Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21742
標題: Study of the effect of FUT4 and FUT6 gene expression on human lung cancer cell line
探討FUT4及FUT6基因表現對人類肺癌細胞株的影響
作者: Chu, Jin-Wei
朱巾微
關鍵字: 岩藻醣;fucosyltransferase;肺癌;多株抗體;IgY;lung cancer
出版社: 分子生物學研究所
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摘要: 
The mechanisms of lung cancer carcinogenesis are quite complicated. One of the mechanisms is closely related to the expression of human glycosyltransferase genes. From the viewpoint of glycobiology, carcinogenesis results from disturbed, hyper- or hypo-glycosylation of cell membrane glycoprotein. Abnormal glycoconjugates interfere with intercellular recognition, and in turn increases metastatic potential through adhesion of tumor cells and vascular endothelial cells.
In order to prove the roles of a-1,3 fucosyltransferase IV and a-1,3 fucosyltransferase VI in lung cancer carcinogenesis, we have successfully transfected either FUT 4 or FUT 6 gene into A549 lung cancer cells.
In addition, we also construct FUT 4 gene into pET-20b plasmid and FUT 6 gene into pET-29a plasmid to produce a large amount of fucosyltransferases. After immunizing the chickens, FUT 4 and FUT 6 polyclonal antibodies were successfully purified from the yolks and confirmed by the western blot. We then transfected A549 lung cancer cells with either FUT4 or FUT6 genes, and conducted experiments with PCR, real-time PCR, western blot, flowcytometry, adhesion test and invasion test . The results show that overexpression of FUT4 or FUT6 gene could elevate mRNA and protein level in A549 lung cancer cells and also promote the expression of A549 lung cancer cells surface antigen Lewisx and sialyl-Lewisx. Moreover, overexpression of FUT4 or FUT6 gene could enhance the A549 lung cancer cells adhesion and invasion . The SCID was used for in vivo experiments, and the results demonstrated that overexpression of FUT4 gene could increase lung tumor formation and metastasis. However, overexpression of FUT6 gene represses lung tumor formation and metastasis. In conclusion, both FUT4 and FUT6 genes, especially FUT4 gene, increase not only the local invasion of A549 lung cancer cells, but also their metastatic potential.

肺癌產生的原因相當複雜,其中與人類醣轉移酶基因的表現有著密不可分的關係。從醣質生物學的角度來看,細胞的癌化是細胞膜上醣質組成和構造產生雜亂、過多或不完整的醣質化現象,因而產生不尋常的醣鏈,導致細胞間的辨識系統失控,最後變成侵略性的轉移。
為了證明醣轉移酶基因FUT 4(a-1,3 fucosyltransferase IV)及FUT 6(a-1,3 fucosyltransferase VI)與肺癌致癌因子間的關係。首先,本實驗室將FUT 4及FUT 6基因分別轉染至低表現的A549肺癌細胞株,建立了穩定表現FUT4及FUT6蛋白的載體系統。除此之外,本論文亦將FUT 4及FUT 6基因分別構築在pET-20b及pET-29a載體上藉以大量產生醣轉移酶,接著從免疫雞後所取得的雞蛋黃中萃取FUT 4及FUT 6蛋白的多株抗體,並經西方墨點法分析,確立FUT4及FUT6基因在A549細胞株中有明顯蛋白質的表現。隨後,利用本實驗室所篩選出不同FUT4及FUT6蛋白表現量之A549肺癌細胞轉染株,分別各以反轉錄聚合酶連鎖反應、即時聚合酶連鎖反應、西方墨點法、流式細胞儀試驗、胞外基質黏附(adhesion)試驗、侵犯(invasion)試驗。結果顯示,在過度表現醣轉移酶的A549轉染株中,其mRNA及醣蛋白表現亦有相對性的增加;此外,過度表現FUT4及FUT6蛋白亦可分別增進肺癌細胞表面的Lewisx及sialyl-Lewisx的表現,也因此促進了A549轉染株對胞外基質及E選凝素的黏附並提升A549轉染株的侵犯能力;最後,本論文利用嚴重免疫缺陷鼠(Severe combined immunodeficiency ,SCID)進行活體腫瘤新生實驗及腫瘤免疫組織染色,証明了提升FUT4蛋白表現可同時增加肺腫瘤顆粒的形成及促進肺癌細胞轉移;然而在活體內過度表現FUT6蛋白卻反而會抑制腫瘤顆粒及轉移的發生。綜合上述, FUT4及FUT6基因不僅會促進A549肺癌細胞向外侵犯作用,更可提升其轉移的能力,尤以FUT4 基因為最。
URI: http://hdl.handle.net/11455/21742
其他識別: U0005-2201200723054300
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