination with 100 μmol/ml LY294002
on GJIC in WB cells.…………………….................................................43
Fig.14 Gap junctional intercellular communication in WB cell line as
measured by the Lucifer yellow scrape-loading/dye transfer technique…………………………………………………………….....44
Fig.15 Effects of 20 μg/ml EDTA（Lane A）, 40 μg/ml EDTA（Lane B）
and 60 μg/ml EDTA（Lane C） in combination with 6 μg/ml leucomalachite green on GJIC in WB cells ............................................45
Fig.16 Gap junctional intercellular communication in WB cell line as
measured by the Lucifer yellow scrape-loading/dye transfer technique…………………………………………………………..……46
Fig.17.Effects of 5 ng/ml BAPT-AM in combination with 6 μg/ml leucomalachite green （Lane C）on GJIC in WB cells .…………..….47
Fig.18. Immunolocalization of connexin43 proteins after exposuring WB
cells to 5μg/ml malachite green(B) or 6μg/ml leucomalachite green
(C) for 1h. Connexin43 proteins were visualized by immunolabeling fixed, permeabilized WB cells with anti-connexin43 antibody and detected with anti-mouse IgG conjucated with Alexa Fluor. DNA was counterstained with DAPI in all images………………………………..48
Fig.19 Immunolocalization of connexin43 proteins after exposuring WB cells
to 0.1 μg/ml malachite green(B) or 6μg/ml leucomalachite green (C)
for 24 h. Connexin43 proteins were visualized by immunolabeling fixed, permeabilized WB cells with anti-connexin43 antibody and detected with anti-mouse IgG conjucated with Alexa Fluor. DNA
was counterstained with DAPI in all images………………………..….49
Fig.20 RT-PCR analysis of connexin43 mRNA (1 h of malachite green or leucomalachite green exposure in WB cells )………………………...50
Fig.21 Western blot analysis of connexin43 in WB cells………………………51
Fig.22 Immunofluorescence analysis of E-cadherin in WB cell treated with 0.1μg/ml, malachite green（MG）after 24h exposure or 5μg/ml
MG after 1hr exposure were clearly altered, indicating modulation of cell-cell adhesion…………………………………………………….....52
Fig.23 Immunofluorescence analysis of E-cadherin in WB cell treated with
6μg/ml leucomalachite green（LMG）after 24h exposure and
6μg/ml LMG after 1h exposure were clearly altered, indicating modulation of cell-cell adhesion………………………………………...53
Fig.24 Western blot analysis of E-cadherin in WB cells…………………….....54
Fig.25 Immunofluorescence analysis ofβ-catenin in WB cell treated with
0.1μg/ml malachite green（MG）after 24h exposure or 5μg/ml MG
after 1hr exposure were clearly altered, indicating modulation of cell-cell adhesion…………..….………………………………………..55
Fig.26 Immunofluorescence analysis of β-catenin in WB cell treated
with 3, 6μg/ml leucomalachite green（LMG）after 24h exposure
and 20μg/ml LMG after 1h exposure were clearly altered,
indicating modulation of cell-cell adhesion……………..………….…56
Fig.27 Western blot analysis ofβ-catenin in WB cell………………………...57
Fig.28.Immunolocalization ofα- tubulin proteins after exposuring WB
cells to 5μg/ml malachite green(B) for 1 h or 0.1μg/ml malachite
green (C) for 24 h. α- tubulin proteins were visualized by
immunolabeling fixed, permeabilized WB cells with anti-α- tubulin antibody and detected with anti-mouse IgG conjucated with Alexa
Fluor. DNA was counterstained with DAPI in all images.……………..58
Fig.29 Immunolocalization ofα- tubulin proteins after exposuring WB
cells to 6 μg/ml leucomalachite green(B) for 1 h or (C) for 24 h.
α- tubulin proteins were visualized by immunolabeling fixed, permeabilized WB cells with anti-α- tubulin antibody and detected
with anti-mouse IgG conjucated with Alexa Fluor .DNA was
counterstained with DAPI in all images……………………………….59
Fig.30 Western blot analysis ofα- tubulin in WB cells………………………..60
Fig.31 Immunolocalization of ZO-1 proteins after exposuring WB cells
to 5 μg/ml malachite green(B) for 1 h or 0.1μg/ml malachite
green (C) for 24 h. ZO-1 proteins were visualized by immunolabeling fixed, permeabilized WB cells with anti- ZO-1 antibody and
detected with anti-mouse IgG conjucated with Alexa Fluor. DNA
was counterstained with DAPI in all images…………...………………61
Fig.32 Immunolocalization of ZO-1 proteins after exposuring WB cells to
6μg/ml leucomalachite green(B) for 1 h or (C) for 24 h. ZO-1 proteins were visualized by immunolabeling fixed, permeabilized WB cells with
anti- ZO-1 antibody and detected with anti-mouse IgG conjucated
with Alexa Fluor. DNA was counterstained with DAPI in all images……………………………………………………..……..….....62
Fig.33 Immunolocalization of Occludin proteins after exposuring WB cells
to 5 μg/ml malachite green(B) for 1 h or 0.1 μg/ml malachite green
(C) for 24 h. Occludin proteins were visualized by immunolabeling
fixed, permeabilized WB cells with anti- Occludin antibody and
detected with anti-mouse IgG conjucated with Alexa Fluor. DNA
was counterstained with DAPI in all images…………………………...63
Fig.34 Immunolocalization of Occludin proteins after exposuring WB
cells to 6 μg/ml leucomalachite green (B) for 1 h or (C) for 24 h. Occludin proteins were visualized by immunolabeling fixed, permeabilized WB cells with anti- Occludin antibody and detected
with anti-mouse IgG conjucated with Alexa Fluor. DNA was counterstained with DAPI in all images……………………………....64

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