臺灣博碩士論文加值系統

癌症所造成的死亡，主要是因為癌細胞轉移至遠處所致。許多癌細胞均具有分泌基質金屬蛋白水解酶 (matrix metalloproteinases, MMPs) 的能力，其中MMP-9扮演重要的角色。MMP-9是一種可以水解細胞外基質 (extracellular matrix, ECM) 的酵素，亦為癌細胞在從原位組織侵入遠處組織的重要因子。nm23-H1基因與乳癌、大腸癌、子宮頸癌、肝癌等癌症的轉移最具相關性。研究發現，提高nm23-H1基因的表現可有效抑制MMPs的分泌，進而抑制腫瘤細胞移動(migration) 以及侵入 (invasion) 等與轉移相關之能力。流行病學研究發現長期補充Vitamin E之族群罹患攝護腺癌機率較低，推測Vitamin E可能具有預防癌症的效果。許多研究也指出生育醇琥珀酸酯 (RRR-α-tocopherol succinate; VES)及生育醇醋酸醚 (RRR-α-tocopherol ether-linked acetic acid; α-TEA) 具有良好的抗癌細胞增生及抗轉移能力，但對於抑制腫瘤的轉移機制仍不確定。因此，在本研究中，我們以具有高轉移活性的人類肝癌細胞SK-Hep-1細胞為模式，探討生育醇(α-tocopherol)、VES、α-TEA對於癌細胞中nm23-H1基因的表現與分泌MMPs能力的影響。結果發現，VES與α-TEA在劑量10 μM下，即可顯著抑制SK-Hep-1細胞的侵入能力 (p<0.05)，且具有劑量關係；VES劑量為50 μM，α-TEA為30 μM時可顯著抑制SK-Hep-1細胞的移動能力 (p<0.05)。VES、α-TEA分別與SK-Hep-1細胞共同培養24小時後，兩者劑量達30 μM時，可顯著抑制SK-Hep-1細胞所分泌之MMP-9活性 (p<0.05)，且具劑量關係。而以生育醇處理則無法抑制MMP-9活性及SK-Hep-1細胞移動、侵入能力。進一步研究發現，α-TEA在劑量10 μM下，可顯著提高nm23-H1的蛋白質表現 (p<0.05)，且具有劑量關係。綜合以上結果，本研究發現VES及α-TEA可抑制人類肝癌細胞SK-Hep-1細胞侵入、移動能力及其所分泌之MMP-9活性。同時，α-TEA抗轉移機制可能是透過提高nm23-H1的蛋白質表現及抑制MMP-9活性，進而降低SK-Hep-1細胞的移動與侵入能力。

Cancer-incurred death is largely due to the distant metastasis of cancerous cells. A large number of cancerous cells has the ability to secrete matrix metalloproteinase (MMPs). Among them, MMP-9, an extractcellular matrix (ECM) enzyme, is a key weapon for cancerous cells to invade distant tissues from in-situ tissues. Nm23-H1 gene is most closely correlated with the metastatic phenotype in human breast carcinoma, colorectal carcinoma, ovarian carcinoma and heptocarcinoma. Nm23-H1 is a potential metastasis suppressor gene that could function on the invasion and migration steps of the metastatic pathway. Epidemiological studies show that the incidence of prostate cancer is low in the population receiving long-term supplements of vitamin E, which suggests a chemopreventive role for vitamin E (The α-Tocopherol, α-Carotene Cancer Prevention Study Group, 1994). Many studies have demonstrated that α-tocopherol succinate (VES) and α-tocopherol ether-linked acetic acid (α-TEA) can effectively inhibit tumor proliferation and metastasis. However, the mechanism underlying such effects on metastasis is still unclear. In this study, we examined the effects of α-tocopherol (α-TOH), VES and a-TEA on invasion, migration, MMP-9 activity and nm23-H1 gene expression of SK-Hep-1 cells. We found that VES and α-TEA significantly inhibited SK-Hep-1 cell invasion and MMP-9 activity released by SK-Hep-1 cells in a dose-dependent manner after incubation for 24 hr (p<0.05). VES and α-TEA also significantly inhibited SK-Hep-1 cell migration after incubation for 5 hr (p<0.05). α-TOH did not affect SK-Hep-1 cell invasion and migration, nor affect MMP-9 activity. Furthermore, the expression of nm23-H1 protein was affected by α-TEA in a dose-dependent manner. In summary, we have demonstrated that VES and α-TEA have significant anti-invasion and anti-migration activities against SK-Hep-1 cells, and that effect is associated with its reduction of MMP-9 activity. At the same time, the effect of α-TEA is associated with its induction of nm23-H1 protein expression.

目錄
縮寫表……………………………………………………………… VI

中文摘要…………………………………………………………… VII

英文摘要…………………………………………………………… IX

文獻整理…………………………………………………………… 1
1.癌症與轉移(metastasis)……………………………………… 1
2.基質金屬蛋白水解酵素………………………………………… 2
3.nm23基因與癌轉移……………………………………………… 4
4.肝癌……………………………………………………………… 5
5.肝癌轉移與nm23-H1及MMPs相關研究…………………………… 6
6.維生素E(vitamin E)…………………………………………… 10
7.生育醇琥珀酸酯(RRR-α-tocopherol succinate；VES)……… 10
8.生育醇醋酸醚(RRR-α-tocopherol ether-linked acetic acid;
α-TEA)…………………………………………………………… 13

研究動機與目的…………………………………………………… 15

實驗架構…………………………………………………………… 17

材料與方法………………………………………………………… 18
一、實驗試劑……………………………………………………… 18
二、細胞解凍……………………………………………………… 19
三、細胞保存……………………………………………………… 19
四、細胞培養……………………………………………………… 19
五、α-TEA合成…………………………………………………… 20
六、生育醇、VES及α-TEA併入SK-Hep-1細胞…………………… 20
七、MTT assay……………………………………………………… 20
八、Cell migration assay……………………………………… 21
九、Cell invasion assay………………………………………… 21
十、Cell adhesion assay………………………………………… 21
十一、蛋白質定量………………………………………………… 22
十二、培養基中MMP-2,-9 activity之測定…………………… 22
十三、西方墨點法………………………………………………… 23
十四、統計分析…………………………………………………… 24

結果………………………………………………………………… 26
一、α-TOH、VES及α-TEA對細胞存活率之影響………………… 26
二、α-TOH、VES及α-TEA的處理對SK-Hep-1細胞侵入能力(invasion)
之影響……………………………………………………………26
三、α-TOH、VES及α-TEA的處理對SK-Hep-1細胞移動能力
(migration)之影響…………………………………………… 26
四、α-TOH、VES及α-TEA的處理對SK-Hep-1細胞MMP-9活性之影響27
五、α-TOH、VES及α-TEA的處理對SK-Hep-1細胞nm23-H1蛋白質表
現之影響……………………………………………………… 27
六、α-TOH、VES及α-TEA的處理對SK-Hep-1細胞黏附能力(adhesion)
之影響……………………………………………………………28
七、MMP-9活性與SK-Hep-1細胞侵入能力之相關性…………… 28
八、Nm23-H1蛋白質表現與SK-Hep-1細胞侵入能力之相關性…… 28

討論………………………………………………………………… 29

圖表………………………………………………………………… 32

文獻整理…………………………………………………………… 45

附錄

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