Galectin-1對亞砷酸鈉毒性影響之研究

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蔣景程(Jiing-Cherng Jeang) 查詢紙本館藏

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生命科學系

論文名稱

Galectin-1對亞砷酸鈉毒性影響之研究
(Effect of Galectin-1 in Arsenite-induced Toxicity)

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摘要(中)

長期暴露在含有砷化物的環境之下，已知會造成肝臟的損傷與周邊血管疾病，如烏腳病及周邊神經病變等。此外，長期暴露於砷化物亦會提高皮膚癌，肺癌及膀胱癌發生的機會。而根據本實驗室之前的研究指出，三價無機砷會與中國倉鼠卵巢細胞中一種特定蛋白-Galectin-1 (GAL1) 產生結合作用。GAL1蛋白是一種β-半乳糖甘結合蛋白，參與細胞貼附、增生與計劃性死亡的過程。本論文進一步研究GAL1蛋白表現和亞砷酸鈉(As(III)毒性間的關係。
本研究發現以As(III)處理KB細胞，對GAL1表現有明顯抑制現象。為了釐清GAL1蛋白表現，對As(III)毒性的影響，利用維他命C調控細胞中GAL1蛋白之表現，維他命C對KB, HeLa S3及3T3細胞不具有毒性，以1-5 mM維他命C處理KB及HeLa S3細胞5天，對GAL1表現量有明顯抑制作用；以維他命C前處理細胞後，再以As(III)共同處理KB及HeLa S3細胞24小時，可以有效降低As(III)引起之細胞毒性；而維他命C對3T3細胞的GAL1表現之影響為雙相作用(biphasic)，3 mM 維他命C對3T3細胞GAL1表現具有抑制作用，與As(III)共同處理24小時，亦可以有效降低As(III)引起之細胞毒性；1.5 mM維他命C處理3T3細胞，可增加GAL1的表現，與As(III)共同處理24小時，則對As(III)引起之細胞毒性具有協力加強之作用；此一結果顯示經由調控細胞GAL1之表現，可以影響As(III)對細胞之毒性。此外，以trichostatin A（TSA，一種組織蛋白去乙醯脢的抑制劑）處理KB細胞，可增加GAL1蛋白的表現量，而同時處理TSA和亞砷酸鈉也會使As(III)的毒性明顯的增加。此一結果顯示GAL1蛋白表現量可以協力增加As(III) 之毒性。

摘要(英)

Chronic exposure to arsenic has been associated with liver damage, peripheral vascular disease and peripheral neuropathy. Furthermore, exposure to excessive levels of arsenic has been correlated with an increased incidence of skin, lung and bladder cancers. The preliminary study in our laboratory showed that galectin-1 (GAL1) is a sodium arsenite (As(III)) binding proteins in CHOA cells. GAL1 is a member of a growing family of animal β-galactoside-binding proteins that are involved in the regulation of cell adhesion and immune function as well as proliferation and apoptosis. In this study, we further investigate the relationship between As(III)-induced toxicity and GAL1 expression.
The present results showed that As(III) treatment inhibits the expression of GAL1 in KB cells. Vitamin C (Vit. C) shows no toxic to KB, HeLa S3 and 3T3 cells. Pretreatment of KB and HeLa S3 cells with Vit. C dose-dependently inhibits GAL1 expression and attenuates the As(III)-induced cytotoxicity. Futhermore, pretreatment of 3T3 cells with 3 mM Vit. C also inhibits GAL1 expression and attenuates As(III)-induced toxicity. In contrast, pretreatment with 1.5 mM Vit. C results in increase of GAL1 in 3T3 cells, and also potentiates As(III)-induced toxicity. This assumption also supported by the fact that trichostatin A (TSA, a histone deacetylase inhibitor) augments GAL1 expression in KB cells and also increases the toxicity of As(III). These results suggest that GAL1 plays a synergistic effect in As(III)-induced toxicity.

關鍵字(中)

★ 維他命C
★ 砷化物
★ 毒性
★ 亞砷酸鈉
★ GAL1蛋白

關鍵字(英)

★ toxicity
★ vitamin C
★ arsenite
★ galectin-1(GAL1)
★ RNAi

論文目次

Contents………………………..………..…………………….……...Ⅰ
Figure Contents…………….………………………………………Ⅳ
Abbreviation……………….…………….…………………………..Ⅵ
Chapter 1. Introduction
I. Arsenic……………………………..……….……...……………..1
II. Galectin-1 ……………………………………………………… 5
III. Preliminary study…………..……………………………………7
IV. Specific aim……………………………………………………..8
Chapter 2. Materials and Methods
Materials
1. Cell line……………………..……..…………………………….9
2. Antibody……………………………………………….………..9
3. Reagent………………………………..……………….………..9
4. Bacteria culture………………………………………………….10
5. Vector……………………………………………………………10
Methods
1. Cell Culture
1.1 Cell Culture………..…….……………..…………………..10
2. Protein Assay and Quantification
2.1 Cell lysates…………………………………………………11
2.2 Protein quantification…………………...………………….11
2.3 Sodium dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis……….……………………………..11
2.4 Western Blot Analysis……………………………………..12
3. Cell survival assay
3.1 MTT Assay…………………………………………………12
3.2 Fluorescence-Activated Cell Sorter (FACS) analysis……...13
4. Construction of Galectin-1 RNAi expression vector
4.1 Oligo Insert Design……………………………………….13
4.2 Anneal Oligos…………………………………………….14
4.3 Addition of 5`-phosphates on oligonucleotides…………..14
4.4 Linearize the Vector………………………………………15
4.5 Extraction of Plasmid DNA Fragment……………………15
4.6 Ligation into pSUPER.retro Vector………………………16
4.7 Transformation……………………………………………17
4.8 Screening………………………………………………….17
4.9 Mini-preparation of plasmid DNA………………………..17
4.10 DNA Sequencing………………………………………….18
Chapter 3. Result….……………………………………………...19
Chapter 4. Discussion…………………………...….……………26
Chapter 5. Perspective…………………………...…..………….31
Figure…..………………………..………………...…….…….………32
Chapter 6. Reference…………………………………………….47
Appendix …………...…………..………………..…………………...54

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指導教授

黃榮南(Rong-Nan Huang)

審核日期

2004-7-16

推文