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研究生:葉士齊
研究生(外文):Yei Shieh-chi
論文名稱:受甲狀腺素正向調控spondin2與細胞移動及入侵之研究
論文名稱(外文):Positive regulation of spondin 2 by thyroid hormone associates with cell migration and invasion
指導教授:林光輝林光輝引用關係
指導教授(外文):Lin Kwang-Huei
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:50
外文關鍵詞:spondin 2thyroid hormone
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甲狀腺素 (3, 3’, 5-triiodo-L-thyronine, T3)是一可調控細胞生長、發育與分化的重要因子,而這些調控是透過甲狀腺受體(Thyroid hormone receptor, TR)來進行。甲狀腺受體屬於固醇類荷爾蒙受體家族之一,是一甲狀腺素依賴型的轉錄因子,而甲狀腺素便是藉由細胞核內的甲狀腺素受體將其訊號傳至細胞內。我們之前利用cDNA microarray分析技術研究在過度表現甲狀腺素受體的肝癌細胞株 (HepG2-TRα1)中,甲狀腺素對下游基因的調控,結果顯示有201個基因受到甲狀腺素正向調控,包含spondin 2。Spondin 2是ECM (extracellular matrix)上的一個分子,可以調控海馬迴神經的生長,且對microbial pathogen上的分子進行辨識,進行清除病菌。
Spondin 2在過去的研究,發現是在肺癌中可能扮演著腫瘤抑制子的角色,並在我們之前的cDNA microarray結果中,是正向受到T3調控較明顯的基因,但其之間的機制和生理意義仍不清楚。利用在過度表現甲狀腺素受體的肝癌細胞株中,經T3 處理48小時後spondin 2 mRNA表現量可上升5~20倍;而蛋白質表現量在經T3處理後表現也有增加15~20倍的情形。為了解spondin 2在細胞內的功能角色,我們在Huh7細胞株中建立knock-down spondin 2表現的細胞株,結果發現在knock-down spondin 2 的細胞株,細胞增生能力提高兩倍,移動和侵入能力也增加三倍以上;在gelatin-zymography實驗中, MMP-2增加兩倍;另外Snail、E-cadherin、Fibronectin和Vimentin這些已知在轉移會有變化的標誌,在Q-RT-PCR中也具有相同變化。根據結果顯示,spondin 2 在肝臟腫瘤生成和發展過程中,可能扮演重要角色,更進一步的研究將是確認spondin 2 在體內的生理意義。
The thyroid hormone, 3, 3', 5-triiodo-L-thyronine (T3) regulates growth, development and differentiation processes in animals. These activities are mediated by the nuclear thyroid hormone receptors (TRs). They belong to the steroid/thyroid hormone receptor superfamily of ligand dependent transcription factors. To study the regulation mechanism after T3 treatment in TRα-overexpressed hepatoma cell line, cDNA microarrays analysis were performed. From the microarray results, 201 of 7600 genes were positively regulated by T3, including spondin 2 (SPON2). SPON2 is a conserved member of a family of secreted extracellular matrix proteins. It not only promotes the hippocampus outgrowth but also acts as an opsonin and pattern-recognition molecule for a range of pathogens.
In the previous studies, SPON2 may play a role of tumor suppressor in lung cancer. However the mechanism and physiological role of T3, SPON2 and cancer are not clear. To confirm the microarray results, SPON2 was further investigated by quantitative RT-PCR and Western blot. After 48 h of T3 treatment in HepG2-TRα1 cell line, SPON2 mRNA and protein level was increased 5- to 20-fold. To explore the effect of SPON2 on cellular function, the SPON2 knock-down cell lines were established in Huh7. The SPON2 knock-down cells had high ability of cell proliferation, migration and invasiveness compared to control cell. Together these experimental these findings indicated SPON2 has an important role in hepatocarcinogenesis and tumor progression. Further study is required to determine the physiological role of SPON2 in vivo.
第一章 緒論
甲狀腺素調控機制…………………….………………………………1
甲狀腺素……………………….………………………………..1
甲狀腺素受體………………….……………………………..…2
甲狀腺素反應元……………….…………………………..……4
Spondin介紹………………………….……………………………….4
Spondin 2的表現和功能 ………….………………………………...5
促進神經細胞生長……………………………………………...5
抑制血管新生………………….………………………………..6
細菌上的病源體的辨認……….………………………………..6
SPON2 和癌症…………………………………………..……..7
第二章 材料與方法
細胞培養(Cell culture)……………………………………………….9
3, 3’, 5-triiodo-L-thyronine (T3)溶液之配製…………………………9
T3-depleted serum (Td)之配製……………………………………….9
萃取RNA和蛋白質的細胞前處理…………………………………10
RNA之萃取…………………………………………………………10
定量反轉錄聚合酶連鎖反應……………………………………….10
北方墨點法(Northern blot)………………………………………….11
蛋白質萃取………………………………………………………….12蛋白質定量………………………………………………………….12
西方墨點法(Western blot)…………………………………….……..13
收集細胞培養液…………………………………………………….13
建立穩定抑制SPON2表現的細胞株………………………………14
細胞增生實驗……………………………………………………….14
細胞移行和侵入實驗(Cell migration and invasion assay)….………14
明膠基質金屬蛋白酵素活性測試(Gelatin Zymography)………….15
結果分析…………………………………………………………….15
第三章 結果
cDNA microarray分析結果………………………………………...17
即時定量聚合酶連鎖反應偵測T3處理後細胞內SPON2表現情形……………………………………………………….……………17
以北方點墨法偵測T3處理後的HepG2-TRα1#1 SPON2表現的情形…………………………………………………….………………17
以西方點墨法偵測T3處理後細胞內SPON2表現情形….………...18
以西方點墨法偵測T3處理後細胞內SPON2分泌到細胞外的表現情形………………………………………………………….…………18
利用西方點墨法偵測肝癌病人肝組織中SPON2的表現量………18
分析T3對SPON2之正向調控機制是否為間接調控………………19
分析T3對SPON2基因上游啟動子之活性影響……………………19
西方點墨法偵測不同的肝癌細胞株中SPON2的表現量…………20
西方點墨法偵測穩定抑制SPON2表現量之細胞株中SPON2的表現量……………………………………………………………….....20
穩定抑制SPON2表現量之細胞株細胞增生的情形………………21
穩定抑制SPON2表現量之細胞株細胞移行的情形………………21
穩定抑制SPON2表現量之細胞株細胞入侵的情形………………21
明膠基質金屬蛋白酵素活性測試(Gelatin Zymography)….………22
利用即時定量聚合酶連鎖反應偵測在細胞進行轉移過程中基因的變化………………………………………………………….………22
第四章 討論…………………………………………………...………23
參考文獻………………………………………………………….……..27
附錄……………………………………………………………….……..32
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