臺灣博碩士論文加值系統

Thymidylate synthase (TS)為合成dTTP中的重要酵素，已有文獻指出腫瘤組織的TS表現量較高，且與病患較差的預後呈正相關，但目前對於TS造成癌化的機制仍不清楚。在過去黃承煒的研究結果中，他發現TS造成癌化現象可被高度表現methylenetetrahydrofolate reductase (MTHFR)所抑制。為避免穩定細胞株的偏差性，我建立不同TS表現量細胞株，其表現量均接近人類大腸癌細胞內TS含量，這些穩定細胞株也具癌化現象，我也建立cystathionine-β-synthase (CBS)表現株，可將homocysteine轉變為cystathionine，在這樣的細胞株加上CBS的co-factor vitamin B6，TS無法造成癌化現象，而大量表現MTHFR也能抑制TS之癌化現象。由這些研究結果，顯示單碳連結folate-methionine循環的改變，參與TS造成之癌化現象。另外，透過microarray分析，發現被TS調控上升的基因當中，有58個基因受CBS調控下降，這些基因與膽固醇生合成和發炎反應相關，顯示TS造成的轉型可能與影響這兩種路徑有關。此外，我亦觀察到MTHFD2在TS細胞中表現量上升，MTHFD2的功能為在粒線體中產生10-formyltetrahydrofolate (10-formyl THF)，此化合物參與purine合成的反應。我的研究發現在人類大腸癌細胞內同時減少MTHFD2及TS表現量，可有效抑制細胞生長，這代表MTHFD2之抑制劑應可和5-FU合併使用，治療大腸癌。

Thymidylate synthase (TS) is an essential enzyme for de novo synthesis of dTTP. Several clinical studies have shown that TS expression is highly elevated in tumor tissues and that high TS expression have been associated with poor prognosis of patients. A previous study has indicated that TS overexpression transforms 3T3 fibroblasts. Cheng-Wei Huang in our laboratory has previously shown that overexpression of methylenetetrahydrofolate reductase (MTHFR) that increases 5-mTHF flux for coupling methionine cycle prevents TS-induced transformation. To avoid the clone variation, I generated other TS stable clones with the expression level in the range similar to that in different cancer cell lines. These clones also developed transformed phenotypes. Overexpression of cystathionine-β-synthase (CBS) that diminishes homocysteine-mediated methylation inhibition also intervened TS-induced transformation. These results reveal that the involvement of disturbing folate-methionine cycle by TS overexpression in transformation. The gene expression profiles in these stable clones were analyzed by microarray. Among genes upregulated by TS, there are 58 genes downregulated in TS+CBS cells. These genes are related to cholesterol biosynthesis and inflammation response. Furthermore, the level of MTHD2, which is responsible for 10-formyltetrahydrofolate formation in mitochondria and participates in purine synthesis, is increased by TS overexpression. Finally, I found that decreasing TS and MTHFD2 suppresses the growth of colon cancer cells. Thus, MTHFD2 inhibition is able to sensitize cancer cells to 5-FdU treatment.

中文摘要................................................I
英文摘要................................................II
目錄...................................................III
緒論....................................................1
I. TS與dTTP合成.....................................1
II. TS與癌症的關聯性..................................2
III. TS與單碳代謝(One-carbon metabolism)...............4
實驗動機..................................................10
實驗材料與方法...........................................11
I. Chemicals and reagents..........................11
II. Plasmids........................................11
III. Antibodies......................................12
IV. Cell culture, transfection, and stable line construction............................................12
V. Virus production and infection..................13
VI. Western blot analysis...........................14
VII. Soft-agar assay.................................14
VIII. Colony formation assay..........................15
IX. Methionine sensitivity assay....................15
X. FdU treatment and WST-1 assay...................15
XI. RNA extraction and qPCR.........................16
XII. cDNA preparation and qPCR.......................16
XIII. dNTP pool measurement...........................17
XIV. Statistical analysis............................17
實驗結果.................................................18
I. 表現CBS可抑制TS誘導的細胞轉型.....................18
II. TS過量表現影響細胞對methionine之敏感度............20
III. 分析TS與TS+CBS影響之基因表現......................21
IV. TS細胞之生長依賴MTHFD2...........................24
V. TS和MTHFD2在癌細胞生長之相互關係..................25
VI. MTHFD2 knockdown影響HCT116與SW620大腸癌細胞株 對5-FdU之敏感度..........................................26
討論....................................................28
圖表....................................................35
參考文獻.................................................54
附錄....................................................59

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